WO2005123127A2

WO2005123127A2 - Methods for treating cancer using prss15 as a target

Info

Publication number: WO2005123127A2
Application number: PCT/US2005/020418
Authority: WO
Inventors: Mary Jo Janatpour; Pablo Garcia; Christoph Reinhard
Original assignee: Novartis Vaccines And Diagnostics Inc.
Priority date: 2004-06-09
Filing date: 2005-06-09
Publication date: 2005-12-29

Abstract

PRSS 15, or human Lon protease, is upregulated in many cancers and contributes to the malignant phenotype. Detection of PRSS15 is useful to diagnose such cancers and modulation of PRSS 15 biological activity is useful to treat such cancers.

Description

METHODS FOR TREATING CANCER USING PRSS15 AS A TARGET

FIELD OF THE INVENTION [0001] This invention relates generally to cancer treatment, prophylaxis and detection, and more specifically to methods of using PRSS 15 as a target for the treatment, prophylaxis and/or detection of cancer.

BACKGROUND OF THE INVENTION [0002] PRSS 15, or human Lon protease enzyme, is a nuclear-encoded mitochondrial matrix protein. It is highly homologous to members of the bacterial Lon protease family and, like bacterial Lon protease, has an ATP-dependent protease activity. PRSS 15 also functions as a chaperone protein, analogous to heat shock proteins, and as a DNA binding protein.

[0003] Mitochondria are semi-autonomous organelles that generate adenosine triphosphate (ATP) through oxidative phosphorylation. Cell viability depends on mitochondria maintaining their integrity and performing this vital role. Mitochondrial proteins, however, are susceptible to oxidative damage, owing to their proximity to electron transport enzymes.

[0004] PRSS 15 appears to protect against this damage by recognizing oxidative changes to protein structure and rapidly degrading oxidized proteins, which could otherwise compromise mitochondrial function and cellular viability. Unlike other proteases, PRSS 15 contains ATPase and proteolytic domains on one molecule. The ATPase activity of PRSS 15 is not required for peptide bond hydrolysis per se, but is required to unfold or remodel target substrates prior to peptide bond hydrolysis. Based on knock-out studies in yeast and bacteria, both ATPase and protease domains are required for PRSS 15 to function properly.

[0005] PRSS 15 also may have other activities unrelated to proteolysis. In yeast, Lon proteins bind mitochondrial DNA directly, thereby stabilizing the mitochondrial genome and regulating mitochondrial gene expression. Lon proteins also facilitate assembly of oligomeric protein complexes.

[0006] Due to their key functions, Lon proteases are ubiquitously expressed in cells. PRSS15, specifically, is ubiquitously expressed in human cells.

SUMMARY OF THE INVENTION [0007] The present inventors have discovered that PRSS 15 is upregulated in certain cancers and that knock-out of PRSS15 inhibits growth of, and causes cytotoxicity to, cancer cells. In particular, the inventors observed these results in colon, prostate, breast and stomach cancers. These discoveries establish PRSS 15 as a target for cancer therapy and diagnostics.

[0008] The inventors further discovered that although cancer cell growth slows and cancer cells die upon treatment with a PRSS15 modulator, normal cells expressing PRSS15 do not respond negatively to such treatment. This fortuitous discovery indicates that PRSS15-targeted cancer therapy will specifically kill malignant cells, without harming normal cells that express PRSS 15.

[0009] Accordingly, the present invention provides a method of treating or preventing a cancer, including a cancer characterized by overexpression and/or upregulation of PRSS 15. The method includes administering a therapeutically or prophylactically effective amount of at least one PRSS 15 modulator, where the modulator decreases or inhibits proliferation of the cancer or causes cancer cell death. The modulator may be a polypeptide, such as an antibody, a polynucleotide, such as an antisense or RNAi oligonucleotide, or a small molecule. Preferably, the cancer is colon cancer, prostate cancer, breast or stomach cancer. In particular embodiments, the method suppresses or inhibits metastasis, aberrant cellular proliferation relative to a normal cell, loss of contact inhibition of cell growth, or loss of anchorage dependent growth.

[0010] In one aspect, the method of treating or preventing a cancer entails administering a therapeutically or prophylactically effective amount of at least one PRSS 15 modulator with at least one other therapeutic agent. The modulator may be co-administered with the other therapeutic agent; alternatively, the modulator and other therapeutic agent may be consecutively administered, in either order. [0011] In another aspect, the invention provides a PRSS 15 modulator suitable for treating or preventing a cancer, where the PRS>S15 modulator decreases or inhibits proliferation of the cancer or causes cancer cell death.

[0012] In yet another aspect, the invention provides a pharmaceutical composition useful for treating or preventing a cancer. The composition comprises a pharmaceutically effective amount of at least one PRSS15 modulator and a pharmaceutically acceptable carrier, where the modulator decreases or inhibits proliferation of the cancer or causes cancer cell death. The modulator may be a polypeptide, such as an antibody, a polynucleotide, such as an antisense or RNAi oligonucleotide, or a small molecule. Preferably, the cancer is colon cancer, prostate cancer, breast or stomach cancer.

[0013] In still another aspect, the invention provides a method of detecting a cancer, particularly a cancer characterized by overexpression and/or upregulation of PRSS15. The method entails (a) determining the level of expression of PRSS 15 in a biological sample obtained from a patient, (b) comparing the level of PRSS 15 expression in the patient biological sample to the level of PRSS 15 expression in a normal biological sample, and (c) correlating the level of PRSS 15 expression to a positive or negative diagnosis of the cancer.

[0014] A related aspect of the invention provides a method for determining a patient's predisposition to a cancer. The method entails (a) determining the level of expression of PRSS 15 in a biological sample obtained from a patient, (b) comparing the level of PRSS 15 expression in said patient biological sample to the level of PRSS 15 expression in a normal biological sample, and correlating the level of PRSS 15 expression to a diagnosis of a predisposition to the cancer.

[0015] The invention also provides a microarray comprising one or more polynucleotide sequences substantially identical to or complementary to the polynucleotide sequences of SEQ ID NO:2 or a portion of the polynucleotide sequence of SEQ ID NO:2.

[0016] The invention also provides a microarray comprising one or more protein- capture agents that bind one or more amino acid sequences encoded by all or a portion of the amino acid sequence SEQ ID NO:l. [0017] In another aspect, the invention provides a method of using a microarray to determine the presence or absence of a cancer. The method includes (a) determining the level of expression of PRSS 15 in a biological sample obtained from a patient, using the microarray, (b) comparing the level of PRSS 15 expression in the patient biological sample to the level of PRSS 15 expression in a normal biological sample, and (c) correlating the level of PRSS 15 expression in the patient biological sample to a positive or negative diagnosis of the cancer.

[0018] In a related aspect, the invention provides a method of using a microarray to determine a patient's predisposition to a cancer. The method includes (a) determining the level of expression of PRSS15 in a biological sample obtained from the patient, using a microarray, (b) comparing the level of PRSS 15 expression in the patient biological sample to the level of PRSS 15 expression in a normal biological sample, and (c) correlating the level of PRSS 15 expression in the patient biological sample to a diagnosis of a predisposition to the cancer.

[0019] In another aspect, the invention provides methods of identifying an agent that binds to PRSS 15 or modulates a biological activity of a PRSS 15 gene product. One such method includes (a) contacting at least one candidate modulator with PRSS 15 under conditions wherein said at least one candidate modulator can bind the PRSS15; and detecting the binding of said at least one candidate modulator to the PRSS 15.

[0020] Another such method includes (a) contacting a candidate modulator with a PRSS 15 gene product, and (b) detecting modulation of a biological activity of said gene product relative to a level of biological activity of said gene product in the absence of said candidate agent.

[0021] Still another such method comprises (a) culturing a cell line transfected with an expression vector comprising a gene encoding PRSS 15 to express the gene in a medium containing at least one candidate modulator of PRSS 15, and (b) measuring binding of the candidate modulator to the PRSS 15 produced by the cell line.

[0022] Yet another screening method includes (a) contacting a candidate modulator with a cancer cell, preferably a cancer cell characterized by overexpression and/or upregulation of PRSS 15, under conditions that allow the candidate modulator to bind the PRSS 15, and (b) detecting a decrease or inhibition of proliferation of the cancer cell relative to proliferation of a cell of the same type that has not contacted the candidate modulator.

[0023] In still another aspect, the invention provides methods of determining the ability of a drug to inhibit substrate cleavage of PRSS 15. One such method comprises the steps of (a) culturing a cell line transfected with an expression vector comprising a gene encoding PRSS 15 to express the gene (i) in the presence of the substrate and (ii) in the presence of both the substrate and the drug; and (b) comparing the level of substrate cleavage that occurs in (a)(i) and (a)(ii), where a lower level of substrate cleavage in the presence of the drug indicates that the drug is an inhibitor of substrate cleavage.

BRIEF DESCRIPTION OF THE DRAWINGS [0024] Figure 1 shows quantitative PCR results demonstrating upregulation of PRSS 15 mRNA in breast, colon and prostate cancers.

[0025] Figure 2 depicts the results of microarray analysis demonstrating upregulation of PRSS 15 mRNA in colon cancers.

[0026] Figure 3 depicts shows results demonstrating that PRSS 15 is ubiquitously expressed in normal tissues.

[0027] Figure 4 shows results demonstrating that PRSS 15 is upregulated in cell lines derived from tumors.

[0028] Figure 5 shows results demonstrating that PRSS 15 knock-out inhibits anchorage dependent growth in PC3 cells.

[0029] Figure 6 shows results demonstrating that PRSS 15 knock-out inhibits anchorage dependent growth in 22rVl cells.

[0030] Figure 7 shows results demonstrating that PRSS 15 knock-out inhibits anchorage dependent growth in HCT116 cells.

[0031] Figure 8 shows results demonstrating that PRSS 15 knock-out inhibits anchorage dependent growth in SW620 cells. [0032] Figure 9 shows results demonstrating that PRSS 15 knock-out inhibits anchorage independent growth in PC3 cells.

[0033] Figure 10 shows results demonstrating that PRSS 15 knock-out inhibits anchorage independent growth in 22rVl cells.

[0034] Figure 11 shows results demonstrating that PRSS 15 knock-out inhibits anchorage independent growth in HCT116 cells.

[0035] Figure 12 shows results demonstrating that PRSS 15 knock-out inhibits anchorage independent growth in SW620 cells.

[0036] Figure 13 shows results demonstrating that PRSS15 knock-out inhibits anchorage independent growth in LNCaP cells.

[0037] Figure 14 shows results demonstrating that PRSS 15 knock-out inhibits anchorage independent growth in 2b cells.

[0038] Figure 15 shows results demonstrating that PRSS 15 knock-out causes cytotoxicity to PC3 cells.

[0039] Figure 16 shows results demonstrating that PRSS 15 knock-out causes cytotoxicity to 22rVl cells.

[0040] Figure 17 shows results demonstrating that PRSS 15 knock-out causes cytotoxicity to HCT116 cells.

[0041] Figure 18 shows results demonstrating that PRSS15 knock-out causes cytotoxicity to SW620 cells.

[0042] Figure 19 shows results demonstrating that PRSS 15 knock-out causes cytotoxicity to DU145 cells.

[0043] Figure 20 shows results demonstrating that PRSS 15 knock-out does not induce significant cytotoxicity to normal cells.

[0044] Figure 21 shows the amino acid sequence of a full length PRSS 15 protein. [0045] Figure 22 shows the cDNA sequence of PRSS15.

[0046] Figure 23 shows results demonstrating that PRSS 15 knockout inhibits anchorage dependent growth in HT29, MDA 435 and HT1080 cells.

DETAILED DESCRIPTION OF THE INVENTION [0047] As noted above, the present inventors have discovered that PRSS15 is upregulated in certain cancers and that knock-out of PRSS 15 inhibits growth of and causes cytotoxicity to cancer cells. In particular, the inventors observed these results in colon, prostate, breast and stomach cancers. The inventors further discovered that although cancer cell growth slows and cancer cells die upon treatment with a PRSS15 modulator, normal cells expressing PRSS 15 do not respond negatively to such treatment. These fortuitous discoveries establish PRSS 15 as a target for cancer treatments and diagnostic, and indicate that PRSS15-targeted cancer therapy will specifically kill malignant cells, without harming normal cells that express PRSS 15.

[0048] The following description outlines the invention related to these discoveries. The invention, however, is not limited to the particular embodiments, methodology, protocols, or reagents described herein. Likewise, the terminology used herein describes particular embodiments only, and is not intended to limit the scope of the invention.

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the relevant art.

[0050] All publications and patents mentioned herein are incorporated herein by reference. Reference to a publication or patent, however, does not constitute an admission as to prior art.

I. Definitions [0051] For convenience, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below.

[0052] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. [0053] "Antisense gene" refers to a gene constructed by reversing the orientation of all or a portion of a gene with respect to its promoter so that the antisense strand is transcribed.

[0054] "Antisense oligonucleotide" refers to a nucleic acid molecule complementary to a portion of a particular gene transcript that can hybridize to the transcript and block its translation. An antisense oligonucleotide may comprise RNA or DNA.

[0055] "Biological activity" refers to the biological behavior and effects of a protein or peptide. The biological activity of a protein may be affected at the cellular level and the molecular level. For example, the biological activity of a protein may be affected by changes at the molecular level. For example, an antisense oligonucleotide may prevent translation of a particular mRNA, thereby inhibiting the biological activity of the protein encoded by the mRNA. In addition, an immunoglobulin may bind to a particular protein and inhibit that protein's biological activity.

[0056] "Biological sample" encompasses a variety of sample types obtained from an organism that may be used in a diagnostic or monitoring assay. The term encompasses blood and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen, or tissue cultures or cells derived therefrom and the progeny thereof. The term specifically encompasses a clinical sample, and further includes cells in cell culture, cell supematants, cell lysates, serum, plasma, urine, amniotic fluid, biological fluids, and tissue samples. The term also encompasses samples that have been manipulated in any way after procurement, such as treatment with reagents, solubilization, or enrichment for certain components.

[0057] "Biomolecular sequence" or "sequence" refers to all or a portion of a polynucleotide or polypeptide sequence.

[0058] "Biomolecule" includes polynucleotides and polypeptides.

[0059] "BLAST" refers to Basic Local Alignment Search Tool, a technique for detecting ungapped sub-sequences that match a given query sequence. "BLASTP" is a BLAST program that compares an amino acid query sequence against a protein sequence database. "BLASTX" is a BLAST program that compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database.

[0060] "Cancer," "neoplasm," "tumor," and "carcinoma," used interchangeably herein, refer to cells or tissues that exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. The methods and compositions of this invention particularly apply to precancerous (i.e., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells.

[0061] A "cancer characterized by the over expression and/or upregulation of PRSS 15" involves cells that exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation and in which PRSS 15 protein or PRSS 15 mRNA is expressed at higher levels than in corresponding normal cells. Preferred embodiments of the present invention relate to cancers characterized by the over expression and/or upregulation of PRSS 15.

[0062] "Cancerous phenotype" refers to any of a variety of biological phenomena that are characteristic of a cancerous cell. The phenomena can vary with the type of cancer, but the cancerous phenotype is generally identified by abnormalities in cell growth or proliferation, regulation of the cell cycle, cell mobility or cell-cell interaction.

[0063] "Cells that express PRSS 15" refers to any cell that expresses detectable levels of PRSS15. PRSS15 protein may be detected using methods such as enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), microarray methods or immunoflourescence. An mRNA encoding PRSS15 protein may be detected by Northern blots, polymerase chain reaction (PCR), microarray methods, or in situ hybridization. Other methods for detecting specific polynucleotides or polypeptides also are discussed herein and are well known to those skilled in the art.

[0064] "Cells that overexpress and/or upregulate PRSS 15" refers to cells wherein the PRSS 15 protein or mRNA transcript is expressed at higher levels than in corresponding normal cells. For example, in a cell that overexpresses and/or upregulates PRSS 15, the mRNA or protein may be produced at levels at least about 20% higher, at least about 25% higher, at least about 30% higher, at least about 35% higher, at least about 40% higher, at least about 45% higher, at least about 50% higher, at least about 55% higher, at least about 60% higher, at least about 65% higher, at least about 70% higher, at least about 75% higher, at least about 80% higher, at least about 85% higher, at least about 90% higher, at least about 95 % higher, at least about 100% or more higher, at least about 1.2-fold higher, at least about 1.5-fold higher, at least about 2-fold higher, at least about 5-fold higher, at least about 10-fold higher, or at least about 50-fold or more higher than that of a corresponding normal cell. The comparison may be made between different tissues or between different cells.

[0065] "Cell type" refers to a cell from a given source (e.g., tissue or organ) or a cell in a given state of differentiation, or a cell associated with a given pathology or genetic makeup.

[0066] "Complementary" refers to the topological compatibility or matching together of the interacting surfaces of a probe molecule and its target. The target and its probe can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other. Hybridization or base pairing between nucleotides or nucleic acids, such as, for example, between the two strands of a double- stranded DNA molecule or between an oligonucleotide probe and a target are complementary.

[0067] "Corresponds to" or "represents" when used in the context of, for example, a polynucleotide or sequence that "corresponds to" or "represents" a gene means that a sequence of the polynucleotide is present in the gene or in the nucleic acid gene product (e.g., mRNA). A subject nucleic acid may also be "identifiable" by a polynucleotide if the polynucleotide corresponds to or represents the gene. The polynucleotide may be wholly present within an exon of a genomic sequence of the gene, or different portions of the sequence of the polynucleotide may be present in different exons (e.g., such that the contiguous polynucleotide sequence is present in an mRNA, either pre- or post-splicing, that is an expression product of the gene).

[0068] "Diagnosis" and "diagnosing" generally includes a determination of a subject's susceptibility to a disease or disorder, a determination as to whether a subject is presently affected by a disease or disorder, a prognosis of a subject affected by a disease or disorder (e.g., identification of pre-metastatic or metastatic cancerous states, stages of cancer, or responsiveness of cancer to therapy), and therametrics (e.g., monitoring a subject's condition to provide information as to the effect or efficacy of therapy).

[0069] "Differential expression" refers to both quantitative as well as qualitative differences in the temporal and tissue expression patterns of a gene. For example, a differentially expressed gene may have its expression activated or completely inactivated in normal versus disease conditions. Such a qualitatively regulated gene may exhibit an expression pattern within a given tissue or cell type that is detectable in either control or disease conditions, but is not detectable in both. "Differentially expressed polynucleotide," as used herein, refers to a polynucleotide sequence that uniquely identifies a differentially expressed gene so that detection of the differentially expressed polynucleotide in a sample is correlated with the presence of a differentially expressed gene in a sample. "Differentially expressed protein," as used herein, refers to an amino acid sequence that uniquely identifies a differentially expressed protein so that detection of the differentially expressed protein in a sample is correlated with the presence of a differentially expressed protein in a sample.

[0070] "Expression" generally refers to the process by which a polynucleotide sequence undergoes successful transcription and translation such that detectable levels of the amino acid sequence or protein are expressed. In certain contexts herein, expression refers to the production of mRNA. In other contexts, expression refers to the production of protein.

[0071] An "expression product" or "gene product" is a biomolecule, such as a protein or mRNA, that is produced when a gene in an organism is transcribed or translated or post-translationally modified.

[0072] A "fragment of a protein" refers to a protein that is a portion of another protein. For example, fragments of proteins may comprise polypeptides obtained by digesting full-length protein isolated from cultured cells. In one embodiment, a protein fragment comprises at least about 6 amino acids. In another embodiment, the fragment comprises at least about 10 amino acids. In yet another embodiment, the protein fragment comprises at least about 16 amino acids.

