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  Mt-sp1 polypeptidesUSPTO Application #: 20080051559Title: Mt-sp1 polypeptides Abstract: This invention provides a novel membrane-type serine protease (designated MT-SP1) elevated expression of which is associated with cancer. In one embodiment, this invention provides a method obtaining a prognosis or of detecting or staging a cancer in an organism. The method involves providing a biological sample from the organism and detecting the level of a membrane type serine protease 1 (MT-SP1) in the sample, where an elevated level of the membrane-type serine protease, as compared to the level of the protease in a biological sample from a normal healthy organism indicates the presence or stage of the cancer. (end of abstract)
Agent: Wilson Sonsini Goodrich & Rosati - Palo Alto, CA, US Inventors: Charles S. Craik, Toshihiko Takeuchi, Marc Shuman USPTO Applicaton #: 20080051559 - Class: 530350000 (USPTO) Related Patent Categories: Chemistry: Natural Resins Or Derivatives; Peptides Or Proteins; Lignins Or Reaction Products Thereof, Proteins, I.e., More Than 100 Amino Acid Residues The Patent Description & Claims data below is from USPTO Patent Application 20080051559. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0002] [Not Applicable] FIELD OF THE INVENTION [0003] This invention relates to the field of serine proteases and associated biology. In particular, this invention relates to the discovery of a new membrane-type serine protease believed to be associated with the etiology of cancer and associated pathologies. BACKGROUND OF THE INVENTION [0004] The serine proteases (SP) are a large family of proteolytic enzymes that include the digestive enzymes, trypsin and chymotrypsin, components of the complement cascade and of the blood-clotting cascade, and enzymes that control the degradation and turnover of macromolecules of the extracellular matrix. Serine proteases are so named because of the presence of a serine residue in the active catalytic site for protein cleavage. Serine proteases have a wide range of substrate specificities and can be subdivided into subfamilies on the basis of these specificities. The main sub-families are trypases (cleavage after arginine or lysine), aspases (cleavage after aspartate), chymases (cleavage after phenylalanine or leucine), metases (cleavage after methionine), and serases (cleavage after serine). [0005] Most proteases are secretory proteins which contain N-terminal signal peptides that serve to export the immature protein across the endoplasmic reticulum and are then cleaved (von Heijne (1986) Nuc. Acid. Res. 14: 5683-5690). Differences in these signal sequences provide one means of distinguishing individual serine proteases. Some serine proteases, particularly the digestive enzymes, exist as inactive precursors or preproenzymes, and contain a leader or activation peptide sequence 3' of the signal peptide. Typically, this activation peptide may be 2-12 amino acids in length, and it extends from the cleavage site of the signal peptide to the N-terminal IIGG sequence of the active, mature protein. Cleavage of this sequence activates the enzyme. This sequence varies in different serine proteases according to the biochemical pathway and/or its substrate (Zunino et al. (1988) Biochimica et. Biophysica Acta 967: 331-340; Sayers, et al. (1992) J. Immunology 148: 292-300). Other features that distinguish various serine proteases are the presence or absence of N-linked glycosylation sites that provide membrane anchors, the number and distribution of cysteine residues that determine the secondary structure of the serine protease and the sequence of a substrate binding sites such as S'. The S' substrate binding region is defined by residues extending from approximately +17 to +29 relative to the N-terminal I (+1). Differences in this region of the molecule are believed to determine serine protease substrate specificities (Zunino et al, supra). [0006] Numerous disease states are caused by and can be characterized by alterations in the activity of specific proteases and their inhibitors. For example emphysema, arthritis, thrombosis, cancer metastasis and some forms of hemophilia result from the lack of regulation of serine protease activities (see, for example, Textbook of Biochemistry with Clinical Correlations, John Wiley and Sons, Inc. N.Y. (1993)). In case of viral infection, the presence of viral proteases have been identified in infected cells. Such viral proteases include, for example, HIV protease associated with AIDS and NS3 protease associated with Hepatitis C. These viral proteases play a critical role in the virus life cycle. [0007] A series of serine proteases have been identified in murine cytotoxic T-lymphocytes (CTL) and natural killer (NK) cells. These serine proteases are involved with CTL and NK cells in the destruction of virally transformed cells and tumor cells and in organ and tissue transplant rejection (Zunino et al. (1990) J. Immunol. 