Abstract:
The disclosed nucleic acid primer sets, used in combination with quantitative amplification (PCR) of tissue cDNA, can indicate the presence of specific proteases in a tissue sample. Specifically, the present invention relates to expression of hepsin protease. The detected proteases are themselves specifically overexpressed in certain cancers, and the presence of their genetic precursors may serve for early detection of associated ovarian and other malignancies, and for the design of interactive therapies for cancer treatment.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]    This is a continuation-in-part application which claims the benefit of priority under 35 USC §120 of U.S. Ser. No. 09/861,966, filed May 21, 2001, which is a divisional application of U.S. Pat. No. 6,268,165, which claims the benefit of priority under 35 USC §120 of U.S. Ser. No. 09/039,211, filed Mar. 14, 1998, which claims benefit of provisional patent application U.S. Ser. No. 60/041,404, filed Mar. 19, 1997, now abandoned. 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0002]    1. Field of the Invention  
           [0003]    Generally, the present invention relates to the fields of molecular biology and medicine. More specifically, the present invention invention is in the field of cancer research, especially ovarian cancer diagnosis.  
         BACKGROUND OF THE INVENTION  
         [0004]    In order for malignant cells to grow, spread or metastasize, they must have the capacity to invade local host tissue, dissociate or shed from the primary tumor, enter and survive in the bloodstream, implant by invasion into the surface of the target organ and establish an environment conducive for new colony growth (including the induction of angiogenic and growth factors). During this progression, natural tissue barriers such as basement membranes and connective tissue have to be degraded. These barriers include collagen, laminin, fibronectin, proteoglycans and extracellular matrix glycoproteins. Degradation of these natural barriers, both those surrounding the primary tumor and at the sites of metastatic invasion, is believed to be brought about by the action of a matrix of extracellular proteases.  
           [0005]    Proteases have been classified into four families: serine proteases, metallo-proteases, aspartic proteases and cysteine proteases. Many proteases have been shown to be involved in human disease processes and these enzymes are targets for the development of inhibitors as new therapeutic agents. Certain individual proteases are induced and overexpressed in a diverse group of cancers, and as such, are potential candidates for markers of early diagnosis and targets for possible therapeutic intervention. A group of examples are shown in Table 1.  
                                                           TABLE 1                           Known proteases expressed in various cancers                Gastric   Brain   Breast   Ovarian                        Serine   uPA   uPA   NES-1   NES-1       Proteases:   PAI-1   PAI-1   uPA   uPA               tPA       PAI-2       Cysteine   Cathepsin B   Cathepsin L   Cathepsin B   Cathepsin B       Proteases:   Cathepsin L       Cathepsin L   Cathepsin L       Metallo-   Matrilysin*   Matrilysin   Stromelysin-3   MMP-2       proteases:   Collagenase*   Stromelysin   MMP-8           Stromelysin-1*   Gelatinase B   MMP-9                   Gelatinase A                                  
 
           [0006]    There is a good body of evidence supporting the downregulation or inhibition of individual proteases and the reduction in invasive capacity or malignancy. In work by Clark et al., inhibition of in vitro growth of human small cell lung cancer was demonstrated using a general serine protease inhibitor. More recently, Torres-Rosedo et al., [ Proc. Natl. Acad. Sci. USA.  90, 7181-7185 (1993)] demonstrated an inhibition of hepatoma tumor cell growth using specific antisense inhibitors for the serine protease hepsin gene. Metastatic potential of melanoma cells has also been shown to be reduced in a mouse model using a synthetic inhibitor (batimastat) of metallo-proteases. Powell et al. [ Cancer Research,  53, 417-422 (1993)] presented evidence to confirm that the expression of extracellular proteases in a non-metastatic prostate cancer cell line enhances their malignant progression. Specifically, enhanced metastasis was demonstrated after introducing and expressing the PUMP-1 metallo-protease gene. There is also a body of data to support the notion that expression of cell surface proteases on relatively non-metastatic cell types increases the invasive potential of such cells.  