[0073] In the context of PRSS 15, the term "functional equivalent" refers to a protein or polynucleotide molecule that possesses functional or structural characteristics that are substantially similar to all or part of the native PRSS 15 protein or native PRSS15-encoding polynucleotides. The term "functional equivalent" is intended to include the "fragments," "mutants," "derivatives," "alleles," "hybrids," "variants," "analogs," or "chemical derivatives" of native PRSS 15.

[0074] "Gene" refers to a polynucleotide sequence that comprises control and coding sequences necessary for the production of a polypeptide or precursor. The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence. A gene may constitute an uninterrupted coding sequence or it may include one or more introns, bound by the appropriate splice junctions. Moreover, a gene may contain one or more modifications in either the coding or the untranslated regions that could affect the biological activity or the chemical structure of the expression product, the rate of expression, or the manner of expression control. Such modifications include, but are not limited to, mutations, insertions, deletions, and substitutions of one or more nucleotides. In this regard, such modified genes may be referred to as "variants" of the "native" gene.

[0075] "Heterologous" means that the materials are derived from different sources, such as from different genes or different species.

[0076] "Host cell" refers to a microorganism, a prokaryotic cell, a eukaryotic cell or cell line cultured as a unicellular entity that may be, or has been, used as a recipient for a recombinant vector or other transfer of polynucleotides, and includes the progeny of the original cell that has been transfected. The progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent due to natural, accidental, or deliberate mutation.

[0077] "Hybridization" refers to any process by which a polynucleotide sequence binds to a complementary sequence through base pairing. Hybridization conditions can be defined by, for example, the concentrations of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art. Hybridization can occur under conditions of various stringency.

[0078] "Individual," "subject," "host," and "patient," used interchangeably herein, refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired. In one preferred embodiment, the individual, subject, host, or patient is a human. Other subjects may include, but are not limited to, cattle, horses, dogs, cats, guinea pigs, rabbits, rats, primates, and mice.

[0079] "Isolated" refers to a polynucleotide, a polypeptide, an immunoglobulin, or a host cell that is in an environment different from that in which the polynucleotide, the polypeptide, the immunoglobulin, or the host cell naturally occurs.

[0080] "Label" refers to agents that are capable of providing a detectable signal, either directly or through interaction with one or more additional members of a signal producing system. Labels that are directly detectable and may find use in the invention include fluorescent labels. Specific fluorophores include fluorescein, rhodamine, BODIPY, cyanine dyes and the like. The invention also contemplates the use of radioactive isotopes, such as ³⁵S, ³²P, ³H, and the like as labels. Colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex) beads may also be utilized. See, e.g., U.S. Patent Nos. 4,366,241; 4,277,437; 4,275,149; 3,996,345; 3,939,350; 3,850,752; and 3,817,837.

[0081] A "liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactant, which is useful for delivery of a drug to a mammal. PRSS 15 modulators and modulators of the invention may be delivered by a liposome. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.

[0082] "Modulate" refers to increasing or decreasing an indicated phenomenon. A PRSS 15 modulator increases or decreases one or more activities of PRSS 15, either directly or indirectly. For example, a PRSS 15 modulator may directly impact protein activity by binding to PRSS 15 or a PRSS 15 substrate, and may indirectly impact protein activity by affecting the transcription, translation or post-translational modification of PRSS 15.

[0083] "Normal," as used in the context of "normal cell," refers to a cell of an untransformed phenotype or exhibiting a morphology of a non-transformed cell of the tissue type being examined.

[0084] "Normal physiological conditions" means conditions that are typical inside a living organism or a cell. Although some organs or organisms provide extreme conditions, the intra-organismal and intra-cellular environment normally varies around pH 7 (i.e., from pH 6.5 to pH 7.5), contains water as the predominant solvent, and exists at a temperature above 0°C and below 50°C. The concentration of various salts depends on the organ, organism, cell, or cellular compartment used as a reference.

[0085] "Oligonucleotide" refers to a polynucleotide sequence comprising, for example, from about 10 nucleotides (nt) to about 1000 nt. Oligonucleotides for use in the invention are preferably from about 15 nt to about 150 nt, more preferably from about 150 nt to about 1000 nt in length. The oligonucleotide may be a naturally occurring oligonucleotide or a synthetic oligonucleotide.

[0086] "Modified oligonucleotide" and "Modified polynucleotide" refer to oligonucleotides or polynucleotides with one or more chemical modifications at the molecular level of the natural molecular structures of all or any of the bases, sugar moieties, internucleoside phosphate linkages, as well as to molecules having added substitutions or a combination of modifications at these sites. The internucleoside phosphate linkages may be phosphodiester, phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone internucleotide linkages, or 3'-3', 5'-3', or 5'-5' linkages, and combinations of such similar linkages. The phosphodiester linkage may be replaced with a substitute linkage, such as phosphorothioate, methylamino, methylphosphonate, phosphoramidate, and guanidine, and the ribose subunit of the polynucleotides may also be substituted (e.g., hexose phosphodiester; peptide nucleic acids). The modifications may be internal (single or repeated) or at the end(s) of the oligonucleotide molecule, and may include additions to the molecule of the internucleoside phosphate linkages, such as deoxyribose and phosphate modifications which cleave or crosslink to the opposite chains or to associated enzymes or other proteins. The terms "modified oligonucleotides" and "modified polynucleotides" also include oligonucleotides or polynucleotides comprising modifications to the sugar moieties (e.g., 3 '-substituted ribonucleotides or deoxyribonucleotide monomers), any of which are bound together via 5' to 3' linkages.

[0087] "Oligonucleotide probe" refers to an oligonucleotide that may recognize a particular target. Depending on context, the term "oligonucleotide probes" refers both to individual oligonucleotide molecules and to a collection of oligonucleotide molecules. In one aspect, an oligonucleotide probe comprises one or more polynucleotide sequences substantially identical to a target polynucleotide sequence or complementary sequence thereof, or portions of the target polynucleotide sequence or complementary sequence thereof.

[0088] "Pharmaceutically acceptable" refers to physiological compatibility. A pharmaceutically acceptable carrier is a carrier that does not adversely affect the potency of a pharmaceutical agent and does not cause an adverse biological reaction within a recipient.

[0089] "Polynucleotide" and "nucleic acid," used interchangeably herein, refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, these terms include, but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. These terms further include, but are not limited to, mRNA or cDNA that comprise intronic sequences. The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidites and thus can be an oligodeoxynucleoside phosphoramidate or a mixed phosphoramidate-phosphodiester oligomer. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars, and linking groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support. The term "polynucleotide" also encompasses peptidic nucleic acids. Polynucleotides may further comprise genomic DNA, cDNA, or DNA-RNA hybrids.

[0090] "Polypeptide" and "protein," used interchangeably herein, refer to a polymeric form of amino acids of any length, which may include translated, untranslated, chemically modified, biochemically modified, and derivatized amino acids. A polypeptide or protein may be naturally occurring, recombinant, or synthetic, or any combination of these. Moreover, a polypeptide or protein may comprise a fragment of a naturally occurring protein or peptide. A polypeptide or protein may be a single molecule or may be a multi-molecular complex. In addition, such polypeptides or proteins may have modified peptide backbones. The terms include fusion proteins, including fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N- terminal methionine residues, immunologically tagged proteins, and the like.

[0091] "Predisposition" to a cancer, disease, or disorder refers to an individual's susceptibility to such cancer, disease, or disorder. Individuals who are susceptible are statistically more likely to have cancer, for example, as compared to normal/wildtype individuals.

[0092] "Prodrug" refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor or other cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. Prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate- containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which may be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use in this invention include, but are not limited to, chemotherapeutic agents described herein.

[0093] A "prophylactically effective amount" refers to an amount of a PRSS 15 modulator or modulator that is effective for preventing a cancer.

[0094] "PRSS 15" is human Lon protease, also designated LonHS, LonP and serine protease 15.

[0095] "PRSS15 agents" refers to a class of molecules that consists of "PRSS15 binding partners" and "PRSS15 modulators." [0096] "PRSS 15 binding partner" refers to a molecule that binds to PRSS 15 polypeptides or PRSS 15 polynucleotides. Exemplary polypeptide binding partners are immunoglobulins. Exemplary polynucleotide binding partners are oligonucleotide probes and antisense molecules. Binding partners may, but need not, modulate a PRSS 15 biological activity.

[0097] "PRSS 15 modulator" refers to a molecule that either increases or decreases a biological activity of PRSS 15. Modulators may act directly on a PRSS15 polynucleotide or polypeptide, or may effect their modulation indirectly. A PRSS 15 modulator may be a "binding partner" that binds to PRSS 15 polypeptides or polynucleotides and inhibits the proliferation of a cancer, preferably a characterized by overexpression and/or upregulation of PRSS15. A PRSS15 modulator may be considered to a be a therapeutic agent. In one embodiment of the invention, a PRSS 15 modulator is a polypeptide, i.e., a polypeptide PRSS 15 modulator. Examples of polypeptide PRSS 15 modulators include immunoglobulins (antibodies), and functional equivalents thereof, peptides generated by rational design, etc. In another embodiment, a PRSS15 modulator is a polynucleotide, i.e., a polynucleotide PRSS15 modulator. Polynucleotide PRSS 15 modulators include antisense oligonucleotides, RNAi oligonucleotides, ribozymes, and peptide nucleic acids. In yet another embodiment, a PRSS 15 modulator may comprise a small molecule, i.e., a small molecule PRSS 15 modulator.

[0098] "RNA interference" (RNAi) refers to sequence-specific or gene specific suppression of gene expression (protein synthesis) that is mediated by short interfering RNA

(siRNA).

[0099] "Sequence Identity" refers to a degree of similarity or complementarity. There may be partial identity or complete identity. A partially complementary sequence is one that at least partially inhibits an identical sequence from hybridizing to a target polynucleotide; it is referred to using the functional term "substantially identical." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially identical sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely identical sequence or probe to the target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence.

[0100] Another way of viewing sequence identity in the context to two nucleic acid or polypeptide sequences includes reference to residues in the two sequences that are the same when aligned for maximum correspondence over a specified region. As used herein, percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0101] "Short interfering RNA" (siRNA) refers to double-stranded RNA molecules from about 10 to about 30 nucleotides long that are named for their ability to specifically interfere with protein expression.

[0102] A "small molecule" comprises a compound or molecular complex, either synthetic, naturally derived, or partially synthetic, composed of carbon, hydrogen, oxygen, and nitrogen, which may also contain other elements, and which may have a molecular weight of less than about 15,000, less than about 14,000, less than about 13,000, less than about 12,000, less than about 11,000, less than about 10,000, less than about 9,000, less than about 8,000, less than about 7,000, less than about 6,000, less than about 5,000, less than about 4,000, less than about 3,000, less than about 2,000, less than about 1,000, less than about 900, less than about 800, less than about 700, less than about 600, less than about 500, less than about 400, less than about 300, less than about 200, or less than about 100. [0103] "Stringent conditions" refers to conditions under which a probe may hybridize to its target polynucleotide sequence, but to no other sequences. Stringent conditions are sequence-dependent (e.g., longer sequences hybridize specifically at higher temperatures). Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength, pH, and polynucleotide concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Typically, stringent conditions will be those in which the salt concentration is at least about 0.01 to about 1.0 M sodium ion concentration (or other salts) at about pH 7.0 to about pH 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.

[0104] "Substantially purified" refers to a compound that is removed from its natural environment and is at least about 60% free, at least about 65% free, at least about 70% free, at least about 75% free, at least about 80% free, at least about 83% free, at least about 85% free, at least about ,88% free, at least about 90% free, at least about 91% free, at least about 92% free, at least about 93% free, at least about 94% free, at least about 95% free, at least about 96% free, at least about 97% free, at least about 98% free, at least about 99% free, at least about 99.9% free, or at least about 99.99% or more free from other components with which it is naturally associated.

[0105] "Therapeutically effective amount" refers to an amount of a PRSS 15 modulator or modulator that is effective for preventing, ameliorating, treating or delaying the onset of a cancer.

[0106] The terms "treatment," "treating," "treat," and the like refer to obtaining a desired pharmacological and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. "Treatment" covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom.

π. PRSS15 Polynucleotides [0107] In one aspect, the invention relates to "PRSS 15 polynucleotides," which refers to polynucleotides that encode a native PRSS 15 polypeptide or a variant thereof. The cDNA polynucleotide sequence for human PRSS 15 is set forth in SEQ ID NO:2. PRSS 15 polynucleotides of the invention include those represented by this sequence and sequences substantially identical to this polynucleotide sequence, by portions of such polynucleotide sequences and by polynucleotide sequences that are complementary to any such sequences. PRSS 15 polynucleotides also include variants of the provided polynucleotides and their corresponding genes, particularly those variants that retain a biological activity of PRSS 15.

[0108] The invention features PRSS15 polynucleotides that are expressed in human tissue and upregulated in human cancers, particularly prostate, colon, stomach and breast cancers. The polynucleotides may comprise a sequence set forth herein or an identifying sequence thereof. An "identifying sequence" is a contiguous sequence of residues at least about 10 nt to about 20 nt in length, usually at least about 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, 100 nt to at least about 200 nt, 300 nt, 400 nt, 500 nt or more in length, that uniquely identifies or defines a PRSS 15 nucleotide sequence, its complements and degenerate variants thereof. An identifying sequence, e.g., exhibits about 100% sequence identity, at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 90%, at least about 85%, at least about 80% or at least about 75% sequence identity to any contiguous nucleotide sequence of more than about 20 nt in SEQ ID NO:2.

[0109] PRSS 15 polynucleotides can be DNAs, including cDNAs and genomic DNAs, or RNAs, including mRNA, as well as fragments thereof, particularly fragments that encode a biologically active gene product and/or are useful in the methods disclosed herein (e.g., in diagnosis, as a unique identifier of a differentially expressed gene of interest, etc.). The term "cDNA" includes all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons and 3' and 5' non-coding regions. Normally mRNA species have contiguous exons, with the intervening introns, when present, being removed by nuclear RNA splicing, to create a continuous open reading frame encoding a polypeptide. mRNA species can also exist with both exons and introns, where the introns may be removed by alternative splicing. Furthermore it should be noted that different species of mRNAs encoded by the same genomic sequence can exist at varying levels in a cell, and detection of these various levels of mRNA species can be indicative of differential expression of the encoded gene product in the cell.

[0110] A genomic sequence of interest comprises the nucleic acid present between the initiation codon and the stop codon, including all of the introns that are normally present in a native chromosome. It can further include the 3' and 5' untranslated regions found in the mature mRNA. It can further include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including about 1 kb, but possibly more, of flanking genomic DNA at either the 5' and 3' end of the transcribed region.

[0111] PRSS15 polynucleotides also include naturally occurring variants, e.g., degenerate variants, allelic variants, and single nucleotide polymorphisms (SNPs), of the sequences provided herein. Additionally, the variants forms of PRSS 15 polynucleotides include mutants and fragments. Mutant PRSS 15 polynucleotide variants may result from nucleotide substitutions, deletions, and insertions. In general, variants of the PRSS 15 polynucleotides described herein have a sequence identity greater than at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 83%, at least about 85%, at least about 88%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9% or may be greater than at least about 99.99% as determined by methods well known in the art, such as the Smith- Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular).

[0112] A fragment of a subject nucleic acid is, for example, a polynucleotide having a nucleic acid sequence which is a portion of a PRSS 15 nucleic acid or its complement. The nucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, at least about 100 nt, at least about 125 nt or at least about 150 nt in length. Of course, larger fragments (e.g., at least 150, 175, 200, 250, 500, 600, 1000, or 2000 nucleotides in length) are also encompassed by the invention. [0113] Sequence identity or sequence similarity may be calculated based on a PRSS 15 reference sequence, which may be a subset of a larger PRSS 15 sequence, such as part of the coding region, flanking region, or a conserved motif. A reference sequence may be at least about 18 contiguous nt long, alternatively at least about 30 nt long, and may extend to the complete sequence that is being compared. Moreover, the reference sequence may comprise nucleotides that encode the amino acids that together constitute an epitope or domain of PRSS 15. Algorithms for sequence analysis are known in the art, such as gapped BLAST, described in Altschul et al., 25 NUCL. ACIDS RES. 3389-3402 (1997).

[0114] Alternatively, sequence analysis may also be based on, but not limited to, the GCG Bestfit and Gap programs, which align two sequences either with the best local alignment (Bestfit) or a global alignment (gap), respectively. Local alignments are an optimal alignment of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman. In contrast, a global alignment considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. Global alignments are created with the algorithm of Needleman and Wunsch, 48 J. MOL. BlOL. 443-53 (1970).

[0115] Probes specific to the PRSS 15 polynucleotides may be generated using the PRSS15 polynucleotide sequences disclosed herein. PRSS15 probes may be designed based on a subset of the PRSS 15 polynucleotide sequence, such as part of the coding region, flanking region, or a conserved motif. A PRSS 15 probe may exhibit about 99.99%, about 99.9%, about 99%, about 98%, about 97%, about 96%, about 95%, about 94, about 93%, about 92%, about 91%, about 90%, about 88%, about 85%, about 83%, about 80%, about 75%, about 70%, or about 65% sequence identity to any contiguous nucleotide sequence of more than about 15 nt. A PRSS 15 probe may comprise a contiguous sequence of nucleotides at least about 10 nt, at least about 12 nt, at least about 15 nt, at least about 16 nt, at least about 18 nt, at least about 20 nt, at least about 22 nt, at least about 24 nt, or at least about 25 nt in length that uniquely identifies a polynucleotide sequence. Moreover, a PRSS 15 probe may be at least about 30 nt, at least about 35 nt, at least about 40 nt, at least about 45, at least about 50 nt, at least about 55nt, at least about 60 nt, at least about 70 nt, at least about 75 nt, at least about 80 nt, at least about 85 nt, at least about 90 nt, at least about 95 nt, at least about 100 nt, at least about 150 nt, at least about 200 nt, at least about 250 nt, at least about 300 nt, at least about 350 nt, at least about 400 nt, at least about 450 nt, at least about 500 nt, at least about 550 nt, at least about 600 nt, at least about 650 nt, at least about 700 nt, at least about 750 nt, at least about 800 nt, at least about 900 nt, at least about 950 nt, at least about 1000 nt, or more nucleotides in length.

[0116] Probes can be synthesized chemically or can be generated from longer polynucleotides using restriction enzymes. The probes can be labeled, for example, with a radioactive, biotinylated, or fluorescent tag.

[0117] The PRSS 15 polynucleotides described herein are useful to produce polypeptides for producing anti- PRSS 15 immunoglobulin, as probes to determine the presence or absence of PRSS 15 polynucleotides or variants thereof in a biological sample, and to generate ribozymes, antisense oligonucleotides, and additional copies of the PRSS 15 polynucleotides.