144:2001-2009; Sayers et al. (1994) J. Immunol. 152: 2289-2297). Human homologs of most of these enzymes have been identified (Trapaniet et al. (1988) Proc. Natl. Acad. Sci. 85: 6924-6928; Caputo et al. (1990) J. Immunol. 145: 737-744). [0008] Proteases have also been implicated in cancer metastasis. Increased synthesis of the protease urokinase has been correlated with an increased ability to metastasize in many cancers. Urokinase activates plasmin from plasminogen which is ubiquitously located in the extracellular space and its activation can cause the degradation of the proteins in the extracellular matrix through which the metastasizing tumor cells invade. Plasmin can also activate the collagenases thus promoting the degradation of the collagen in the basement membrane surrounding the capillaries and lymph system thereby allowing tumor cells to invade into the target tissues (Dano, et al. (1985) Adv. Cancer. Res., 44: 139). [0009] The discovery of a new serine protease precursor and the polynucleotides encoding it satisfies a need in the art by providing new prognostic and diagnostic methods and, therapeutic compositions useful in the treatment or prevention of cancer. SUMMARY OF THE INVENTION [0010] This invention pertains to the discovery of a new serine protease associated with cancer cells. In particular, nucleic acid cDNAs derived from PC-3 mRNA were sequenced that encoded a novel serine protease referred to herein as membrane-type serine protease 1 (MT-SP1). The MT-SP1 polypeptide encoded by the nucleic acid(s) is localized in tumor tissues (e.g. prostatic cancers, gastric cancers, breast cancers, etc.), and in preferred embodiments is identified in blood vessels associated with tumors. Inhibition of MT-SP1 inhibits cancer growth in relevant animal models. Without being bound to a particular theory it is believed that MT-SP1 is implicated in tumor proliferation and/or growth and/or tumor angiogenesis. MT-SP1 is also demonstrated herein to be a good diagnostic, and more preferably, a good prognostic for various cancers. MT-SP1 can be used to detect the presence or absence of a cancer, to determine the location and/or size and/or morphology of a cancer, and to make a prediction regarding the severity and/or outcome of a cancer or a particular therapeutic regimen. [0011] In one embodiment, this invention provides nucleic acids encoding MT-SP1 and/or probes suitable for amplification of MT-SP1 nucleic acids (e.g. from a PC-3 mRNA template). These nucleic acids include, but are not limited to: (a) a nucleic acid comprising a nucleic acid encoding a serine protease domain having the sequence of SEQ ID NO: 2; (b) a nucleic acid comprising a nucleic acid encoding a serine protease domain having the sequence of amino acids 615 through 855 of SEQ ID NO: 2; (c) a nucleic acid that specifically hybridizes to the nucleic acid of SEQ ID NO: 1 or a fragment thereof under stringent conditions and is of sufficient length that said nucleic acid can uniquely indicate the presence or absence of a nucleic acid encoding a membrane-type serine protease in a total genomic DNA pool, a total cDNA pool or a total mRNA pool sample from a PC-3 cell; (d) a nucleic acid comprising a sequence that has the same sequence as a nucleic acid amplified from a PC-3 cDNA template using PCR primers corresponding to nucleotides 37-54 of SEQ ID NO: 1 and 2604-2583 of the complement of SEQ ID NO: 1; (e) a DNA encoding an mRNA that, when reverse transcribed, produces the cDNA of SEQ ID NO: 1; (f) a DNA encoding an mRNA that, when reverse transcribed, produces the cDNA encoding amino acids 615-855 of SEQ ID NO: 2; (g) a pair of primers that, when used in a nucleic acid amplification reaction with PC-3 cDNA template specifically amplifies a nucleic acid encoding the polypeptide of SEQ ID NO: 2; (h) a pair of primers that, when used in a nucleic acid amplification reaction with mRNA template from a PC-3 cell specifically amplify a nucleic acid encoding the polypeptide having the sequence of amino acids 615 through 855 of SEQ ID NO: 2; and (i) a nucleic acid encoding a membrane-type serine protease, wherein said nucleic acid encodes a consensus sequence shown in FIG. 4 and does not encode TRYB_human, ENTK-Human, HEPS_human, TRY2_Human, and CTRB_human. Preferred nucleic acids encode a polypeptide having the sequence of amino acids 615 through 855 of SEQ ID NO: 2, while other preferred nucleic acids encode a polypeptide having the sequence of SEQ ID NO: 2. In one embodiment the nucleic acid has the sequence of SEQ ID NO: 1. The nucleic acid(s) are optionally