[0118] PRSS 15 polynucleotides are provided in a non-naturally occurring environment, i.e., are separated from their naturally occurring environment. In certain embodiments, PRSS15 protein is present in a substantially purified form.

m. PRSS 15 Polypeptides [0119] In another aspect, the invention relates to "PRSS 15 polypeptides," which includes polypeptides encoded by PRSS15 nucleic acids. SEQ ID NO:l contains the amino acid sequence of a full length PRSS15 protein. In general, "PRSS15 polypeptide" refers to a full length PRSS15 protein encoded by a PRSS15 gene, as well as portions or fragments thereof. PRSS 15 polypeptides also include variants of the naturally occurring proteins, where such variants are identical or substantially similar to the naturally occurring protein. In general, variant polypeptides have a sequence that has at least about 80%, usually at least about 90%, and more usually at least about 98% sequence identity with a PRSS 15 polypeptide described herein, as measured by BLAST. Variant polypeptides can be naturally or non-naturally glycosylated.

[0120] In general, variants of the PRSS 15 polypeptides described herein have a sequence identity greater than at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 83%, at least about 85%, at least about 88%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9% or may be greater than at least about 99.99% as determined by methods well known in the art, such as BLAST.

[0121] In one embodiment, a variant PRSS 15 polypeptide may be a mutant polypeptide. The mutations in the PRSS 15 polypeptide may result from, but are not limited to, amino acid substitutions, additions or deletions. The amino acid substitutions may be conservative amino acid substitutions or substitutions to eliminate non-essential amino acids. In general, conservative amino acid substitutions are those that preserve the general charge, hydrophobicity, hydrophilicity, and/or steric bulk of the amino acid substituted.

[0122] In some mutant PRSS 15 polypeptides, amino acids may be substituted to alter a glycosylation site, a phosphorylation site or an acetylation site. In a specific embodiment, the substitution or deletion of one or more cysteine residues that are not necessary for function may help to minimize misfolding of the PRSS 15 polypeptide.

[0123] Importantly, variant polypeptides may be designed so as to retain or have enhanced biological activity of a particular region of the protein (e.g., a functional domain and/or, where the polypeptide is a member of a protein family, a region associated with a consensus sequence). Selection of amino acid alterations for production of variants may be based upon the accessibility (interior vs. exterior) of the amino acid, the thermostability of the variant polypeptide, desired glycosylation sites, desired disulfide bridges, desired metal binding sites, and desired substitutions within proline loops. Cysteine-depleted muteins can be produced as disclosed in USPN 4,959,314.

[0124] Variants also include fragments of the PRSS 15 polypeptides disclosed herein, particularly biologically active fragments and fragments corresponding to functional domains. Fragments of interest will typically be at least about 10 aa to at least about 15 aa in length, usually at least about 50 aa in length, and can be as long as 300 aa in length or longer. The protein variants described herein are encoded by polynucleotides that are within the scope of the invention.

[0125] PRSS 15 polypeptides of the invention are provided in a non-naturally occurring environment, e.g., are separated from their naturally occurring environment. In certain embodiments, PRSS 15 protein is present in a substantially purified form. IV. PRSS 15 Agents: Modulators and Binding Partners [0126] In another aspect, the invention provides a "PRSS 15 agent," which refers to a class of molecules that consists of "PRSS 15 binding partners" and "PRSS 15 modulators."

[0127] PRSS15 binding partners are molecules that bind to PRSS15 polypeptides or PRSS 15 polynucleotides. Exemplary polypeptide binding partners are immunoglobulins. Exemplary polynucleotide binding partners are oligonucleotide probes and antisense molecules. Binding partners may, but need not, modulate a PRSS 15 biological activity.

[0128] PRSS 15 modulators are molecules that either increase or decrease a biological activity of PRSS15. Modulators may act directly on a PRSS15 polynucleotide or polypeptide, or may effect their modulation indirectly. A PRSS 15 modulator may also be a binding partner. In one embodiment of the invention, a PRSS 15 modulator is a polypeptide, i.e., a polypeptide PRSS15 modulator. Examples of polypeptide PRSS15 modulators include immunoglobulins (antibodies), and functional equivalents thereof, peptides generated by rational design, etc. In another embodiment, a PRSS15 modulator is a polynucleotide, i.e., a polynucleotide PRSS15 modulator. Polynucleotide PRSS15 modulators include antisense oligonucleotides, RNAi oligonucleotides, ribozymes, and peptide nucleic acids. In yet another embodiment, a PRSS 15 modulator may comprise a small molecule, i.e., a small molecule PRSS 15 modulator. Preferred modulators are suitable for treating or preventing a cancer, more preferably a cancer characterized by overexpression and/or upregulation of PRSS15.

A. Immuno globulins [0129] PRSS 15 agents include immunoglobulins and functional equivalents of immunoglobulins that specifically bind to PRSS 15 polypeptides. The terms "immunoglobulin" and "antibody" are used interchangeably and in their broadest sense herein. Thus, they encompass intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. In one embodiment, the subject immunoglobulins comprise at least one human constant domain. In another embodiment, the PRSS 15 immunoglobulins comprise a constant domain that exhibits at least about 90-95% sequence identity with a human constant domain and yet retains human effector function. An immunoglobulin PRSS 15 agent or functional equivalent thereof may be human, chimeric, humanized, murine, CDR-grafted, phage-displayed, bacteria-displayed, yeast-displayed, transgenic-mouse produced, mutagenized, and randomized. i. Antibodies Generally [0130] The terms "antibody" and "immunoglobulin" cover fully assembled antibodies and antibody fragments that can bind antigen ( e.g., Fab', F'(ab)₂, Fv, single chain antibodies, diabodies), including recombinant antibodies and antibody fragments. Preferably, the immunoglobulins or antibodies are chimeric, human, or humanized.

[0131] The variable domains of the heavy and light chain recognize or bind to a particular epitope of a cognate antigen. The term "epitope" is used to refer to the specific binding sites or antigenic determinant on an antigen that the variable end of the immunoglobulin binds. Epitopes can be linear, i.e., be composed of a sequence of amino acid residues found in the primary PRSS 15 sequence. Epitopes also can be conformational, such that an immunoglobulin recognizes a 3-D structure found on a folded PRSS 15 molecule. Epitopes can also be a combination of linear and conformational elements. Further, carbohydrate portions of a molecule, as expressed by the target bearing tumor cells can also be epitopes.

[0132] Immunoglobulins are said to be "specifically binding" if: 1) they exhibit a threshold level of binding activity, and/or 2) they do not significantly cross-react with known related polypeptide molecules. The binding affinity of an immunoglobulin can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949). In some embodiments, the immunoglobulins of the present invention bind to PRSS 15 at least 10³, more preferably at least IO⁴, more preferably at least 10⁵, and even more preferably at least IO⁶ fold higher than to other proteins ii. Polyclonal and Monoclonal Antibodies [0133] Immunoglobulins of the invention may be polyclonal or monoclonal, and may be produced by any of the well known methods in this art.

[0134] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc), intraperitoneal (ip) or intramuscular (im) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, In addition, aggregating agents such as alum are suitably used to enhance the immune response.

[0135] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen.

[0136] In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized while uncontaminated by other immunoglobulins. For example, monoclonal antibodies may be produced by the hybridoma method or by recombinant DNA methods. Monoclonal antibody PRSS 15 agents also may be isolated from phage antibody libraries. iii. Chimeric and Humanized Antibodies [0137] PRSS15-binding immunoglobulins or antibodies can be "chimeric" in the sense that a variable region can come from a one species, such as a rodent, and the constant region can be from a second species, such as a human.

[0138] "Humanized" forms of non-human PRSS 15-binding antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody.

[0139] In general, the humanized antibody may comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In one embodiment, humanized antibodies comprise a humanized FR that exhibits at least 65% sequence identity with an acceptor (non-human) FR, e.g., murine FR. The humanized antibody also may comprise at least a portion of an immunoglobulin constant region (Fc), particularly a human immunoglobulin.

[0140] Methods for humanizing non-human antibodies have been described in the art. Preferably, a humanized antibody has one or more amino acid residues introduced into it from a source, which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization may be essentially performed by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity.

[0141] Other methods generally involve conferring donor CDR binding affinity onto an antibody acceptor variable region framework. One method involves simultaneously grafting and optimizing the binding affinity of a variable region binding fragment. Another method relates to optimizing the binding affinity of an antibody variable region. iv. Antibody Fragments [0142] "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab') , Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0143] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment. The Fab fragments also contain the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. [0144] Pepsin treatment yields an F(ab')² fragment that has two antigen-binding sites and is still capable of crosslinking antigen. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CFfl domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear at least one free thiol group. F(ab')² antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are well known in the art.

[0145] "Fv" is the minimum antibody fragment that contains a complete antigen- recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen- binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0146] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. See PLUCKTHUN, 113 THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES 269-315 (Rosenburg and Moore eds. 1994). See also WO 93/16185; U.S. Patent Nos. 5,587,458 and 5,571,894.

[0147] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies. However, these fragments may now be produced directly by recombinant host cells. v. Conjugation and Labeling [0148] Anti-PRSS15 antibodies may be administered in their "naked" or unconjugated form, or may have other agents conjugated to them. [0149] For examples the antibodies may be in detectably labeled form. Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, and the like. Procedures for accomplishing such labeling are well known in the art. vi. Bispecific Antibodies [0150] Bispecific antibodies of the invention are small antibody fragments with two antigen-binding sites. Each fragment comprises a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites.

[0151] Methods for making bispecific antibodies are well known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities.

[0152] In another approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) may be fused to immunoglobulin constant domain sequences. Specifically, the variable domains are fused with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. In one embodiment, the fusion protein comprises the first heavy-chain constant region (CHI) because it contains the site necessary for light chain binding. Polynucleotides encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, may be inserted into separate expression vectors and co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance. [0153] Bispecific antibodies also have been produced using leucine zippers. And single-chain Fv (sFv) dimers.

B. Non-Immuno lobulin Polypeptide Agents [0154] In another embodiment, a PRSS 15 agent may be a peptide generated by rational design or by phage display. For example, the peptide may be a "CDR mimic" or immunoglobulin analogue based on the CDRs of an immunoglobulin. Even though such peptides may have the ability, by themselves, to decrease or inhibit the proliferation of a cancer, the peptide may be fused to a therapeutic agent to add or enhance the properties of the peptide.

C. Antisense Oligonucleotides [0155] Polynucleotide PRSS 15 agents may comprise one or more antisense oligonucleotide PRSS15 agents. In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or variants thereof. Oligonucleotides may comprise naturally occurring nucleotides, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non- naturally occurring portions that function similarly. Such modified or substituted oligonucleotides possess desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for polynucleotide target and increased stability in the presence of nucleases.

[0156] In general, antisense oligonucleotides specifically hybridize with one or more polynucleotides encoding PRSS 15 and interfere with the normal function of the polynucleotides. In one embodiment, an antisense oligonucleotide PRSS 15 agent may target DNA encoding PRSS 15 and interfere with its replication and/or transcription. In another embodiment, an antisense oligonucleotide PRSS 15 agent specifically hybridizes with RNA, including pre-mRNA and mRNA. Such antisense oligonucleotide PRSS 15 agents may affect, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity that may be engaged in or facilitated by the RNA. The overall effect of such interference with target PRSS 15 polynucleotide function is to modulate, decrease, or inhibition PRSS 15 expression. [0157] There are several sites within the PRSS 15 gene that may be utilized in designing an antisense oligonucleotide. For example, an antisense oligonucleotide PRSS 15 agent may bind the region encompassing the translation initiation codon, also known as the start codon, of the open reading frame of PRSS 15. In this regard, "start codon and "translation initiation codon" generally refer to the portion of such mRNA or gene that encompasses from at least about 25 to at least about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon.

[0158] Another intragenic site for antisense interaction to occur is the termination codon of the open reading frame. The terms "stop codon region" and "translation termination codon region" refer generally to a portion of such a mRNA or gene that encompasses from at least about 25 to at least about 50 contiguous nucleotides in either direction form a translation termination codon.

[0159] The open reading frame or coding region is also a region that may be targeted effectively. The open reading frame is generally understood to refer to the region between the translation initiation codon and the translation termination codon. Another target region is the 5' untranslated region, which is the portion of a mRNA in the 5' direction from the translation initiation codon. It includes the nucleotides between the 5' cap site and the translation initiation codon of a mRNA or corresponding nucleotides on the gene.

[0160] Similarly, the 3' untranslated region may be used as a target for antisense oligonucleotide PRSS 15 agents. The 3' untranslated region is that portion of the mRNA in the 3' direction from the translation termination codon, and thus includes the nucleotides between the translation termination codon and the 3' end of a mRNA or corresponding nucleotides of the gene.

[0161] An antisense oligonucleotide PRSS15 agent may also target the 5' cap region of a PRSS 15 mRNA. The 5' cap comprises an N7-methylated guanosine residue joined to the 5'-most residue of the mRNA via 5'-5' triphosphate linkage. The 5' cap region is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap.

[0162] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more intron regions, which are excised from a transcript before it is translated. The remaining (and therefore translated) exon regions are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, represent possible target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Moreover, aberrant fusion junctions due to rearrangements or deletions are also possible targets for antisense oligonucleotide PRSS 15 agents.

[0163] With these various target sites in mind, antisense oligonucleotide PRSS 15 agents that are sufficiently complementary to the target PRSS 15 polynucleotides must be chosen. There must be a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the PRSS 15 polynucleotide target. Importantly, the sequence of an antisense oligonucleotide PRSS 15 agent need not be 100% complementary to that of its target PRSS 15 polynucleotide to be specifically hybridizable. An antisense oligonucleotide PRSS 15 agent is specifically hybridizable when binding of the antisense oligonucleotide to the target PRSS 15 polynucleotide interferes with the normal function of the target PRSS 15 polynucleotide to cause a loss of utility, and there is a sufficient degree of complementarity to avoid nonspecific binding of the antisense oligonucleotide PRSS 15 agent to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.

[0164] The antisense oligonucleotide PRSS 15 agents may be at least about 8 nt to at least about 50 nt in length. In one embodiment, the antisense oligonucleotide PRSS15 binding partners may be about 12 to about 30 nt in length. As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.

[0165] For those nucleosides that include a pentofuranosyl sugar, the phosphate group may be linked to the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

[0166] Specific examples of antisense oligonucleotide PRSS 15 agents useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As used herein, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0167] Modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 '-5' linkages, 2 '-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. See, e.g., U.S. Patent Nos. 5,625,050; 5,587,361; 5,571,799; 5,563,253; 5,550,111; 5,541,306; 5,536,821; 5,519,126; 5,476,925; 5,466,677; 5,455,233; 5,453,496; 5,405,939; 5,399,676; 5,321,131; 5,286,717.

[0168] Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts. See, e.g., U.S. Patent Nos.: 5,677,439; 5,677,437; 5,633,360; 5,663,312; 5,623,070; 5,618,704; 5,610,289; 5,608,046; 5,602,240.

[0169] In other oligonucleotide "mimetics," the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate polynucleotide target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. See, e.g., U.S. Patent Nos. 5,539,082; 5,714,331; 5,719,262; Nielsen et al., 254 SCIENCE 1497- 1500 (1991).

[0170] Modified oligonucleotides may also contain one or more substituted sugar moieties. See, e.g., U.S. Patent Nos. 5,700,920; 5,670,633; 5,658,873; 5,646,265; 5,639,873; 5,627,053; 5,610,300; 5,597,909; 5,591,722.

[0171] Antisense oligonucleotide PRSS 15 agents may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleotides include other synthetic and natural nucleotides such as 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5- propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. See, e.g., U.S. Patent Nos. 5,750,692; 5,681,941; 5,614,617; 5,596,091; 5,594,121; 5,587,469; 5,552,540; 5,525,711; 5,502,177; 5,484,908; 5,459,255; 5,457,187; 5,432,272; 5,367,066; 5,175,273; 5,134,066. [0172] Another modification of the antisense oligonucleotide PRSS15 agents involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., 86 PROC. NATL. ACAD. SCI. USA 6553-56 (1989)), cholic acid (Manoharan et al, 4 BIOORG. MED. CHEM. LET. 1053-60(1994)), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., 660 ANN. N.Y. ACAD. SCI. 306-09 (1992); Manoharan et al., 3 BIOORG. MED. CHEM. LET. 2765-70 (1993)), a thiocholesterol (Oberhauser et al., 20 NUCL. ACIDS RES. 533-38 (1992)), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., 10 EMBO J. 1111-18 (1991); Kabanov et al., 259 FEBS LETT. 327-30 (1990); Svinarchuk et al., 75 BIOCHΠVΠE 49-54 (1993)), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l,2-di-o-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., 36 TETRAHEDRON LETT. 3651-54 (1995); Shea et al., 18 NUCLEIC. ACIDS RES. 3777-83 (1990)), a polyamine or a polyethylene glycol chain (Manoharan et al., 14 NUCLEOSIDES & NUCLEOTIDES 969-73 (1995)), or adamantane acetic acid (Manoharan et al., 36 TETRAHEDRON LETT. 3651-54 (1995)), a palmityl moiety (Mishra et al., 1264 BIOCHEM. BIOPHYS. ACTA 229-37 (1995)), or an octadecylamine or hexylamino-carbonyl- oxycholesterol moiety (Crooke et al., 277 J. PHARMACOL. EXP. THER.923-37 (1996). See generally U.S. Patent Nos. 5,688,941; 5,599,928; 5,599,923; 5,597,696; 5,595,726; 5,587,371; 5,585,481; 5,574,142; 5,567,810; 5,565,552; 5,514,785; 5,512,667; 5,510,475; 5,451,463; 5,416,203, 5,391,723; 5,371,241, 5,317,098; 5,292,873; 5,272,250; 5,262,536; 5,258,506; 5,254,469; 5,245,022; 5,214,136; 5,112,963; 5,082,830.

[0173] It is not necessary for all positions in a given antisense oligonucleotide PRSS 15 agent to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.

[0174] The invention also includes chimeric antisense oligonucleotide PRSS 15 agents. "Chimeric" antisense oligonucleotides or "chimeras," in the context of this invention, are antisense oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target polynucleotide.

[0175] Chimeric antisense oligonucleotide PRSS 15 agents may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. See, e.g., U.S. Patent Nos. 5,700,922; 5,652,356; 5,652,355; 5,623,065; 5,565,350; 5,491,133; 5,403,711.

[0176] An additional region of the antisense oligonucleotide PRSS 15 agents may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of antisense oligonucleotide inhibition of PRSS 15 gene expression. Consequently, comparable results may be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same PRSS 15 target region. Cleavage of the RNA target may be routinely detected by gel electrophoresis and, if necessary, associated polynucleotide hybridization techniques known in the art.

[0177] The antisense oligonucleotide PRSS 15 agents used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0178] Table 2, in the examples section below, contains the sequences of several preferred antisense oligonucleotides.

D. Ribozymes [0179] The invention further embraces other polynucleotide PRSS 15 agents, such as, ribozymes, that inhibit PRSS15 expression. Ribozymes are RNA molecules having an enzymatic activity that is able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence-specific manner. Such enzymatic RNA molecules may be targeted to virtually any RNA transcript, and efficient cleavage achieved in vitro. See generally Kim et al., 84 PROC. NATL. ACAD. SCI. USA 8788 (1987); Haseloff & Gerlach, 334 NATURE 585 (1988); Cech, 260 JAMA 3030 (1988); Jefferies et al., 17 NUCL. ACIDS RES. 1371 (1989).

[0180] Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. In general, enzymatic polynucleotides act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic polynucleotide which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic polynucleotide first recognizes and then binds a target RNA through complementary base- pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic polynucleotide has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

[0181] The enzymatic nature of a ribozyme may be advantageous over other technologies, such as antisense technology (where a polynucleotide molecule simply binds to a polynucleotide target to block its translation) because the effective concentration of ribozyme necessary to effect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA.

[0182] In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. In other words, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, it is likely that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site. [0183] The ribozyme PRSS 15 agents may comprise one of several motifs including hammerhead (Rossi et al., 8 AIDS RESEARCH AND HUMAN RETROVIRUSES 183 (1992), hairpin (Hampel and Tritz, 28 BIOCHEM. 4929 (1989); Hampel et al., NUCL. ACIDS RES. 299 (1990)), hepatitis delta virus motif (Perrotta and Been, 31 BIOCHEM. 16 (1992), group I intron (U.S. Patent No. 4,987,071), RNaseP RNA in association with an RNA guide sequence (Guerrier- Takada et al., 35 CELL 849 (1983)), and Neurospora VS RNA (Saville & Collins, 61 CELL 685-96 (1990); Saville & Collins, 88 PROC. NATL. ACAD. SCI. USA 8826-30 (1991); Collins & Olive, 32 BlOCHEM. 2795-99 (1993)). These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic polynucleotide molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.

[0184] As in the antisense approach, the ribozyme PRSS 15 agents may be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and may be delivered to cells that express PRSS 15 polypeptides in vivo. Polynucleotide constructs encoding the ribozyme PRSS 15 agent may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA. In one embodiment, delivery involves using a DNA construct "encoding" the ribozyme PRSS15 agent under the control of a strong constitutive promoter, such as, for example, RNA Polymerase II or RNA Polymerase HI promoter, so that transfected cells will produce sufficient quantities of the ribozyme PRSS 15 agent to destroy endogenous messages and inhibit translation. Because ribozymes, unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

[0185] Ribozyme PRSS 15 agents may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by ionophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. Furthermore, ribozyme PRSS 15 agents may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the RN A/vehicle combination may be locally delivered by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. See generally WO 94/02595; WO 93/23569.

E. RNAi Molecules [0186] Yet another form of PRSS 15 agent is short interfering RNA (siRNA) useful for performing RNA interference (RNAi).

[0187] siRNA refers to double-stranded RNA molecules from about 10 to about 30 nucleotides long that are named for their ability to specifically interfere with protein expression. Preferably, siRNA molecules are 12-28 nucleotides long, more preferably 15-25 nucleotides long, still more preferably 19-23 nucleotides long and most preferably 21-23 nucleotides long. Therefore, preferred siRNA molecules are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 28 or 29 nucleotides in length.

[0188] The length of one strand designates the length of an siRNA molecule. For instance, an siRNA that is described as 21 ribonucleotides long (a 21-mer) could comprise two opposite strands of RNA that anneal together for 19 contiguous base pairings. The two remaining ribonucleotides on each strand would form an "overhang." When an siRNA contains two strands of different lengths, the longer of the strands designates the length of the siRNA. For instance, a dsRNA containing one strand that is 21 nucleotides long and a second strand that is 20 nucleotides long, constitutes a 21-mer.

[0189] siRNAs that comprise an overhang are desirable. The overhang may be at the 5' or the 3' end of a strand. Preferably, it is at the 3' end of the RNA strand. The length of an overhang may vary, but preferably is about 1 to about 5 bases, and more preferably is about 2 nucleotides long. Preferably, the siRNA of the present invention will comprise a 3' overhang of about 2 to 4 bases. More preferably, the 3' overhang is 2 ribonucleotides long. Even more preferably, the 2 ribonucleotides comprising the 3' overhang are uridine (U).

[0190] siRNAs of the present invention are designed to interact with a target PRSS 15 ribonucleotide sequence, meaning they complement a target sequence sufficiently to bind to the target sequence. In one embodiment, the invention provides an siRNA molecule comprising a ribonucleotide sequence at least 80% identical to a PRSS 15 ribonucleotide sequence. Preferably, the siRNA molecule is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the PRSS 15 ribonucleotide sequence. Most preferably, an siRNA will be 100% identical to the target nucleotide sequence. However, siRNA molecules with insertions, deletions or single point mutations relative to a target may also be effective. Tools to assist siRNA design are readily available to the public. For example, a computer-based siRNA design tool is available on the internet at www.dharmacon.com.

F. Small Molecules [0191] Small molecules constitute another type of PRSS 15 agents. The small molecules may be binding agents and/or modulators. In general, small molecules comprise a compound or molecular complex, either synthetic, naturally derived, or partially synthetic, composed of carbon, hydrogen, oxygen, and nitrogen, which may also contain other elements, and which may have a molecular weight of less than about 15,000, less than about 14,000, less than about 13,000, less than about 12,000, less than about 11,000, less than about 10,000, less than about 9,000, less than about 8,000, less than about 7,000, less than about 6,000, less than about 5,000, less than about 4,000, less than about 3,000, less than about 2,000, less than about 1,000, less than about 900, less than about 800, less than about 700, less than about 600, less than about 500, less than about 400, less than about 300, less than about 200, or less than about 100.

[0192] Particularly useful small molecules are competitors of ATP binding. Methods of identifying additional small molecule agents are described below.

G. Use and Formulation of PRSS 15 Agents [0193] The many practical uses of PRSS 15 agents are apparent to those of skill in the art. For example, they may be used to purify, detect, and target PRSS 15 polypeptides, including both in vitro and in vivo diagnostic and therapeutic methods. Many of the agents act as agonists or antagonists of PRSS 15 polypeptides.

[0194] As discussed in more detail below, the agents may be used either alone or in combination with other compositions. For example, the agents may be fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins.

[0195] PRSS 15 agents generally are "substantially purified," meaning separated and/or recovered from a component of their natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the PRSS 15 agent, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. Ordinarily, an isolated agent will be prepared by at least one purification step. In one embodiment, the agent is purified to at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 88%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or at least about 99.99% by weight of PRSS 15 agent.

[0196] The PRSS 15 agents may be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material which, when combined with the agent, retains the agent's function and is nonreactive with the subject's immune systems. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like.

V. Methods of Treating or Preventing Cancer and Pharmaceutical Compositions [0197] In still another aspect, the invention provides a method of treating or preventing a cancer, preferably a characterized by overexpression and/or upregulation of PRSS 15. The method includes administering a therapeutically or prophylactically effective amount of at least one PRSS 15 modulator, where the modulator decreases or inhibits proliferation of the cancer or causes cancer cell death. The modulator may be a polypeptide, such as an antibody, a polynucleotide, such as an antisense or RNAi oligonucleotide, or a small molecule. Preferably, the cancer is colon cancer, prostate cancer, breast or stomach cancer. In particular embodiments, the method suppresses or inhibits metastasis, aberrant cellular proliferation relative to a normal cell, loss of contact inhibition of cell growth, or loss of anchorage dependent growth.

[0198] A reduction of cancer cell growth resulting from methods of treatment can be assessed using any of a variety of known diagnostic assays for cancer, including physical examination, biopsy, contrast radiographic studies, CAT scan, and detection of a tumor marker. A. Formulations [0199] For treating and preventing cancer, PRSS15 modulators are formulated in pharmaceutical compositions. Thus, in yet another aspect, the invention provides a pharmaceutical composition useful for treating or preventing a cancer. The composition comprises a pharmaceutically effective amount of at least one PRSS 15 modulator and a pharmaceutically acceptable carrier, where the modulator decreases or inhibits proliferation of the cancer or causes cancer cell death. The PRSS 15 modulator comprises a polynucleotide PRSS 15 modulator, which may include, but is not limited to, an antisense oligonucleotide, a ribozyme, or an siRNA. In an alternative embodiment, the PRSS 15 modulator comprises a polypeptide PRSS 15 modulator, such as an anti-PRSS15 immunoglobulin or functional equivalent thereof. In another alternative embodiment, a pharmaceutical composition comprises a small molecule PRSS 15 modulator.

[0200] Pharmaceutical compositions comprising PRSS 15 modulators may be formulated according to known methods, such as by the admixture of a pharmaceutically acceptable carrier. Specifically, the PRSS 15 modulators may be admixed with pharmaceutical diluents, excipients, or other carriers suitably selected with respect to the intended form of administration, e.g., oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

[0201] For example, for oral administration in the form of a tablet or capsule, the PRSS 15 modulator may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents may also be incorporated into the mixture. Suitable binders include, without limitation, starch; gelatin; natural sugars such as glucose or beta-lactose; corn sweeteners; natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose; polyethylene glycol; waxes and the like. Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

[0202] For liquid forms, the PRSS 15 modulator may be combined in suitably flavored suspending or dispersing agents such as synthetic and natural gums, including tragacanth, acacia and methyl-cellulose. Other dispersing agents that may be employed include glycerin and the like.

[0203] The pharmaceutical compositions may be administered parenterally via injection of a formulation consisting of the active ingredient dissolved in an inert liquid carrier. The term "parenteral" as used herein includes subcutaneous injections, intravenous, intramuscular, intraperitoneal injections, or infusion techniques. Acceptable liquid carriers include, for example, the vegetable oils such as, for example, peanut oil, cotton seed oil, sesame oil and the like as well as organic solvents such as, for example, solketal, glycerol formal and the like. The formulations may be prepared by dissolving or suspending the active ingredient in the liquid carrier such that the final formulation contains from about 0.005% to 10% by weight of the active ingredient.

[0204] Topical compositions containing the PRSS 15 modulator may be admixed with a variety of carrier materials well known in the art including alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate and the like to form, for example, alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations. Examples of such carriers and methods of formulation may be found in REMINGTON'S PHARMACEUTICAL SCIENCES (1990). Pharmaceutical formulations may contain from about 0.005% to about 10% by weight of the active ingredient. In one embodiment, the pharmaceutical formulations contain from about 0.01% to 5% by weight of the PRSS 15 modulator.

[0205] Furthermore, the pharmaceutical compositions of the invention may be administered in intranasal form. See, e.g., WO 01/41782. Alternatively, the pharmaceutical compositions may be administered via pulmonary inhalation. The methods of the invention relate to preparing a pharmaceutical compositions for subsequence delivery as an aqueous or nonaqueous solution or suspension or a dry powder form. See, e.g., WO 01/49274.

[0206] In another embodiment, the pharmaceutical compositions of the invention may be administered via transdermal routes using forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration may be, for example, continuous rather than intermittent throughout the dosage regimen. [0207] The PRSS 15 modulators disclosed herein may also be formulated as liposomes. Liposomes containing the PRSS 15 modulator are prepared by methods known in the art. See, e.g., Epstein et al., 82 PROC. NATL. ACAD. SCI. USA 3688 (1985); Hwang et al., 77 PROC. NATL. ACAD. SCI. USA 4030 (1980); U.S. Patent Nos. 5,013,556; 4,485,045 4,544,545; WO 97/38731.

[0208] Sustained-release compositions may be prepared. Suitable examples of sustained-release compositions include semipermeable matrices of solid hydrophobic polymers containing the PRSS 15 modulator, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid- glycolic acid copolymers such as the LUPRON DEPOT® (Tap Pharmaceuticals, Inc., Chicago, IL) (injectable microspheres composed of lactic acid glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[0209] The PRSS 15 modulators may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. REMINGTON'S PHARMACEUTICAL SCIENCES (A. Osol ed., 16th ed. 1980).

B. Administrations [0210] The invention embraces treatment of any cancer where the administration of a PRSS 15 modulator modulates, decreases, or inhibits cancer cell proliferation, cancer cell migration, cancer cell adhesion, and/or metastasis. In particular embodiments, the cancer is colon cancer, prostate cancer, high grade prostate cancer, breast cancer, or stomach cancer.

[0211] The phrase "treating or preventing a cancer" includes, but is not limited to, reducing proliferation of cancer cells and reducing the incidence of a non-cancerous cell becoming a cancerous cell. Whether a reduction in cancer cell growth has been achieved may be readily determined using any known assay including, but not limited to, [³H]- thymidine incorporation; counting cell number over a period of time; detecting and/or measuring a marker associated with cancer (e.g., colon cancer biomarkers include CEA, CAI9-9, and LASA).

[0212] In general, the pharmaceutical compositions disclosed herein may be used alone at appropriate dosages defined by routine testing in order to obtain optimal inhibition of the proliferation of a cancer, while minimizing any potential toxicity. The dosage regimen utilizing the PRSS 15 modulators may be selected in accordance with a variety of factors including type, age, weight, sex, medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular modulator thereof employed. A physician of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the condition.

[0213] Optimal precision in achieving concentrations of modulator within the range that yields maximum efficacy with minimal toxicity may require a regimen based on the kinetics of the modulator's availability to PRSS 15 target sites. Distribution, equilibrium, and elimination of a modulator may be considered when determining the optimal concentration for a treatment regimen. The dosages of the PRSS15 modulators may be adjusted when combined to achieve desired effects. On the other hand, dosages of these various agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone.

[0214] In particular, toxicity and therapeutic efficacy of PRSS 15 modulators may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index and it may be expressed as the ration LD50/ED50. Compounds exhibiting large therapeutic indices are preferred. Although compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. Generally, the PRSS 15 modulators of the invention may be administered in a manner that maximizes efficacy and minimizes toxicity. [0215] Data obtained from cell culture assays and animal studies may be used in formulating a range of dosage for use in humans. The dosages of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods of the invention, the therapeutically effective dose may be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (the concentration of the test compound that achieves a half -maximal inhibition of symptoms) as determined in cell culture. Such information may be used to accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0216] Moreover, the dosage administration of the pharmaceutical compositions may be optimized using a pharmacokinetic/pharmacodynamic modeling system. For example, one or more dosage regimens may be chosen and a pharmacokinetic/pharmacodynamic model may be used to determine the pharmacokinetic/pharmacodynamic profile of one or more dosage regimens. Next, one of the dosage regimens for administration may be selected which achieves the desired pharmacokinetic/pharmacodynamic response based on the particular pharmacokinetic/pharmacodynamic profile. See, e.g., WO 00/67776.

[0217] Specifically, the pharmaceutical compositions may be administered at least once a week over the course of several weeks. In one embodiment, the pharmaceutical compositions are administered at least once a week over several weeks to several months. In another embodiment, the pharmaceutical compositions are administered once a week over four to eight weeks. In yet another embodiment, the pharmaceutical compositions are administered once a week over four weeks.

[0218] More specifically, the pharmaceutical compositions may be administered at least once a day for about 2 days, at least once a day for about 3 days, at least once a day for about 4 days, at least once a day for about 5 days, at least once a day for about 6 days, at least once a day for about 7 days, at least once a day for about 8 days, at least once a day for about 9 days, at least once a day for about 10 days, at least once a day for about 11 days, at least once a day for about 12 days, at least once a day for about 13 days, at least once a day for about 14 days, at least once a day for about 15 days, at least once a day for about 16 days, at least once a day for about 17 days, at least once a day for about 18 days, at least once a day for about 19 days, at least once a day for about 20 days, at least once a day for about 21 days, at least once a day for about 22 days, at least once a day for about 23 days, at least once a day for about 24 days, at least once a day for about 25 days, at least once a day for about 26 days, at least once a day for about 27 days, at least once a day for about 28 days, at least once a day for about 29 days, at least once a day for about 30 days, or at least once a day for about 31 days.

[0219] Alternatively, the pharmaceutical compositions may be administered about once every day, about once every 2 days, about once every 3 days, about once every 4 days, about once every 5 days, about once every 6 days, about once every 7 days, about once every 8 days, about once every 9 days, about once every 10 days, about once every 11 days, about once every 12 days, about once every 13 days, about once every 14 days, about once every 15 days, about once every 16 days, about once every 17 days, about once every 18 days, about once every 19 days, about once every 20 days, about once every 21 days, about once every 22 days, about once every 23 days, about once every 24 days, about once every 25 days, about once every 26 days, about once every 27 days, about once every 28 days, about once every 29 days, about once every 30 days, or about once every 31 days or more.

[0220] The pharmaceutical compositions of the invention may alternatively be administered about once every week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, about once every 5 weeks, about once every 6 weeks, about once every 7 weeks, about once every 8 weeks, about once every 9 weeks, about once every 10 weeks, about once every 11 weeks, about once every 12 weeks, about once every 13 weeks, about once every 14 weeks, about once every 15 weeks, about once every 16 weeks, about once every 17 weeks, about once every 18 weeks, about once every 19 weeks, about once every 20 weeks or more.

[0221] Alternatively, the pharmaceutical compositions of the invention may be administered about once every month, about once every 2 months, about once every 3 months, about once every 4 months, about once every 5 months, about once every 6 months, about once every 7 months, about once every 8 months, about once every 9 months, about once every 10 months, about once every 11 months, or about once every 12 months or more. [0222] Alternatively, the pharmaceutical compositions may be administered at least once a week for about 2 weeks, at least once a week for about 3 weeks, at least once a week for about 4 weeks, at least once a week for about 5 weeks, at least once a week for about 6 weeks, at least once a week for about 7 weeks, at least once a week for about 8 weeks, at least once a week for about 9 weeks, at least once a week for about 10 weeks, at least once a week for about 11 weeks, at least once a week for about 12 weeks, at least once a week for about 13 weeks, at least once a week for about 14 weeks, at least once a week for about 15 weeks, at least once a week for about 16 weeks, at least once a week for about 17 weeks, at least once a week for about 18 weeks, at least once a week for about 19 weeks, or at least once a week for about 20 weeks or more.

[0223] Alternatively the pharmaceutical compositions may be administered at least once a week for about 1 month, at least once a week for about 2 months, at least once a week for about 3 months, at least once a week for about 4 months, at least once a week for about 5 months, at least once a week for about 6 months, at least once a week for about 7 months, at least once a week for about 8 months, at least once a week for about 9 months, at least once a week for about.10 months, at least once a week for about 11 months, or at least once a week for about 12 months or more.

[0224] Alternatively, the pharmaceutical compositions may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily. In the case of oral administration, the daily dosage of the compositions may be varied over a wide range from about 0.0001 to about 1,000 mg per patient, per day. The range may more particularly be from about 0.001 mg/kg to 10 mg/kg of body weight per day, preferably usually about 0.1-100 mg, preferably about 1.0-50 mg or, more preferably, about 1.0-20 mg per day for adults (as 60 kg).

[0225] For oral administration, the pharmaceutical compositions may preferably be provided in a form of scored or unscored tablets containing about 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, or 50.0 mg of the active ingredient for the symptomatic adjustment of the dosage for the patient to be treated.

[0226] In the case of injections, it is usually convenient to give by an intravenous route in an amount of about 0.01-30 mg, preferably about 0.1-20 mg or, more preferably, about 0.1-10 mg per day to adults (at about 60 kg). In the case of other animals, the dose calculated for 60 kg may be administered as well.

[0227] In addition to these methods and formulations, the PRSS 15 modulators may be effectively introduced via gene therapy. Specifically, appropriate vectors encoding these compounds and/or isolated polynucleotides may be introduced into a patient. See, e.g., W096/07321 (concerning the use of gene therapy to generate intracellular antibodies.) There are two major approaches to shuttling a polynucleotide into a patient's cells, ex vivo and in vivo.

[0228] For ex vivo treatment, the patient's cells are removed, the polynucleotide is introduced into these isolated cells and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes that are implanted into the patient. See, e.g., U.S. Patent Nos. 4,892,538 and 5,283,187. Techniques suitable for the transfer of polynucleotide into mammalian cells ex vivo include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, and the like. A commonly used vector for ex vivo delivery of the gene is a retrovirus.