present in an expression cassette and/or a vector and are optionally labeled with a detectable label. Also provided are host cells comprising such vectors and a process producing a polypeptide comprising expressing from such host cells a polypeptide encoded an MT-SP1 DNA. This invention also includes a process for producing a cell that expresses an MT-SP1 polypeptide. The process involves comprising transforming or transfecting the cell with the vector encoding an MT-SP1 such that the cell expresses the MT-SP1 polypeptide. [0012] In another embodiment this invention provides isolated MT-SP1 polypeptides (e.g. as encoded by the nucleic acids described above). Preferred polypeptides comprise a protease domain of SEQ ID NO: 2 or the polypeptide of SEQ ID NO: 2. Preferred polypeptides also include, but are not limited to polypeptides that have serine protease activity and that are specifically bound by an antibody raised against the polypeptide of SEQ ID NO: 2 and/or polypeptides having protease activity and having 95% or greater sequence identity to a polypeptide having the sequence of SEQ ID NO: 2; and/or having protease activity and having 95% or greater identity to a polypeptide having the sequence of amino acids 615 through 855 of SEQ ID NO: 2. [0013] Also provided are antibodies that specifically bind to the MT-SP1 polypeptides of this invention (e.g. a polypeptide encoded by SEQ ID NO: 2). The antibodies can be monoclonal, polyclonal, antibody fragments or single-chain antibodies. [0014] This invention also provides diagnostic assays for cancer(s). Such assays involve providing a biological sample from an organism; and detecting the level of a membrane type serine protease 1 (MT-SP1) in the sample, where an elevated level of the membrane-type serine protease, as compared to the level of the protease in a biological sample from a normal healthy organism indicates the presence of the cancer. The method can involve determining the copy number of MT-SP1 genes in the cells of the biological sample (e.g. using FISH or Comparative Genomic Hybridization (CGH)). In another embodiment, the method can involve measuring the level of MT-SP1 mRNA in the biological sample, wherein an increased level of MT-SP1 RNA in the sample compared to MT-SP1 RNA in a control sample indicates the presence (or significant probability of the presence) of the cancer. The mRNA determination can involve hybridizing (e.g. using a Northern blot, a Southern blot, an array hybridization, an affinity chromatography, an in situ hybridization, etc.) the mRNA to one or more probes that specifically hybridize (under stringent conditions) to a nucleic acid encoding the MT-SP1 protein. A probe used in such measurements can optionally include a plurality of probes that form an array of probes. Preferred detection methods involve quantifying MT-SP1 mRNA. In still another embodiment, the level of MT-SP1 mRNA is measured using a nucleic acid amplification reaction. In addition, or alternatively, the method can involve determining the level (e.g. via a method selected from the group consisting of capillary electrophoresis, a Western blot, mass spectroscopy, ELISA, immunochromatography, and immunohistochemistry) or activity of an MT-SP1 protein in the biological sample. Preferred biological samples for these assays include, but are not limited to excised tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, and urine. [0015] In certain embodiments, it is desired to pre-screen test agents for the ability to bind to an MT-SP1 nucleic acid and/or protein. Such pre-screening methods typically involve (a) contacting a nucleic acid encoding an MT-SP1 serine protease or an MT-SP1 serine protease protein with a test agent; and (b) detecting specific binding of the test agent to the MT-SP1 protein or nucleic acid. Preferred test agents do not include antibodies, and/or nucleic acids. In particularly preferred assay formats the MT-SP1 nucleic acid and/or protein is immobilized on a solid support, while in other preferred assay formats, the test agent is immobilized (e.g. in a 96 well plate, etc.). Preferred methods of detecting binding utilize detectable labels (e.g. fluorescent labels) and a particular preferred detection methods utilizes fluorescent resonance energy transfer (FRET). [0016] MT-SP1 levels are also good prognostic indicators for various cancers as described herein. This invention therefore also provides methods (prognostic assays) for evaluating the severity or outcome of a cancer (e.g. for estimating length of survival of a cancer patient). The methods preferably involve (a) obtaining a biological sample from a cancer patient having at least a preliminary diagnosis of cancer; (b) measuring MT-SP1 in said sample and comparing the sample MT-SP1 level to the