[0229] For in vivo delivery, the polynucleotide may be injected directly into the patient, usually at the site where the modulator is required. Alternatively, the in vivo polynucleotide transfer techniques that include transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno-associated virus) and lipid-based systems (useful lipids for lipid mediated transfer of the gene are DOTMA, DOPE and DC-Choi, for example) are used for such delivery. In some situations it is desirable to provide the polynucleotide source with an agent that targets the target cells, such as an immunoglobulin specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, and so forth. In the case of liposomes, proteins that bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, immunoglobulins for proteins that undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor- mediated endocytosis is described, for example, by Wu et al., 262 J. BIOL. CHEM. 4429-32 (1987); and Wagner et al., 87 PROC. NATL. ACAD. SCI. 3410-14 (1990). For review of gene marking and gene therapy protocols, see Anderson et al., 256 SCIENCE 808-13 (1992). See also WO 93/25673 and the references cited therein.

[0230] Administration of polynucleotide therapeutic composition agents includes local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration. In general, the therapeutic polynucleotide composition contains an expression construct comprising a promoter operably linked to a polynucleotide of at least 12, 22, 25, 30, or 35 contiguous nt of the polynucleotide disclosed herein. Various methods can be used to administer the therapeutic composition directly to a specific site in the body. For example, a small metastatic lesion is located and the therapeutic composition injected several times in several different locations within the body of the tumor. Alternatively, arteries which serve a tumor are identified, and the therapeutic composition injected into such an artery, in order to deliver the composition directly into the tumor. A tumor that has a necrotic center is aspirated and the composition injected directly into the now empty center of the tumor. An antisense or RNAi composition may be directly administered to the surface of the tumor, for example, by topical application of the composition. X-ray imaging may be used to assist in certain of the above delivery methods.

[0231] Targeted delivery of therapeutic compositions containing an antisense polynucleotide, siRNA, ribozymes, subgenomic polynucleotides, or antibodies to specific tissues can also be used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J.A. Wolff, ed.) (1994); Wu et al, J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. (USA) (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338. Therapeutic compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 :g of DNA can also be used during a gene therapy protocol. Factors such as method of action (e.g., for enhancing or inhibiting levels of the encoded gene product) and efficacy of transformation and expression are considerations that will affect the dosage required for ultimate efficacy of the antisense subgenomic polynucleotides.

[0232] Where greater expression is desired over a larger area of tissue, larger amounts of polynucleotides or the same amounts readministered in a successive protocol of administrations, or several administrations to different adjacent or close tissue portions of, for example, a tumor site, may be required to effect a positive therapeutic outcome. In all cases, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect.

[0233] Therapeutic polynucleotides and polypeptides of the present invention can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non- viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated.

[0234] Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; USPN 5, 219,740; WO 93/11230; WO 93/10218; USPN 4,777,127; GB Patent No. 2,200,651; EP 0 345 242; and WO 91/02805), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532), and adeno- associated virus (AAV) vectors (see, e.g., WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administration of DNA linked to killed adenovirus is described in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.

[0235] Non- viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles cells (see, e.g., USPN 5,814,482; WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in WO 90/11092 and USPN 5,580,859. Liposomes that can act as gene delivery vehicles are described in USPN 5,422,120; WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional approaches are described in Philip, Mol. Cell Biol. (1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) Pi: 1581.

[0236] Further non- viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al., Proc. Natl. Acad. Sci. USA (1994) 91 (24):11581. Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials or use of ionizing radiation (see, e.g., USPN 5,206,152 and WO 92/11033). Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun (see, e.g., USPN 5,149,655); use of ionizing radiation for activating transferred gene (see, e.g., USPN 5,206,152 and WO 92/11033).

C. Co-Administration with Other Therapeutic Agents [0237] In another aspect, the method of treating or preventing a cancer entails administering a therapeutically or prophylactically effective amount of at least one PRSS 15 modulator with at least one other therapeutic agent. The modulator may be co-administered with the other therapeutic agent; alternatively, the modulator and other therapeutic agent may be consecutively administered, in either order.

[0238] Other therapeutic agents that may be co-administered or sequentially administered include chemotherapeutic agents, immunosuppressive agents, cytokines, cytotoxic agents, nucleolytic compounds, radioactive isotopes, receptors, and pro-drug activating enzymes, which may be naturally occurring or produced by recombinant methods. The combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. [0239] In one embodiment, the therapeutic agent administered simultaneously or sequentially, in either order and at various times, comprises a chemotherapeutic agent. A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiehin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitroureas such as cannustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromoinycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idambicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, rnepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofrran; spirogermanium; tenuazonic acid; triaziquone; 2, 2' ,2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4 hydroxytamoxifen, trioxifene, keoxifene, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0240] In another embodiment, the therapeutic agent comprises a cytokine. The term "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (GCSF); interleukins (LLs) such as LL-1, IL-la, IL-2, LL-3, LL-4, LL-5, IL-6, LL-7, LL-8, LL-9, LL-11, IL-12, IL-15; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the tern cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines. [0241] In another embodiment, the therapeutic agent comprises a small molecule toxin, including maytansine, calicheamicin, trichothene, and CC 1065. In a specific embodiment, the therapeutic agent may comprise one more calicheamicin molecules. The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. Structured analogues of calicheamicin are also known. See Hinman et al., 53 CANCER RESEARCH 3336-42 (1993); Lode et al., 58 CANCER RESEARCH 2925-28 (1998).

[0242] In yet another embodiment, the therapeutic agent may comprise one or more enzymatically active toxins and fragments thereof. Examples of such toxins include nonbinding active fragments of diphtheria toxin, diphtheria A chain, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, dianthin proteins, Phytolaca americana proteins (PAPI, PAP AH, and PAP-S), momordica charantia inhibitor, curcin, crotin sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictoein, phenomvcin, enomycin and the tricothecenes. See, e.g., WO 93/21232.

[0243] The invention further contemplates therapeutic agents that have nucleolytic activity such as a ribonuclease and a deoxyribonuclease. In addition, a variety of radioactive isotopes are available for the production of radioconjugated modulators. Examples include Y⁹⁰, At²²², Ret⁸⁶, Re¹⁸⁶, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes of Lu.

[0244] The therapeutic agents may administered as prodrugs and subsequently activated by a prodrug-activating enzyme that converts a prodrug like a peptidyl chemotherapeutic agent to an active anti-cancer drug. See, e.g., WO 88/07378; WO 81/01145; U.S. Patent No. 4,975,278. In general, the enzyme component includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.

[0245] Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5- fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate cleaving enzymes such as β-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; β-lactamase useful for converting drugs derivatized with β-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G, amidase, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs.

[0246] Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes," may be used to convert the prodrugs of the invention into free active drugs. See, e.g., Massey, 328 NATURE 457-48 (1987).

[0247] In one embodiment of the invention, the PRSS 15 modulator is administered before the therapeutic agent. The administration of the PRSS15 modulator may occur anytime from several minutes to several hours before the adminstration of the therapeutic agent. The PRSS 15 modulator may alternatively be administered anytime from several hours to several days, possibly several weeks up to several months before the therapeutic agent.

[0248] More specifically, the PRSS 15 modulator may be administered at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, or at least about 24 hours before the therapeutic agent.

[0249] Moreover, the PRSS 15 modulator may be administered at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days or at least about 31 days before the administration of the therapeutic agent.

[0250] In yet another embodiment, the PRSS 15 modulator may be administered at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, at least about 15 weeks, at least about 16 weeks, at least about 17 weeks, at least about 18 weeks, at least about 19 weeks, or at least about 20 weeks or more before the therapeutic agent.

[0251] In a further embodiment, the PRSS 15 modulator may be administered at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about nine months, at least about ten months, at least about eleven months, or at least about twelve months before the therapeutic agent.

[0252] In another embodiment, the PRSS 15 modulator is administered after the therapeutic agent. The administration of the PRSS 15 modulator may occur anytime from several minutes to several hours after the adminstration of the therapeutic agent. The PRSS 15 modulator may alternatively be administered anytime from several hours to several days, possibly several weeks up to several months after the therapeutic agent.

[0253] More specifically, the PRSS 15 modulator may be administered at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, or at least about 24 hours or more after the therapeutic agent.

[0254] Moreover, the PRSS 15 modulator may be administered at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days or at least about 31 days or more after the administration of the therapeutic agent.

[0255] In yet another embodiment, the PRSS 15 modulator may be administered at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, at least about 15 weeks, at least about 16 weeks, at least about 17 weeks, at least about 18 weeks, at least about 19 weeks, or at least about 20 weeks or more after the therapeutic agent.

[0256] In a further embodiment, the PRSS 15 modulator may be administered at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about nine months, at least about ten months, at least about eleven months, or at least about twelve months after the therapeutic agent.

VI. Methods of Detecting Cancer or Determining a Patient's Predisposition to Cancer [0257] In still another aspect, the invention provides a method of detecting a cancer, preferably a characterized by overexpression and/or upregulation of PRSS 15. The method entails (a) determining the level of expression of PRSS 15 in a biological sample obtained from a patient, (b) comparing the level of PRSS 15 expression in the patient biological sample to the level of PRSS 15 expression in a normal biological sample, and (c) correlating the level of PRSS 15 expression to a positive or negative diagnosis of the cancer.

[0258] A related aspect of the invention provides a method for determining a patient's predisposition to a cancer. The method entails (a) determining the level of expression of PRSS 15 in a biological sample obtained from a patient, (b) comparing the level of PRSS 15 expression in said patient biological sample to the level of PRSS 15 expression in a normal biological sample, and (c) correlating the level of PRSS 15 expression to a diagnosis of a predisposition to the cancer.

[0259] Specifically, in diagnosing a patient, the level of a PRSS 15 gene product in a biological sample obtained from a patient may be determined. Next, the level of PRSS 15 expression in the patient's biological sample may be compared to the PRSS15 gene product level from a normal biological sample and correlated to a positive or negative diagnosis of cancer. A patient's predisposition to a cancer may be determined using a similar method.

[0260] The PRSS 15 polynucleotides and polypeptides described herein are useful to diagnose cancer. In specific non-limiting embodiments, the methods are useful for detecting PRSS15-associated cancer cells, facilitating diagnosis of cancer and the severity of a cancer (e.g., tumor grade) in a subject, facilitating a determination of the prognosis of a subject, determining the susceptibility to cancer in a subject, and assessing the responsiveness of the subject to therapy. Such methods may involve detection of levels of PRSS 15 polynucleotides or PRSS 15 polypeptides in a patient biological sample, e.g., a suspected or prospective cancer tissue or cell. The detection methods of the invention may be conducted in vitro or in vivo, on isolated cells, or in whole tissues or a bodily fluid, e.g., blood, plasma, serum, urine, and the like.

[0261] In one embodiment, the aggressive nature and/or the metastatic potential of a cancer may be determined by comparing PRSS 15 polynucleotide and/or polypeptide levels to polynucleotide and/or polypeptide levels of another gene known to vary in cancerous tissue, e.g., expression of p53, DCC, ras, FAP. See, e.g., Fearon, 768 ANN. N.Y. ACAD. SCI. 101(1995); Bodmer et al., 4(3) NAT. GENET. 217 (1994); Hamilton et al., 72 CANCER 957 (1993); and Fearon et al., 61(5) CELL 759 (1990). Thus, the expression of PRSS15 polynucleotides and PRSS 15 polypeptides may be used to discriminate between normal and cancerous tissue, to discriminate between cancers with different cells of origin, and to discriminate between cancers with different potential metastatic rates, etc. For a review of cancer biomarkers, see Hanahan et al., 100 CELL 57-70 (2000).

[0262] In one embodiment, the PRSS 15 polynucleotides, PRSS 15 polypeptides and PRSS 15 modulators may be used to detect, assess, and treat colon cancer, prostate cancer, especially high grade prostate cancer, breast cancer or stomach cancer. A. Detecting a PRSS 15 Polynucleotide in a Cell [0263] Methods are provided for detecting expression of a PRSS15 polynucleotide in a cell. Any of a variety of known methods may be used for detection, including, but not limited to, detection of a PRSS 15 transcript by hybridization with a polynucleotide specific for a PRSS 15 transcript; detection of a PRSS15 transcript by a polymerase chain reaction using specific oligonucleotide primers (RT-PCR); in situ hybridization of a cell using as a probe a polynucleotide that hybridizes to PRSS 15 that is differentially expressed in a colon cancer cell. The methods may be used to detect and/or measure PRSS 15 mRNA levels in a cancer cell. In some embodiments, the methods comprise a) contacting a sample with a PRSS 15 polynucleotide under conditions that allow hybridization; and b) detecting hybridization.

[0264] Detection of differential hybridization, when compared to a suitable control, is an indication of the presence in the sample of a PRSS 15 polynucleotide that is differentially expressed in a cancer cell. Appropriate controls include, for example, a sample which is known not to contain a PRSS 15 polynucleotide. Conditions that allow hybridization are known in the art. Detection may also be accomplished by any known method, including, but not limited to, in situ hybridization, PCR (polymerase chain reaction), RT-PCR (reverse transcription-PCR), and "Northern" or RNA blotting, or combinations of such techniques, using a suitably labeled polynucleotide. A variety of labels and labeling methods for polynucleotides are also known in the art and may be used in the assay methods of the invention. Specific hybridization may be determined by comparison to appropriate controls.

[0265] Polynucleotides generally comprising at least 12 contiguous nt of the PRSS 15 polynucleotides provided herein are used for a variety of purposes, such as probes for detection of and/or measurement of, transcription levels of other PRSS 15 polynucleotides. A probe that hybridizes specifically to a PRSS 15 polynucleotide disclosed herein should provide a detection signal at least about 0.3-fold higher, at least about 0.5- fold higher, at least about 0.7- fold higher, at least about 0.8-fold higher, at least about 0.9-fold higher, at least about 1.0-fold higher, at least about 1.2-fold higher, at least about 1.4-fold higher, at least about 1.6-fold higher, at least about 1.8-fold higher, at least about 2-fold higher, at least about 2.5-fold higher, at least about 3.0-fold higher, at least about 3.5-fold higher, at least about 4.0-fold higher, at least about 4.5-fold higher, at least about 5-fold higher, at least about 10-fold higher, or at least about 20-fold or more higher than the background hybridization provided with other unrelated sequences. It should be noted that "probe" as used herein is meant to refer to a polynucleotide sequence used to detect a PRSS 15 gene product in a test sample. The probe may be detectably labeled and contacted with, for example, a microarray comprising immobilized polynucleotides obtained from a test sample. Alternatively, the probe may be immobilized on a microarray and the test sample detectably labeled.

[0266] Nucleotide probes may be used to detect expression of a gene corresponding to the provided PRSS 15 polynucleotide. In Northern blots, mRNA is separated electrophoretically and contacted with a probe. A probe is detected as hybridizing to an mRNA species of a particular size. The amount of hybridization may be quantitated to determine relative amounts of expression, for example under a particular condition. Probes are used for in situ hybridization to cells to detect expression. Probes may also be used in vivo for diagnostic detection of hybridizing sequences. Probes are typically labeled with a radioactive isotope. Other types of detectable labels may be used such as chromophores, fluorophores, and enzymes. Other examples of nucleotide hybridization assays are described in U.S. Patent No. 5,124,246 and WO 92/02526.

[0267] PCR is another means for detecting small amounts of target PRSS 15 polynucleotides. Two primer polynucleotides that hybridize with the target PRSS 15 polynucleotides may be used to prime the reaction. The primers may comprise sequences within or 3' and 5' to the PRSS 15 polynucleotides described herein. Alternatively, if the primers are 3' and 5' to these polynucleotides, they need not hybridize to the polynucleotides or the complements. After amplification of the target with a thermostable polymerase, the amplified target polynucleotides may be detected by methods known in the art, e.g., Southern blot. PRSS15 mRNA or cDNA may also be detected by traditional blotting techniques (e.g., Southern blot, Northern blot, etc.) described in SAMBROOKET AL., MOLECULAR CLONING: A LAB. MANUAL (2001) (e.g., without PCR amplification). In general, mRNA or cDNA generated from mRNA using a polymerase enzyme may be purified and separated using gel electrophoresis, and transferred to a solid support, such as nitrocellulose. The solid support is exposed to a labeled probe, washed to remove any unhybridized probe, and duplexes containing the labeled probe are detected.

[0268] Methods using PCR amplification may be performed on the DNA from a single cell, although it is convenient to use at least about 10⁵ cells. A detectable label may be included in the amplification reaction. Suitable detectable labels include fluorochromes, (e.g., fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2',7'-dimethoxy 4',5'-dichloro-6- carboxyfluorescein, 6-carboxy-X-rhodamine (ROX), 6-carboxy2',4',7',4,7- hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA)), radioactive labels, (e.g., 32P, 35S' 3H, etc.), and the like. The label may be a two stage system whereby the polynucleotides are conjugated to biotin, haptens, etc. having a high affinity modulator, e.g., avidin, specific antibodies, etc., whereby the modulator is conjugated to a detectable label. The label may be conjugated to one or both of the primers. Alternatively, the pool of nucleotides used in the amplification is labeled, so as to incorporate the label into the amplification product.

B. Detecting a PRSS 15 Polypeptide in a Cell [0269] Methods are provided for detecting a PRSS15 polypeptide in a cell. Any of a variety of known methods may be used for detection, including, but not limited to, immunoassay, using antibody specific for the encoded polypeptide, e.g., by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and the like; and functional assays for the encoded polypeptide, e.g., biological activity.

[0270] For example, an immunofiuorescence assay may be easily performed on cells without first isolating the encoded PRSS 15 polypeptide. The cells are first fixed onto a solid support, such as a microscope slide or microtiter well. This fixing step may permeabilize the cell membrane. The permeablization of the cell membrane permits the polypeptide-specific antibody to bind. Next, the fixed cells are exposed to an antibody specific for the encoded PRSS 15 polypeptide. To increase the sensitivity of the assay, the fixed cells may be further exposed to a second antibody, which is labeled and binds to the first antibody, which is specific for the encoded polypeptide. Typically, the secondary antibody is detectably labeled, e.g., with a fluorescent marker. The cells that express the encoded polypeptide will be fluorescently labeled and easily visualized under the microscope. See, e.g., Hashido et al., 187 BIOCHEM. BIOPHYS. RES. COMM. 1241-48 (1992).

[0271] As will be readily apparent to the ordinarily skilled artisan upon reading the present specification, the detection methods and other methods described herein may be readily varied. Such variations are within the intended scope of the invention. For example, in the above detection scheme, the probe for use in detection may be immobilized on a solid support, and the test sample contacted with the immobilized probe. Binding of the test sample to the probe may then be detected in a variety of ways, e.g., by detecting a detectable label bound to the test sample to facilitate detected of test sample-immobilized probe complexes.

[0272] The invention further provides methods for detecting the presence of and/or measuring a level of PRSS 15 polypeptide in a biological sample, using an antibody specific for PRSS15. Specifically, the method for detecting the presence of PRSS15 polypeptides in a biological sample may comprise the step of contacting the sample with a monoclonal antibody and detecting the binding of the antibody with the PRSS 15 in the sample. More specifically, the antibody may be labeled so as to produce a detectable signal using compounds including, but not limited to, a radiolabel, an enzyme, a chromophore and a fluorophore.

[0273] Detection of specific binding of an antibody specific for PRSS 15, or a functional equivalent thereof, when compared to a suitable control, is an indication that PRSS 15 polypeptides are present in the sample. Suitable controls include a sample known not to contain PRSS 15 polypeptides and a sample contacted with an antibody not specific for the encoded polypeptide, e.g., an anti-idiotype antibody. A variety of methods to detect specific antibody-antigen interactions are known in the art and may be used in the method, including, but not limited to, standard immunohistological methods, immunoprecipitation, an enzyme immunoassay, and a radioimmunoassay. In general, the specific antibody will be detectably labeled, either directly or indirectly. Direct labels include radioisotopes; enzymes whose products are detectable (e.g., luciferase, 3-galactosidase, and the like); fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, and the like); fluorescence emitting metals (e.g., 112Eu, or others of the lanthanide series, attached to the antibody through metal chelating groups such as EDTA); chemiluminescent compounds (e.g., luminol, isoluminol, acridinium salts, and the like); bioluminescent compounds (e.g., luciferin, aequorin (green fluorescent protein), and the like). The antibody may be attached (coupled) to an insoluble support, such as a polystyrene plate or a bead. Indirect labels include second antibodies specific for antibodies specific for the encoded polypeptide ("first specific antibody"), wherein the second antibody is labeled as described above; and members of specific binding pairs, e.g., biotin-avidin, and the like. The biological sample may be brought into contact with and immobilized on a solid support or carrier, such as nitrocellulose, that is capable of immobilizing cells, cell particles, or soluble proteins. The support may then be washed with suitable buffers, followed by contacting with a detectably-labeled first specific antibody. Detection methods are known in the art and will be chosen as appropriate to the signal emitted by the detectable label. Detection is generally accomplished in comparison to suitable controls and to appropriate standards.

[0274] In some embodiments, the methods are adapted for use in vivo, e.g., to locate or identify sites where PRSS15-associated cancer cells are present. In these embodiments, a detectably-labeled moiety, e.g., an antibody, which is specific for PRSS 15 is administered to an individual (e.g., by injection), and labeled cells are located using standard imaging techniques, including, but not limited to, magnetic resonance imaging, computed tomography scanning, and the like. In this manner, PRSS 15 expressing cells are differentially labeled.

C Kits [0275] The detection methods may be provided as part of a kit. Thus, the invention further provides kits for detecting the presence and/or a level of a PRSS 15 polynucleotide expressed in a cancer cell (e.g., by detection of an mRNA encoded by the differentially expressed gene of interest), and/or a polypeptide encoded thereby, in a biological sample. Procedures using these kits may be performed by clinical laboratories, experimental laboratories, medical practitioners, or private individuals. The kits of the invention for detecting a polypeptide encoded by a PRSS 15 polynucleotide may comprise a PRSS 15 binding partner, such as an antibody. The kits of the invention for detecting a PRSS 15 polynucleotide may comprise a PRSS 15 probe that specifically hybridizes to such a polynucleotide. The kit may provide additional components that are useful in procedures, including, but not limited to, buffers, developing reagents, labels, reacting surfaces, means for detection, control samples, standards, instructions, and interpretive information.

D. Arrays [0276] In relation to methods of detection, the invention also provides microarrays comprising one or more polynucleotide sequences substantially identical to or complementary to the polynucleotide sequence of SEQ ID NO: 2 or a portion of the polynucleotide sequence of SEQ ID NO:2. [0277] The invention also provides microarrays comprising one or more protein- capture agents that bind one or more amino acid sequences encoded by all or a portion of the amino acid sequence SEQ ID NO: 1.

[0278] In another aspect, the invention provides a method of using a microarray to determine the presence or absence of a cancer. The method includes (a) determining the level of expression of PRSS 15 in a biological sample obtained from a patient, using the microarray, (b) comparing the level of PRSS 15 expression in the patient biological sample to the level of PRSS 15 expression in a normal biological sample, and (c) correlating the level of PRSS 15 expression in the patient biological sample to a positive or negative diagnosis of the cancer.

[0279] In a related aspect, the invention provides a method of using a microarray to determine a patient's predisposition to a cancer. The method includes (a) determining the level of expression of PRSS 15 in a biological sample obtained from the patient, using a microarray, (b) comparing the level of PRSS 15 expression in the patient biological sample to the level of PRSS 15 expression in a normal biological sample, and (c) correlating the level of PRSS 15 expression in the patient biological sample to a diagnosis of a predisposition to the cancer.

[0280] Polynucleotide arrays provide a high throughput technique that can assay a large number of polynucleotides or polypeptides in a sample. This technology can be used as a tool to test for differential expression.

[0281] A variety of methods of producing arrays, as well as variations of these methods, are known in the art and contemplated for use in the invention. For example, arrays can be created by spotting polynucleotide probes onto a substrate (e.g., glass, nitrocellulose, etc.) in a two-dimensional matrix or array having bound probes. The probes can be bound to the substrate by either covalent bonds or by non-specific interactions, such as hydrophobic interactions.

[0282] Samples of polynucleotides can be detectably labeled (e.g. , using radioactive or fluorescent labels) and then hybridized to the probes. Double stranded polynucleotides, comprising the labeled sample polynucleotides bound to probe polynucleotides, can be detected once the unbound portion of the sample is washed away. Alternatively, the polynucleotides of the test sample can be immobilized on the array, and the probes detectably labeled. Techniques for constructing arrays and methods of using these arrays are described in, for example, Schena et al. (1996) Proc Natl Acad Sci U S A. 93(20): 10614-9; Schena et al. (1995) Science 270(5235):467-70; Shalon et al. (1996) Genome Res. 6(7):639-45, USPN 5,807,522, EP 799 897; WO 97/29212; WO 97/27317; EP 785 280; WO 97/02357; USPN 5,593,839; USPN 5,578,832; EP 728 520; USPN 5,599,695; EP 721 016; USPN 5,556,752; WO 95/22058; and USPN 5,631,734. In most embodiments, the "probe" is detectably labeled. In other embodiments, the probe is immobilized on the array and not detectably labeled.

[0283] Arrays can be used, for example, to examine differential expression of genes and can be used to determine gene function. For example, arrays can be used to detect differential expression of a gene corresponding to a polynucleotide described herein, where expression is compared between a test cell and control cell (e.g., cancer cells and normal cells). For example, high expression of a particular message in a cancer cell, which is not observed in a corresponding normal cell, can indicate a cancer specific gene product. Exemplary uses of arrays are further described in, for example, Pappalarado et al., Sent. Radiation Oncol. (1998) 8:211; and Ramsay, Nature Biotechnol. (1998) i<5:40. Furthermore, many variations on methods of detection using arrays are well within the skill in the art and within the scope of the present invention. For example, rather than immobilizing the probe to a solid support, the test sample can be immobilized on a solid support which is then contacted with the probe.

E. Managing Cancer and Methods of Assessing Therapy and Prognosis [0284] Detection methods of the invention also are useful for assessing therapy, prognosing cancer, and making decisions about managing cancer in an individual. Accordingly, the invention also provides a method of assessing therapy and prognosing cancer, comprising determining the level of a PRSS 15 gene product in a biological sample obtained from a patient, where the level of PRSS 15 gene product indicates the effect of therapy on, or prognosis of a patient. [0285] PRSS15 polynucleotides and polypeptides are of particular interest as markers (e.g., in blood or tissues) that will detect changes along the carcinogenesis pathway and/or monitor the efficacy of various therapies and preventive interventions.

[0286] For example, the level of expression of PRSS 15 polynucleotides can be indicative of a poorer prognosis, and therefore warrant more aggressive chemo- or radiotherapy for a patient or vice versa.

[0287] Determining expression of PRSS 15 polynucleotides and comparison of a patient's profile with known expression in normal tissue and variants of the disease allows a determination of the best possible treatment for a patient, both in terms of specificity of treatment and in terms of comfort level of the patient. Surrogate tumor markers, such as polynucleotide expression, can also be used to better classify, and thus diagnose and treat, different forms and disease states of cancer. Two classifications widely used in oncology that can benefit from identification of the expression levels of PRSS 15 polynucleotides are staging of the cancerous disorder, and grading the nature of the cancerous tissue.

[0288] The PRSS 15 polynucleotides and polypeptides can be useful to monitor patients having or susceptible to cancer to detect potentially malignant events at a molecular level before they are detectable at a gross morphological level. In addition, PRSS 15 polynucleotides and polypeptides can be useful as therametrics, e.g., to assess the effectiveness of therapy by using the polynucleotides or their encoded gene products before, during, and after therapy.

[0289] Furthermore, a polynucleotide identified as corresponding to a gene that is differentially expressed in, and thus is important for, one type of cancer can also have implications for development or risk of development of other types of cancer, e.g., where a polynucleotide represents a gene differentially expressed across various cancer types. Thus, for example, expression of a polynucleotide corresponding to a gene that has clinical implications for high grade prostate cancer can also have clinical implications for breast cancer, or ovarian cancer.

1. Staging [0290] Staging is a process used by physicians to describe how advanced the cancerous state is in a patient. Staging assists the physician in determining a prognosis, planning treatment and evaluating the results of such treatment. Staging systems vary with the types of cancer, but generally involve the following "TNM" system: the type of tumor, indicated by T; whether the cancer has metastasized to nearby lymph nodes, indicated by N; and whether the cancer has metastasized to more distant parts of the body, indicated by M. Generally, if a cancer is only detectable in the area of the primary lesion without having spread to any lymph nodes it is called Stage I. If it has spread only to the closest lymph nodes, it is called Stage II. In Stage HI, the cancer has generally spread to the lymph nodes in near proximity to the site of the primary lesion. Cancers that have spread to a distant part of the body, such as the liver, bone, brain or other site, are Stage IV, the most advanced stage.

[0291] Detection of PRSS 15 polynucleotides and polypeptides can facilitate the staging process.

2. Grading of cancers [0292] Grade is a term used to describe how closely a tumor resembles normal tissue of its same type. The microscopic appearance of a tumor is used to identify tumor grade based on parameters such as cell morphology, cellular organization, and other markers of differentiation. As a general rule, the grade of a tumor corresponds to its rate of growth or aggressiveness, with undifferentiated or high-grade tumors generally being more aggressive than well-differentiated or low-grade tumors. The following guidelines are generally used for grading tumors: 1) GX Grade cannot be assessed; 2) Gl Well differentiated; 3) G2 Moderately well differentiated; 4) G3 Poorly differentiated; 5) G4 Undifferentiated. For prostate cancer, the Gleason Grading/Scoring system is most commonly used. A prostate biopsy tissue sample is examined under a microscope and a grade is assigned to the tissue based on: 1) the appearance of the cells, and 2) the arrangement of the cells. Each parameter is assessed on a scale of one (cells are almost normal) to five (abnormal), and the individual Gleason Grades are presented separated by a "+" sign. Alternatively, the two grades are combined to give a Gleason Score of 2-10. Thus, for a tissue sample that received a grade of 3 for each parameter, the Gleason Grade would be 3+3 and the Gleason Score would be 6. A lower Gleason Score indicates a well-differentiated tumor, while a higher Gleason Score indicates a poorly differentiated cancer that is more likely to spread. The majority of biopsies in general are Gleason Scores 5, 6 and 7.

[0293] PRSS 15 polynucleotides and polypeptides can be especially valuable in determining the grade of the tumor.

VU. Vectors. Host cells and Protein production [0294] The present invention also provides vectors containing PRSS 15 polynucleotides, host cells containing such vectors, and the production of PRSS 15 polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

[0295] PRSS 15 polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells. [0296] The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0297] As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria.

[0298] Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. 5 Appropriate culture mediums and conditions for the above-described host cells are known in the art.

[0299] Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNHSA, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, ρKK233-3, pDR540, pRITS available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHLL-D2, pHTL-Sl, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carload, CA). Other suitable vectors will be readily apparent to the skilled artisan. [0300] Nucleic acids of interest may be cloned into a suitable vector by route methods. Suitable vectors include plasmids, cosmids, recombinant viral vectors e.g. retroviral vectors, YACs, BACs and the like, phage vectors.

[0301] Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.

[0302] A PRSS 15 polypeptide can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.

[0303] PRSS 15 polypeptides can also be recovered from products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the PRSS 15 polypeptides may be glycosylated or may be non-glycosylated. In addition, PRSS 15 polypeptides may also include an initial modified methionine residue, in some cases as a result of host-5 mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.

[0304] In one embodiment, the yeast Pichia pastoris is used to express PRSS 15 polypeptides in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O2. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for 02. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOXl) is highly active. In the presence of methanol, alcohol oxidase produced from the AOXl gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See, Ellis, S.B., et ai, Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P.J, et ai, Yeast 5:167-77 (1989); Tschopp, J.F., et al, Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOXl regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.

[0305] In one example, the plasmid vector pPIC9K is used to express DNA encoding a PRSS 15 polypeptide, as set forth herein, in a Pichea yeast system essentially as described in "Pichia Protocols: Methods in Molecular Biology," D.R. Higgins and J. Cregg, eds. The Humana Press, Totowa, NJ, 1998. This expression vector allows expression and secretion of a PRSS 15 polypeptide by virtue of the strong AOXl promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.

[0306] Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pP!C9, pPIC3.5, pHJL-D2, pHTL-Sl, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required.

[0307] In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a PRSS 15 polynucleotide, may be achieved by cloning the heterologous PRSS 15 polynucleotide into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol. [0308] In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with PRSS 15 polynucleotides, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination (see, e.g., U.S. Patent No. 5,641,670, issued June 24, 1997; International Publication No. WO 96/29411, published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).

[0309] Examples of useful mammalian host cell lines are monkey kidney C V 1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad.Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather. Biol. Reprod. 23:243-251 (1980)),- monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0310] In addition, PRSS 15 polypeptides can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D- isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4- aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2- amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, 5 hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

[0311] Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see, e.g., Carter et al., Nucl. Acids Res. 73:4331 (1986); and Zoller et al., Nucl. Acids Res. 70:6487 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315 (1985)), restriction selection mutagenesis (see, e.g., Wells et al., Philos. Trans. R. Soc. London SerA 377:415 (1986)).

[0312] Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant. Alanine is also typically preferred because it is the most common amino acid. If alanine substituting does not yield adequate amounts of variant, an isoteric amino acid can be used.

Vm. Identifying PRSS 15 Agents [0313] In another aspect, the invention provides methods of identifying an agent that binds to PRSS 15 or modulates a biological activity of a PRSS 15 gene product.

[0314] One such method includes (a) contacting at least one candidate modulator with PRSS 15 under conditions wherein said at least one candidate modulator can bind the PRSS 15; and detecting the binding of said at least one candidate modulator to the PRSS 15.

[0315] Another such method includes (a) contacting a candidate modulator with a PRSS 15 gene product, and (b) detecting modulation of a biological activity of said gene product relative to a level of biological activity of said gene product in the absence of said candidate agent.

[0316] Still another such method comprises (a) culturing a cell line transfected with an expression vector comprising a gene encoding PRSS 15 to express the gene in a medium containing at least one candidate modulator of PRSS 15, and (b) measuring binding of the candidate modulator to the PRSS 15 produced by the cell line.

[0317] Yet another screening method includes (a) contacting a candidate modulator with a cancer cell characterized by overexpression and/or upregulation of PRSS 15 under conditions that allow the candidate modulator to bind the PRSS 15, and (b) detecting a decrease or inhibition of proliferation of the cancer cell relative to proliferation of a cell of the same type that has not contacted the candidate modulator.

[0318] In still another aspect, the invention provides methods of determining the ability of a drug to inhibit substrate cleavage of PRSS 15. One such method comprises the steps of (a) culturing a cell line transfected with an expression vector comprising a gene encoding PRSS 15 to express the gene (i) in the presence of the substrate and (ii) in the presence of both the substrate and the drug; and (b) comparing the level of substrate cleavage that occurs in (a)(i) and (a)(ii), where a lower level of substrate cleavage in the presence of the drug indicates that the drug is an inhibitor of substrate cleavage.

[0319] Identification of PRSS 15 agents can be accomplished using any of a variety of drug screening techniques. Such agents are candidates for development of cancer therapies. Of particular interest are screening assays for agents that have tolerable toxicity for normal, non-cancerous human cells. The screening assays of the invention are generally based upon the ability of the agent to modulate an activity of a PRSS 15 gene product and/or to inhibit or suppress phenomenon associated with cancer (e.g., cell proliferation, colony formation, cell cycle arrest, metastasis, and the like).

[0320] Screening assays can be based upon any of a variety of techniques readily available and known to one of ordinary skill in the art. In general, the screening assays involve contacting a cancerous cell (preferably a cancerous cell such as a cancerous prostate cell) with a candidate agent, and assessing the effect upon biological activity of a differentially expressed gene product. The effect upon a biological activity can be detected by, for example, detection of expression of a PRSS15 gene product (e.g., a decrease in mRNA or polypeptide levels, would in turn cause a decrease in biological activity of the gene product). Alternatively or in addition, the effect of the candidate agent can be assessed by examining the effect of the candidate agent in a functional assay. For example, the effect upon protease activity can be assessed. Agents of interest primarily are those that decrease activity of PRSS15.

[0321] Assays described herein can be readily adapted in the screening assay embodiments of the invention. Exemplary assays useful in screening candidate agents include, but are not limited to, hybridization-based assays (e.g., use of nucleic acid probes or primers to assess expression levels), antibody-based assays (e.g., to assess levels of polypeptide gene products), binding assays (e.g., to detect interaction of a candidate agent with a differentially expressed polypeptide, which assays may be competitive assays where a natural or synthetic ligand for the polypeptide is available), and the like. Additional exemplary assays include, but are not necessarily limited to, cell proliferation assays, antisense knockout assays, assays to detect inhibition of cell cycle, assays of induction of cell death/apoptosis, and the like. Generally such assays are conducted in vitro, but many assays can be adapted for in vivo analyses, e.g., in an animal model of the cancer.

[0322] In certain embodiments, the methods of screening of candidate agents involves producing a composition with a certain activity e.g. a PRSS 15 protease activity, and determining whether a candidate agent can modulate (i.e., increase or decrease) the activity of the composition. Activity-modulating agents may then be "counter-screened" against other, compositions that have related activities, such as other types of protease, in order to determine the specificity of the agent. Agents with modulatory activity are usually agents that specifically modulate a particular protease activity, or activities, with respect to other activities

[0323] PRSS 15 modulatory agents are agents that modulate (i.e. increase or decrease) PRSS 15 protease activity with respect to the activity of other proteases. In many embodiments, a modulatory agent increases or decreases the activity of PRSS 15 by greater than about 20%, greater than about 40%, greater than about 60%, greater than about 80%, or greater than about 90% or more, as compared to the activity of a another protease. [0324] In certain embodiments, a PRSS 15 modulator has a cytotoxic activity and/or a cytostatic activity. A cytotoxic activity is an activity that reduces the viability of a cell, and may result in cell death. In certain embodiments, greater than about 20%, greater than about 40%, greater than about 60%, greater than about 80%, or greater than about 90% of cells are killed by such an agent. A cytostatic activity is an activity that reduces cell division without reducing viability of the cell.

A. Candidate agents [0325] Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

[0326] Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts (including extracts from human tissue to identify endogenous factors affecting differentially expressed gene products) are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

[0327] Exemplary candidate agents of particular interest include, but are not limited to, antisense and RNAi polynucleotides, antibodies, soluble receptors, and the like. Antibodies and soluble receptors are of particular interest as candidate agents where the target differentially expressed gene product is secreted or accessible at the cell-surface (e.g., receptors and other molecule stably-associated with the outer cell membrane).