MT-SP1 level in normal healthy humans wherein a sample MT-SP1 level in excess of MT-SP1 levels in normal healthy humans indicates a reduced survival expectancy compared to patients with normal MT-SP1 level. Particular embodiments include a preliminary diagnosis of prostate cancer, a cancer of the digestive tract, a breast cancer, and/or a urogential cancer. Preferred biological samples, include, but are not limited to a primary tumor or a tissue affected by the cancer (e.g. a tumor biopsy) and/or samples selected from the group consisting of whole blood, plasma, serum, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural effusion, urine, or tumor tissue. As indicated above, MT-SP1 can be evaluated by copy number of MT-SP1 genomic DNA, MT-SP1 mRNA levels, levels of nucleic acid(s) derived from MT-SP1 mRNA (e.g. cDNAs, RT-PCR products, etc.), MT-SP1 protein levels and/or MT-SP1 activity levels. In a preferred embodiments the level of MT-SP1 is measured by immunohistochemical staining of cells comprising the biological sample (e.g. tumor tissue cells) and/or via an immunoassay (e.g., ELISA using an anti-MT-SP1 antibody as described above). [0017] Also provided are methods of treating a cancer in a patient. The methods involve performing one or more of the prognostic assays described herein on a cancer patient having at least a preliminary diagnosis of a cancer; and (c) selecting a patient identified with an MT-SP1 level excess of MT-SP1 levels in normal healthy humans and providing an adjuvant cancer therapy (e.g. a therapy selected from the group consisting of chemotherapy, radiation therapy, reoperation, antihormone therapy, and immunotherapy). [0018] This invention also affords methods of screening for recurrence of a cancer after removal of a primary tumor. These methods involve performing one or more of the assays described herein on a biological sample from a cancer patient following removal of a primary tumor. The assay can be repeated at a multiplicity of instances after removal of the primary tumor. [0019] Similarly the assays of this invention provide methods of monitoring the effectiveness of cancer treatment in patients. This involves obtaining a first biological sample from said patient prior to or following one or more treatments of a cancer; obtaining a second biological sample from said cancer patient during or after said one or more treatments; evaluating the samples for MT-SP1 level as described herein where a lower level of MT-SP1 in the second sample as compared to the MT-SP1 level in the first sample indicates efficacy of the treatment(s). Typically the treatments involve chemotherapy, radiation therapy, immunotherapy, anti hormone therapy, or surgery. [0020] It was also a discovery of this invention that MT-SP1 provides a good target for specifically delivering an effector (e.g. a liposome, a cytotoxin, a detectable label) to a cell (e.g. a tumor cell) expressing MT-SP1. The methods involve providing a chimeric moiety comprising an effector (e.g. an effector molecule) attached to an anti-MT-SP1 antibody; and contacting the cell with said chimeric moiety whereby the chimeric moiety binds (e.g. specifically binds) to the "target" cell. In preferred embodiments, the cell (e.g. tumor cell) internalizes at least a portion (e.g. a detectable or measurable amount) of the molecule. Preferred cells include cancer cells, e.g., cells from a prostate cancer, a cancer of the digestive tract, a breast cancer, and a urogential cancer. Preferred effectors include a cytotoxin, a detectable label, a radionuclide, a drug, a liposome, a ligand, and an antibody. In particularly preferred embodiments, the chimeric moiety is a fusion protein. Preferred fusion proteins include effectors selected from the group consisting of ricin, abrin, Diphtheria toxin, and Pseudomonas exotoxin. In other preferred embodiments, the effector is cytotoxic and/or a liposome comprising an anti-cancer drug (e.g. doxirubicin, vinblastine, vincristine, taxol, doxirubicin, and genistein). [0021] This invention also provides chimeric moieties (e.g. chimeric molecules) comprising an effector attached to an anti-MT-SP1 antibody. Preferred effectors include cytotoxin, a detectable label, a radionuclide, a drug, a liposome, a ligand, and an antibody. Preferred chimeric moieties are fusion proteins and particularly preferred fusion proteins have cytotoxic effectors (e.g., ricin, abrin, Diphtheria toxin, Pseudomonas exotoxin, etc.). Other preferred effectors include a liposome comprising an anti-cancer drug as described above. The chimeric moieties described herein can be formulated as pharmaceutical compositions comprising the chimeric moiety and pharmaceutically acceptable excipient. Continue reading... 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