B. RNAi Agents [0328] For methods that involve RNAi (RNA interference), a double stranded RNA (dsRNA) molecule is usually used. The dsRNA is prepared to be substantially identical to at least a segment of a subject polynucleotide (e.g. a cDNA or gene). In general, the dsRNA is selected to have at least 70%, 75%, 80%, 85% or 90% sequence identity with the subject polynucleotide over at least a segment of the candidate gene. In other instances, the sequence identity is even higher, such as 95%, 97% or 99%, and in still other instances, there is 100% sequence identity with the subject polynucleotide over at least a segment of the subject polynucleotide. The size of the segment over which there is sequence identity can vary depending upon the size of the subject polynucleotide. In general, however, there is substantial sequence identity over at least 15, 20, 25, 30, 35, 40 or 50 nucleotides. In other instances, there is substantial sequence identity over at least 100, 200, 300, 400, 500 or 1000 nucleotides; in still other instances, there is substantial sequence identity over the entire length of the subject polynucleotide, i.e., the coding and non-coding region of the candidate gene.

[0329] Because only substantial sequence similarity between the subject polynucleotide and the dsRNA is necessary, sequence variations between these two species arising from genetic mutations, evolutionary divergence and polymorphisms can be tolerated. Moreover, as described further infra, the dsRNA can include various modified or nucleotide analogs.

[0330] Usually the dsRNA consists of two separate complementary RNA strands. However, in some instances, the dsRNA may be formed by a single strand of RNA that is self -complementary, such that the strand loops back upon itself to form a hairpin loop. Regardless of form, RNA duplex formation can occur inside or outside of a cell.

[0331] The size of the dsRNA that is utilized varies according to the size of the subject polynucleotide whose expression is to be suppressed and is sufficiently long to be effective in reducing expression of the subject polynucleotide in a cell. Generally, the dsRNA is at least 10-15 nucleotides long. In certain applications, the dsRNA is less than 20, 21, 22, 23, 24 or 25 nucleotides in length. In other instances, the dsRNA is at least 50, 100, 150 or 200 nucleotides in length. The dsRNA can be longer still in certain other applications, such as at least 300, 400, 500 or 600 nucleotides. Typically, the dsRNA is not longer than 3000 nucleotides. The optimal size for any particular subject polynucleotide can be determined by one of ordinary skill in the art without undue experimentation by varying the size of the dsRNA in a systematic fashion and determining whether the size selected is effective in interfering with expression of the subject polynucleotide.

[0332] dsRNA can be prepared according to any of a number of methods that are known in the art, including in vitro and in vivo methods, as well as by synthetic chemistry approaches.

[0333] In vitro methods. Certain methods generally involve inserting the segment corresponding to the candidate gene that is to be transcribed between a promoter or pair of promoters that are oriented to drive transcription of the inserted segment and then utilizing an appropriate RNA polymerase to carry out transcription. One such arrangement involves positioning a DNA fragment corresponding to the candidate gene or segment thereof into a vector such that it is flanked by two opposable polymerase-specific promoters that can be same or different. Transcription from such promoters produces two complementary RNA strands that can subsequently anneal to form the desired dsRNA. Exemplary plasmids for use in such systems include the plasmid (PCR 4.0 TOPO) (available from Invitrogen). Another example is the vector pGEM-T (Promega, Madison, WI) in which the oppositely oriented promoters are T7 and SP6; the T3 promoter can also be utilized.

[0334] In a second arrangement, DNA fragments corresponding to the segment of the subject polynucleotide that is to be transcribed is inserted both in the sense and antisense orientation downstream of a single promoter. In this system, the sense and antisense fragments are cotranscribed to generate a single RNA strand that is self-complementary and thus can form dsRNA.

[0335] Various other in vitro methods have been described. Examples of such methods include, but are not limited to, the methods described by Sadher et al. (Biochem. Int. 14:1015, 1987); by Bhattacharyya (Nature 343:484, 1990); and by Livache, et al. (U.S. Patent No. 5,795,715), each of which is incorporated herein by reference in its entirety.

[0336] Single-stranded RNA can also be produced using a combination of enzymatic and organic synthesis or by total organic synthesis. The use of synthetic chemical methods enable one to introduce desired modified nucleotides or nucleotide analogs into the dsRNA.

[0337] In vivo methods. dsRNA can also be prepared in vivo according to a number of established methods (see, e.g., Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, 2^nd ed.; Transcription and Translation (B.D. Hames, and S.J. Higgins, Eds., 1984); DNA Cloning, volumes I and LT (D.N. Glover, Ed., 1985); and Oligonucleotide Synthesis (M.J. Gait, Ed., 1984, each of which is incorporated herein by reference in its entirety).

[0338] Once the single-stranded RNA has been formed, the complementary strands are allowed to anneal to form duplex RNA. Transcripts are typically treated with DNAase and further purified according to established protocols to remove proteins. Usually such purification methods are not conducted with phenol: chloroform. The resulting purified transcripts are subsequently dissolved in RNAase free water or a buffer of suitable composition.

[0339] dsRNA is generated by annealing the sense and anti-sense RNA in vitro. Generally, the strands are initially denatured to keep the strands separate and to avoid self- annealing. During the annealing process, typically certain ratios of the sense and antisense strands are combined to facilitate the annealing process. In some instances, a molar ratio of sense to antisense strands of 3:7 is used; in other instances, a ratio of 4:6 is utilized; and in still other instances, the ratio is 1:1.

[0340] The buffer composition utilized during the annealing process can in some instances affect the efficacy of the annealing process and subsequent transfection procedure. While some have indicated that the buffered solution used to carry out the annealing process should include a potassium salt such as potassium chloride (e.g. at a concentration of about 80 mM). In some embodiments, the buffer is substantially postassium free. Once single- stranded RNA has annealed to form duplex RNA, typically any single-strand overhangs are removed using an enzyme that specifically cleaves such overhangs (e.g., RNAase A or RNAase T).

[0341] Once the dsRNA has been formed, it is introduced into a reference cell, which can include an individual cell or a population of cells (e.g., a tissue, an embryo and an entire organism). The cell can be from essentially any source, including animal, plant, viral, bacterial, fungal and other sources. If a tissue, the tissue can include dividing or nondividing and differentiated or undifferentiated cells. Further, the tissue can include germ line cells and somatic cells. Examples of differentiated cells that can be utilized include, but are not limited to, neurons, glial cells, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, adipocytes, osteoblasts, osteoclasts, hepatocytes, cells of the endocrine or exocrine glands, fibroblasts, myocytes, cardiomyocytes, and endothelial cells. The cell can be an individual cell of an embryo, and can be a blastocyte or an oocyte.

[0342] Certain methods are conducted using model systems for particular cellular states (e.g., a disease). For instance, certain methods provided herein are conducted with a cancer cell lines that serves as a model system for investigating genes that are correlated with various cancers.

[0343] A number of options can be utilized to deliver the dsRNA into a cell or population of cells such as in a cell culture, tissue or embryo. For instance, RNA can be directly introduced intracellularly. Various physical methods are generally utilized in such instances, such as administration by microinjection (see, e.g., Zernicka-Goetz, et al. (1997) Development 124:1133-1137; and Wianny, et al. (1998) Chromosoma 107: 430-439).

[0344] Other options for cellular delivery include permeabilizing the cell membrane and electroporation in the presence of the dsRNA, liposome-mediated transfection, or transfection using chemicals such as calcium phosphate. A number of established gene therapy techniques can also be utilized to introduce the dsRNA into a cell. By introducing a viral construct within a viral particle, for instance, one can achieve efficient introduction of an expression construct into the cell and transcription of the RNA encoded by the construct.

[0345] If the dsRNA is to be introduced into an organism or tissue, gene gun technology is an option that can be employed. This generally involves immobilizing the dsRNA on a gold particle which is subsequently fired into the desired tissue. Research has also shown that mammalian cells have transport mechanisms for taking in dsRNA (see, e.g., Asher, et al. (1969) Nature 223:715-717). Consequently, another delivery option is to administer the dsRNA extracellularly into a body cavity, interstitial space or into the blood system of the mammal for subsequent uptake by such transport processes. The blood and lymph systems and the cerebrospinal fluid are potential sites for injecting dsRNA. Oral, topical, parenteral, rectal and intraperitoneal administration are also possible modes of administration.

[0346] The composition introduced can also include various other agents in addition to the dsRNA. Examples of such agents include, but are not limited to, those that stabilize the dsRNA, enhance cellular uptake and/or increase the extent of interference. Typically, the dsRNA is introduced in a buffer that is compatible with the composition of the cell into which the RNA is introduced to prevent the cell from being shocked. The minimum size of the dsRNA that effectively achieves gene silencing can also influence the choice of delivery system and solution composition.

[0347] Sufficient dsRNA is introduced into the tissue to cause a detectable change in expression of a taget gene (assuming the candidate gene is in fact being expressed in the cell into which the dsRNA is introduced) using available detection methodologies. Thus, in some instances, sufficient dsRNA is introduced to achieve at least a 5-10% reduction in candidate gene expression as compared to a cell in which the dsRNA is not introduced. In other instances, inhibition is at least 20, 30, 40 or 50%. In still other instances, the inhibition is at least 60, 70, 80, 90 or 95%. Expression in some instances is essentially completely inhibited to undetectable levels.

[0348] The amount of dsRNA introduced depends upon various factors such as the mode of administration utilized, the size of the dsRNA, the number of cells into which dsRNA is administered, and the age and size of an animal if dsRNA is introduced into an animal. An appropriate amount can be determined by those of ordinary skill in the art by initially administering dsRNA at several different concentrations for example, for example. In certain instances when dsRNA is introduced into a cell culture, the amount of dsRNA introduced into the cells varies from about 0.5 to 3 μg per IO⁶ cells. [0349] A number of options are available to detect interference of candidate gene expression (i.e., to detect candidate gene silencing). In general, inhibition in expression is detected by detecting a decrease in the level of the protein encoded by the candidate gene, determining the level of mRNA transcribed from the gene and/or detecting a change in phenotype associated with candidate gene expression.

C. Use of Polypeptides to Screen for Peptide Analogs and Antagonists [0350] Polypeptides encoded by PRSS 15 polynucleotides can be used to screen peptide libraries to identify modulators, such as receptors, from among the encoded polypeptides. Peptide libraries can be synthesized according to methods known in the art (see, e.g., USPN 5,010,175 and WO 91/17823).

[0351] Agonists or antagonists of the PRSS 15 polypeptides can be screened using any available method known in the art, such as signal transduction, antibody binding, receptor binding, mitogenic assays, chemotaxis assays, etc. The assay conditions ideally should resemble the conditions under which the native activity is exhibited in vivo, that is, under physiologic pH, temperature, and ionic strength. Suitable agonists or antagonists will exhibit strong inhibition or enhancement of the native activity at concentrations that do not cause toxic side effects in the subject. Agonists or antagonists that compete for binding to the native polypeptide can require concentrations equal to or greater than the native concentration, while inhibitors capable of binding irreversibly to the polypeptide can be added in concentrations on the order of the native concentration.

[0352] Such screening and experimentation can lead to identification of a polypeptide modulator and at least one peptide agonist or antagonist of the modulator. Such agonists and antagonists can be used to modulate, enhance, or inhibit PRSS15 function in cells.

LX. Examples [0353] Without further elaboration, it is believed that one skilled in the art, using the preceding description, can fully utilize the invention. The following examples are therefore illustrative only, and not limiting of the remainder of the disclosure in any way whatsoever. Example 1 : Detection of Differential Expression using Arrays [0354] This example demonstrates that the PRSS 15 gene is upregulated in many colon, prostate, breast and stomach cancers, as evidenced by mRNA levels.

[0355] Patients diagnosed with colon cancer (n=76), prostate cancer (n=106), breast cancer (n=23) and stomach cancer (n=7) were analyzed for upregulation of the PRSS 15 gene. Normal and cancerous tissues were collected from patients using laser capture microdissection (LCM) techniques, which are well known in the art (see, e.g., Ohyama et al. (2000) Biotechniques 29:530-6; Curran et al. (2000) Mol. PathoL 53:64-8; Suarez-Quian et al. (1999) Biotechniques 26:328-35; Simone et al. (1998) Trends Genet 14:272-6; Conia et al. (1997) J. Clin. Lab. Anal. 11:28-38; Emmert-Buck et al. (1996) Science 274:998-1001).

[0356] cDNA probes were prepared from total RNA isolated from the patient cells. Because LCM provides for the isolation of specific cell types to provide a substantially homogenous cell sample, this provided for a similarly pure RNA sample.

[0357] Total RNA was first reverse transcribed into cDNA using a primer containing a T7 RNA polymerase promoter, followed by second strand DNA synthesis. cDNA was then transcribed in vitro to produce antisense RNA using the T7 promoter- mediated expression (see, e.g., Luo et al. (1999) Nature Med 5:117-122), and the antisense RNA was then converted into cDNA. The second set of cDNAs were again transcribed in vitro, using the T7 promoter, to provide antisense RNA. Optionally, the RNA was again converted into cDNA, allowing for up to a third round of T7 -mediated amplification to produce more antisense RNA. Thus the procedure provided for two or three rounds of in vitro transcription to produce the final RNA used for fluorescent labeling.

[0358] Fluorescent probes were generated by first adding control RNA to the antisense RNA mix, and producing fluorescently labeled cDNA from the RNA starting material. Fluorescently labeled cDNAs prepared from the tumor RNA sample were compared to fluorescently labeled cDNAs prepared from normal cell RNA sample. For example, the cDNA probes from the normal cells were labeled with Cy3 fluorescent dye (green) and the cDNA probes prepared from the tumor cells were labeled with Cy5 fluorescent dye (red), and vice versa. [0359] Polynucleotides for use on the arrays were obtained from both publicly available sources and from cDNA libraries generated from selected cell lines and patient tissues. PCR products from these sources were spotted onto the array using a Molecular Dynamics Gen in spotter according to the manufacturer's recommendations. The array included control spots, including negative control spots, and test polynucleotides. The test polynucleotides were spiked into each sample before the labeling reaction with a range of concentrations from 2-600 pg/slide and ratios of 1:1.

[0360] The differential expression assay was performed by mixing equal amounts of probes from tumor cells and normal cells of the same patient. The arrays were prehybridized by incubation for about 2 hrs at 60°C in 5X SSC/0.2% SDS/1 mM EDTA, and then washed three times in water and twice in isopropanol. Following prehybridization of the array, the probe mixture was then hybridized to the array under conditions of high stringency (overnight at 42°C in 50% formamide, 5X SSC, and 0.2% SDS. After hybridization, the array was washed at 55°C three times as follows: 1) first wash in IX SSC/0.2% SDS; 2) second wash in 0.1X SSC/0.2% SDS; and 3) third wash in 0.1X SSC.

[0361] The arrays were then scanned for green and red fluorescence using a Molecular Dynamics Generation HI dual color laser-scanner/detector. The images were processed using BioDiscovery Autogene software, and the data from each scan set normalized to provide for a ratio of expression relative to normal. Data from the microarray experiments was analyzed according to the algorithms described in U.S. application serial no. 60/252,358, filed November 20, 2000, by E.J. Moler, M.A. Boyle, and F.M. Randazzo, and entitled "Precision and accuracy in cDNA microarray data," which application is specifically incorporated herein by reference.

[0362] The experiment was repeated, labeling the probes with the opposite color in order to perform the assay in both "color directions." The level of fluorescence for each sequence on the array was expressed as a ratio of the geometric mean of replicate spots/genes from the different arrays or replicate spots/gene from different arrays or some other permutation. The data were normalized using the spiked positive controls present in each duplicated area, and the precision of this normalization was included in the final determination of the significance of each differential. The fluorescent intensity of each spot was also compared to the negative controls in each duplicated area to determine which spots have detected significant expression levels in each sample.

[0363] A statistical analysis of the fluorescent intensities was applied to each set of duplicate spots to assess the precision and significance of each differential measurement, resulting in a p-value testing the null hypothesis that there is no differential in the expression level between the tumor and normal samples of each patient. During initial analysis of the microarrays, the hypothesis was accepted if p > 10^"3, and the differential ratio was set to 1.000 for those spots. All other spots have a significant difference in expression between the tumor and normal sample. If the tumor sample has detectable expression and the normal does not, the ratio is truncated at 1000 since the value for expression in the normal sample would be zero, and the ratio would not be a mathematically useful value (e.g., infinity). If the normal sample has detectable expression and the tumor does not, the ratio is truncated to 0.001, since the value for expression in the tumor sample would be zero and the ratio would not be a mathematically useful value. These latter two situations are referred to herein as "on/off." Database tables were populated using a 95% confidence level (p>0.05).

[0364] Table 1 summarizes the results of this study.

Table 1

[0365] PRSS 15 mRNA was upregulated to more than twice its normal level in 39% of colon cancers and to more than five times its normal level in 5-9% of colon cancers. PRSS 15 mRNA was not down-regulated in any of the colon cancers.

[0366] In prostate cancer, PRSS 15 mRNA was upregulated to more than twice its normal level in 15% of cases and down-regulated in 4% of cases. However, 36.4% of high grade prostate cancer (Gleason grade 4p3) showed upregulation of PRSS 15 mRNA to more than twice its normal level, and no cases of high grade prostate case showed down-regulation of PRSS 15 mRNA.

[0367] Breast and stomach cancers showed more variation, some cases had upregulation and others had downregulation of PRSS 15 mRNA.

[0368] The results of this study show that PRSS 15 is differentially expressed in colon, prostate (particularly high grade prostate), breast and stomach cancers.

Example 2: Detection of Differential Expression using Quantitative PCR [0369] This example further demonstrates that the PRSS15 gene is upregulated in many colon, prostate, breast and stomach cancers, as evidenced by mRNA levels.

[0370] Quantitative PCR was performed to examine the level of PRSS 15 mRNA in various colon, prostate and breast cancers relative to corresponding normal tissues. Quantitative real-time PCR was performed by first isolating RNA from cells using a Roche RNA Isolation kit according to manufacturer's directions. One microgram of RNA was used to synthesize a first-strand cDNA using MMLV reverse transcriptase (Ambion) using the manufacturer's buffer and recommended concentrations of oligo dT, nucleotides and Rnasin.

[0371] First, primers were designed. The primers were biased against known genes and sequences to confirm the specificity of the primers to the target. The sequences of the primers are Forward: TAAGCGGCTGTACAAGG, and Reverse: GGTGGGTCTGCTTGATC. These primers were used in a test qPCR using the primers against normal RTd tissue, as well as a mock RT to pick up levels of possible genomic contamination.

[0372] Quantitative PCR of a panel of normal tissue and LCM tissue were used to determine the expression levels of PRSS 15. Quantitative PCR was performed by first isolating the RNA from the above mentioned tissue/cells using a Qiagen RNeasy mini prep kit. In the case of the LCM tissue, RNA was amplified via PCR to increase concentration after initial RNA isolation. 0.5 micrograms of RnA was used to generate a first strand cDNA using Stratagene MuLV Reverse Transcriptase, using recommended concentrations of buffer, enzyme, and Rnasin. Concentrations and volumes of dNTP, and oligo dT, or random hexamers were lower than recommended to reduce the level of background primer dimerization in the qPCR.

[0373] The cDNA was then used for qPCR to determine the levels of expression of PRSS 15 using the GeneAmp 7000 by ABI as recommended by the manufacturer. Primers for housekeeping were also run in order to normalize the values and eliminate possible variations in cDNA template concentrations, pipetting error, etc.

[0374] Figure 1 shows the results, which are expressed as a relative value normalized to GusB. All three types of cancer showed significant upregulation relative to normal controls.

Example 3: Detection of Differential Expression in Colon Cancer using Arrays [0375] The methods of Example 1 were used to evaluate mRNA expression of PRSS 15 in colon cancer. Samples of 8 primary colon tumors, 29 metastatic colon tumors and 4 normal colon tissues were analyzed. [0376] The results are shown in Figure 2, with each measurement being expressed as a percentage of the highest PRSS 15 mRNA level detected. The bold vertical line marks the average value for normal colon samples. PRSS 15 mRNA levels in all but one of the tumor samples was higher than this average normal value.

Example 4: Detection of PRSS15 in Normal Human Tissues [0377] This example shows that PRSS 15 mRNA is ubiquitously expressed in normal human tissues. The methods of Example 1 were used to evaluate PRSS 15 mRNA levels in diverse normal human tissues. Thirty five different types of normal tissue were tested.

[0378] Figure 3 shows the results. Every normal tissue contained PRSS 15 mRNA. This is expected because all human tissues contain mitochondria. The level of PRSS 15 mRNA in adrenal tissue is exceptionally high, owing to the large number of mitochondria in adrenal tissue. On a per mitochondria basis, however, the level of PRSS 15 in adrenal glands is on par with other tissues. Thus, cancer treatments that target PRSS 15 should not be more toxic to adrenals that other organs.

Example 5: PRSS15 mRNA is Upregulated in Tumor Cell Lines [0379] This example shows that PRSS 15 mRNA is upregulated in cell lines derived from tumors.

[0380] Quantitative PCR was performed on normal prostate epithelial cells (PrEC), prostate epithelial cells transformed with HPV-18 (RWPE-1 and RWPE-2), prostate cancer cells derived from metastatic prostate tumors (Dul45, PC3, LnCap, MDAPca2b, and 22rVl) and breast cancer cells (MDA231, MDA435, and MCF7).

[0381] Results are shown in Figure 4 as relative values normalized to beta actin expression. All of the cancer cells had markedly more PRSS 15 mRNA expression than normal prostate epithelial cells or normal prostate epithelial cells transformed with HPV-18.

Example 7: Antisense Oligonucleotides Knock Down PRSS15 mRNA in LNCaP Cells [0382] This example demonstrates antisense regulation of PRSS15 expression. A number of different oligonucleotides complementary to PRSS 15 mRNA were designed as potential antisense oligonucleotides and tested for their ability to suppress expression of PRSS 15.

[0383] The antisense oligomers were designed using the sequences of the PRSS 15 polynucleotide sequence and the software program HYBsimulator Version 4 (available for Windows 95/Windows NT or for Power Macintosh, RNAture, Inc. 1003 Health Sciences Road, West, Irvine, CA 92612 USA). Factors that were considered when designing the antisense oligonucleotides included: 1) the secondary structure of oligonucleotides; 2) the secondary structure of the target gene; 3) the specificity with no or minimum cross- hybridization to other expressed genes; 4) stability; 5) length and 6) terminal GC content. The antisense oligonucleotides were designed so that they will hybridize to their target sequences under conditions of high stringency at physiological temperatures (e.g., an optimal temperature for the cells in culture to provide for hybridization in the cell, e.g., about 37°C), but with minimal formation of homodimers. A reverse control of antisense oligomers, designated -12RC, also were designed.

[0384] The sequences of antisense oligonucleotides, which were used throughout the examples provided herein, are listed in Table 2 (below).

Table 2

[0385] For each transfection mixture, a carrier molecule, preferably a lipitoid or cholesteroid, was prepared to a working concentration of 0.5 mM in water, sonicated to yield a uniform solution, and filtered through a 0.45 um PVDF membrane. The antisense or control oligonucleotide was then prepared to a working concentration of 100 uM in sterile Millipore water. The oligonucleotide was further diluted in OptiMEM™ (Gibco/BRL), in a microfuge tube, to 2 μM, or approximately 20 μg oligo/ml of OptiMEM™. In a separate microfuge tube, lipitoid or cholesteroid, typically in the amount of about 1.5-2 nmol lipitoid/μg antisense oligonucleotide, was diluted into the same volume of OptiMEM™ used to dilute the oligonucleotide. The diluted antisense oligonucleotide was immediately added to the diluted lipitoid and mixed by pipetting up and down. Oligonucleotide was added to the cells to a final concentration of 30 nM.

[0386] The level of target mRNA (PRSS 15) in the transfected cells was quantitated in the cancer cell lines using the methods described above. Values for the target mRNA were normalized versus an internal control (e.g., beta-actin or Gus B). For each 20 μl reaction, extracted RNA (generally 0.2-1 μg total) was placed into a sterile 0.5 or 1.5 ml microcentrifuge tube, and water was added to a total volume of 12.5 μl. To each tube was added 7.5 μl of a buffer/enzyme mixture, prepared by mixing (in the order listed) 2.5 μl H₂O, 2.0 μl 10X reaction buffer, 10 μl oligo dT (20 pmol), 1.0 μl dNTP mix (10 mM each), 0.5 μl RNAsin® (20u) (Ambion, Inc., Hialeah, FL), and 0.5 μl MMLV reverse transcriptase (50u) (Ambion, Inc.). The contents were mixed by pipetting up and down, and the reaction mixture was incubated at 42°C for 1 hour. The contents of each tube were centrifuged prior to amplification. [0387] An amplification mixture was prepared by mixing in the following order: IX PCR buffer LT, 3 mM MgCl₂, 140 μM each dNTP, 0.175 pmol each oligo, 1:50,000 dil of SYBR® Green, 0.25 mg/ml BSA, 1 unit Taq polymerase, and H₂O to 20 μl. (PCR buffer LT is available in 10X concentration from Perkin-Elmer, Norwalk, CT). In IX concentration it contains 10 mM Tris pH 8.3 and 50 mM KC1. SYBR® Green (Molecular Probes, Eugene, OR) is a dye which fluoresces when bound to double stranded DNA. As double stranded PCR product is produced during amplification, the fluorescence from SYBR® Green increases. To each 20 μl aliquot of amplification mixture, 2 μl of template RT was added, and amplification was carried out according to standard protocols.

[0388] Tables 3 and 4 (below) show the results obtained with several antisense oligonucleotides.

Table 3

Table 4

Example 8: PRSS15 Knock-Out Inhibits Anchorage Dependent Growth in Prostate and Colon Cancer Cell Lines [0389] This example shows that PRSS 15 contributes to anchorage dependent in prostate and colon cancer cells.

[0390] The ability of the antisense oligonucleotides to inhibit cell proliferation was assessed in PC3 prostate cancer cells, 22rVl prostate cancer cells, HCT116 colon cancer cells, and SW620 colon cancer cells. [0391] Cells were transfected overnight at 37°C and the transfection mixture was replaced with fresh medium the next morning. Transfection was performed as follows. For each transfection mixture, a carrier molecule (such as a lipid, lipid derivative, lipid-like molecule, cholesterol, cholesterol derivative, or cholesterol-like molecule) was prepared to a working concentration of 0.5 mM in water, sonicated to yield a uniform solution, and filtered through a 0.45 μm PVDF membrane. The antisense or control oligonucleotide was then prepared to a working concentration of 100 μM in sterile Millipore water. The oligonucleotide was further diluted in OptiMEM™ (Gibco/BRL), in a microfuge tube, to 2 μM, or approximately 20 μg oligo/ml of OptiMEM™. In a separate microfuge tube, the carrier molecule, typically in the amount of about 1.5-2 nmol carrier/μg antisense oligonucleotide, was diluted into the same volume of OptiMEM™ used to dilute the oligonucleotide. The diluted antisense oligonucleotide was immediately added to the diluted carrier and mixed by pipetting up and down. Oligonucleotide was added to the cells to a final concentration of 30 nM.

[0392] Cells were plated to approximately 60-80% confluency in 96-well dishes. Antisense or reverse control oligonucleotide was diluted to 2 μM in OptiMEM™. The oligonucleotide-OptiMEM™ was then added to a delivery vehicle, which was optimized for the particular cell type used in the assay. The oligo/delivery vehicle mixture was then further diluted into medium with serum on the cells. The final concentration of oligonucleotide for all experiments was about 300 nM.

[0393] Alternatively, cells were plated in T75 flasks an transfected under conditions that were scaled up relative to increased surface area. Cells were then detached post- transfection, counted and seeded onto 96-well plates.

[0394] Cells were grown for four days with regular changes of media.

[0395] Cell proliferation was assessed using commercially available kits such as Cyquant® (Molecular Probes, Inc., Eugene, OR) and Quantos™ (Stratagene, Inc., La Jolla, CA). The plates were read using a microtiter plate-reading fluorometer with filters appropriate for fluorescence wavelength, for example, 355 nm and 460 nm emission. [0396] The PRSS 15 antisense oligonucleotides effectively inhibited growth in all four cancer cell lines. Figures 5-8 summarize the results in PC3, 22rVl, HCT116 and SW620 cells, respectively. The results are expressed in fluorescence units. UTl and UT2 represent untreated cells. CHTR109-3 is a positive control antisense molecule.

Example 9: PRSS15 Knock-Out also Inhibits Anchorage Dependent Growth in Other Cancer Cell Lines [0397] The methods of Example 8 were used to study the effect of PRSS 15 antisense oligonucleotides on HT29, MDA435 and HT1080 cells. Results are shown in Figure 23.

Example 10: PRSS15 Knock-Out Inhibits Anchorage Independent Growth in Prostate and Colon Cancer Cell Lines [0398] This example shows that PRSS 15 contributes to anchorage independent growth in prostate and colon cancer cell lines.

[0399] The effect of gene expression upon colony formation of PC3 cells, 22Rvl cells, HCT116 cells, SW620 cells, LNCaP cells and 2b cells was tested in a soft agar assay. Soft agar assays were conducted by first establishing a bottom layer of 2 ml of 0.6% agar in media plated fresh within a few hours of layering on the cells. The cell layer was formed on the bottom layer by removing cells transfected as described above from plates using 0.05% trypsin and washing twice in media. The cells were counted in a Coulter counter, and resuspended to IO⁶ per ml in media. 10 μl aliquots were placed with media in 96-well plates (to check counting with WST1), or diluted further for the soft agar assay. 2000 cells were plated in 800 μl 0.4% agar in duplicate wells above 0.6% agar bottom layer. After the cell layer agar solidified, 2 ml of media was dribbled on top and antisense or reverse control oligo (as described above) was added without delivery vehicles. Fresh media and oligos were added every 3-4 days.

[0400] After colonies appeared, 20 μl undiluted Alamar blue dye was added to each well. The plate was then placed on a shaker for about 10 to about 15 minutes to insure even penetration in the agar mesh. The plates were returned to the incubator and fluorescence (excitation 530 nm, emission 590 nm) was monitored after several hours (generally multiples readings are recommended around 3, 5 and 24 hours). [0401] The PRSS 15 antisense oligonucleotides effectively inhibited growth in all four cancer cell lines. Figures 9-14 summarize the results in PC3, 22rVl, HCT116, SW620 cells, LNCaP cells and 2b cells respectively. The results are expressed in fluorescence units. UTl and UT2 represent untreated cells. CHTR109-3 is a positive control antisense molecule.

Example 11: PRSS15 Knock-Out Induces Prostate and Colon Cancer Cell Death [0402] This example shows that knock-out of PRSS 15 induces prostate and colon cancer cell death.

[0403] Transfection of PC3, 22rVl, HCT116, SW620 and DU145 cells was performed as described above. The cells were plated one day before transfection in four 96- well plates, one plate for "day 0" and three plates for days 1-3. Cells were transfected with antisense oligonucleotides and reverse control oligonucleotides After transfection, the plates were incubated at 37°C overnight.

[0404] About 8 mis of warmed alpha MEM LDH lysis buffer (2% Triton X100) and about 8 mis of culture media (1/2 dilution) were combined. Two 96-well v-bottom plates were prepared, one labeled "lysis" and one labeled "supernatant." About 100 μl of alpha MEM was added to the wells containing 100 μl of the transfection mix without mixing. The plates were tilted and all 200 μl of the supernatant was transferred to supernatant plate.

[0405] About 200 μl Alpha MEM lysis buffer (diluted Vi) was added to the plate from which supernatant was removed, causes lysis of the cells. The solution was mixed 4-5 times then about 200 μl of lysed cells were transferred to the lysis plate. Both the lysis and supernatant plates were centrifuged at about 1600 rpm for about 10 min. As before, two 96- well flat-bottomed plates were labeled as "lysate" and "supernatant." After centrifugation, about 100 μl of both the supernatant or lysate was transferred to the corresponding 96-well flat-bottomed plate.

[0406] The assay was developed using a commercially available kit, the Cytotoxicity Detection Kit (LDH) from Roche Diagnostics Corp. (Indianapolis, IN). Briefly, the lyophilisate of the catalyst (bottle 1, blue cap) was reconstituted in about 1 ml redistilled water for about 10 minutes and mixed thoroughly. The dye was warmed in a 37°C waterbath (if it is frozen). For 2 (11X6) plates, about 310 μl of the catalyst (bottle 1) was added to about 14 ml of the Dye (bottle 2) and mixed well. About 100 μl of the reaction mixture (catalyst and dye) was added to each well of the lysate and supernatant plate and incubated for up to 20-25 min at 15-25°C in the dark. A lag period of about 1 to about 2 minutes between plates was left before adding the dye so there was a comparable incubation time for the supernatant and the lysate.

[0407] The antisense oligonucleotides caused cytotoxicity in all four cell lines. Figures 15-19 summarize the results in PC3, 22rVl, HCT116, SW620, andDU145 cells, respectively. The results are expressed as a fraction of dead cells. UTl and UT2 represent untreated cells. CHTR109-3 is a positive control antisense molecule. A positive control using antisense and reverse control oligonucleotides for BCL2 (a known anti-apoptotic gene) also was included in some assays; loss of message for BCL2 leads to an increase in cell death compared with treatment with the control oligonucleotide (background cytotoxicity due to transfection).

Example 12: PRSS15 Knock-Out Does Not Induce Significant Cytotoxicity in Normal Cells [0408] The methods of Example 11 were used to evaluate the effect of PRSS 15 knock-out in "normal" fibroblast cells of the MRC9 cell line.

[0409] Results are shown in Figure 20.

Example 13: Summary of Effects of PRSS15 Knock-Out on Cell Lines [0410] Table 5 (below) summarizes the effects of PRSS 15 knock-out on cell lines tested in the studies outlined above.

Table 5

Claims

WE CLAIM:

1. A method of treating or preventing a cancer, including a cancer characterized by overexpression and/or upregulation of PRSS 15, the method comprising administering a therapeutically or prophylactically effective amount of at least one PRSS 15 modulator, where the modulator decreases or inhibits proliferation of the cancer or causes cancer cell death.

2. The method of claim 1, wherein the modulator is a polypeptide, such as an antibody, a polynucleotide, such as an antisense or RNAi oligonucleotide, or a small molecule.

3. The method of claim 1, wherein the cancer is colon cancer, prostate cancer, breast or stomach cancer.

4. The method of claim 1 which suppresses or inhibits metastasis, aberrant cellular proliferation relative to a normal cell, loss of contact inhibition of cell growth, or loss of anchorage dependent growth.

5. The method of claim 1 further comprising treating or preventing a cancer by administering at least one other therapeutic agent with the PRSS 15 modulator.

6. The method of claim 5 wherein the PRSS 15 modulator is co-administered with the other therapeutic agent, or the PRSS 15 modulator and other therapeutic agent are consecutively administered, in either order.

7. A PRSS 15 modulator suitable for treating or preventing a cancer, wherein the PRSS 15 modulator decreases or inhibits proliferation of the cancer or causes cancer cell death.

8. A pharmaceutical composition useful for treating or preventing a cancer, the composition comprising a pharmaceutically effective amount of at least one PRSS 15 modulator and a pharmaceutically acceptable carrier, where the modulator decreases or inhibits proliferation of the cancer or causes cancer cell death.

9. The composition according to claim 8, wherein the PRSS 15 modulator is a polypeptide, such as an antibody, a polynucleotide, such as an antisense or RNAi oligonucleotide, or a small molecule.

10. The composition according to claim 9 which is effective in treating colon cancer, prostate cancer, breast cancer or stomach cancer.

11. A method of detecting a cancer, particularly a cancer characterized by overexpression and/or upregulation of PRSS 15, the method comprising: (a) determining the level of expression of PRSS 15 in a biological sample obtained from a patient, (b) comparing the level of PRSS 15 expression in the patient biological sample to the level of PRSS 15 expression in a normal biological sample, and (c) correlating the level of PRSS 15 expression to a positive or negative diagnosis of the cancer.

12. A method for determining a patient's predisposition to a cancer, the method comprising: (a) determining the level of expression of PRSS 15 in a biological sample obtained from a patient, (b) comparing the level of PRSS 15 expression in said patient biological sample to the level of PRSS 15 expression in a normal biological sample, and (c) correlating the level of PRSS 15 expression to a diagnosis of a predisposition to the cancer.

13. A microarray comprising one or more polynucleotide sequences substantially identical to or complementary to the polynucleotide sequences of SEQ ID NO:2 or a portion of the polynucleotide sequence of SEQ ID NO:2.

14. A microarray comprising one or more protein-capture agents that bind one or more amino acid sequences encoded by all or a portion of the amino acid sequence SEQ JO NO: 1.

15. The method of claim 11, wherein determining the level of expression of PRSS 15 in a biological sample obtained from the patient is performed using a microarray.

16. The method of claim 12, wherein determining the level of expression of PRSS15 in a biological sample obtained from the patient is performed using a microarray.

17. A method of identifying an agent that binds to PRSS 15 or modulates a biological activity of a PRSS 15 gene product, the method comprising: (a) contacting at least one candidate modulator with PRSS15 under conditions wherein said at least one candidate modulator can bind the PRSS15, and (b) detecting the binding of said at least one candidate modulator to the PRSS 15.

18. A method of identifying an agent that binds to PRSS15 or modulates a biological activity of a PRSS 15 gene product, the method comprising: (a) contacting a candidate modulator with a PRSS 15 gene product, and (b) detecting modulation of a biological activity of said gene product relative to a level of biological activity of said gene product in the absence of said candidate agent.

19. A method of identifying an agent that binds to PRSS 15 or modulates a biological activity of a PRSS 15 gene product, the method comprising: (a) culturing a cell line transfected with an expression vector comprising a gene encoding PRSS 15 to express the gene in a medium containing at least one candidate modulator of PRSS 15, and (b) measuring binding of the candidate modulator to the PRSS 15 produced by the cell line.

20. A method of identifying an agent that binds to PRSS 15 or modulates a biological activity of a PRSS15 gene product, the method comprising: (a) contacting a candidate modulator with a cancer cell, preferably a cancer cell characterized by overexpression and/or upregulation of PRSS 15, under conditions that allow the candidate modulator to bind the PRSS 15, and (b) detecting a decrease or inhibition of proliferation of the cancer cell relative to proliferation of a cell of the same type that has not contacted the candidate modulator.

21. A method of determining the ability of a drug to inhibit substrate cleavage of PRSS 15, the method comprising: (a) culturing a cell line transfected with an expression vector comprising a gene encoding PRSS 15 to express the gene (i) in the presence of the substrate and (ii) in the presence of both the substrate and the drug, and (b) comparing the level of substrate cleavage that occurs in (a)(i) and (a)(ii), where a lower level of substrate cleavage in the presence of the drug indicates that the drug is an inhibitor of substrate cleavage.