           [0007]    To date, ovarian cancer remains the number one killer of women with gynecologic malignant hyperplasia. Approximately 75% of women diagnosed with such cancers are already at an advanced stage (III and IV) of the disease at their initial diagnosis. During the past 20 years, neither diagnosis nor five-year survival rates have greatly improved for these patients. This is substantially due to the high percentage of high-stage initial detection of the disease. Therefore, the challenge remains to develop new markers that improve early diagnosis and thereby reduce the percentage of high-stage initial diagnoses. The ability to disengage from one tissue and re-engage the surface of another tissue is what provides for the morbidity and mortality associated with this disease. Therefore, extracellular proteases may be good candidates for markers of malignant ovarian hyperplasia.  
           [0008]    Thus, the prior art is deficient in a tumor marker useful as an indicator of early disease, particularly for ovarian cancers. The present invention fulfills this long-standing need and desire in the art.  
         SUMMARY OF THE INVENTION  
         [0009]    This invention allows for the detection of cancer, especially ovarian cancer, by screening for hepsin mRNA in tissue, which is indicative of the hepsin protease, which is shown herein to be specifically associated with the surface of 80 percent of ovarian and other tumors. Proteases are considered to be an integral part of tumor growth and metastasis, and therefore, markers indicative of their presence or absence are useful for the diagnosis of cancer. Furthermore, the present invention is useful for treatment (i.e., by inhibiting hepsin or expression of hepsin), for targeted therapy, for vaccination, etc.  
           [0010]    In one embodiment of the present invention, there is provided a method for detecting malignant hyperplasia in a biological sample by detecting hepsin mRNA in the sample. The presence of the hepsin mRNA in the sample is indicative of the presence of malignant hyperplasia, and the absense of the hepsin mRNA in the sample is indicative of the absence of malignant hyperplasia.  
           [0011]    In another embodiment of the present invention, there are provided methods of inhibiting expression of hepsin in a cell by introducing into a cell a vector encoding an antisense hepsin mRNA or an antibody that binds the hepsin protein.  
           [0012]    In yet another embodiment of the present invention, there is provided a method of targeted therapy to an individual, comprising the step of administering a compound to an individual, wherein the compound has a targeting moiety and a therapeutic moiety, wherein the targeting moiety is specific for hepsin.  
           [0013]    In still yet another embodiment of the present invention, there are provided methods of vaccinating an individual against hepsin or produce immune-activated cells directed toward hepsin by inoculating an individual with a hepsin protein or fragment thereof, wherein the hepsin protein or fragment thereof lack hepsin protease activity.  
           [0014]    In still another embodiment of the present invention, there are provided compositions comprising immunogenic fragments of hepsin protein or an oligonucleotide having a sequence complementary to SEQ ID No. 188. Also embodied is a method of treating a neoplastic state in an individual in need of such treatment with an effective dose of the above-described oligonucleotide.  
           [0015]    In another embodiment of the present invention, there is provided a method of screening for compounds that inhibit hepsin activity, comprising the steps of contacting a sample with a compound, wherein the sample comprises hepsin protein; and assaying for hepsin protease activity. A decrease in the hepsin protease activity in the presence of the compound relative to hepsin protease activity in the absence of the compound is indicative of a compound that inhibits hepsin activity.  
           [0016]    Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0017]    The appended drawings have been included herein so that the above-recited features, advantages and objects of the invention will become clear and can be understood in detail. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and should not be considered to limit the scope of the invention.  
         [0018]    [0018]FIG. 1 shows agarose gel comparison of PCR products derived from normal and carcinoma cDNA.  
         [0019]    [0019]FIG. 2 shows Northern blot analysis of ovarian tumors using hepsin, SCCE, PUMP-1, TADG-14 and β-tubulin probes.  
         [0020]    [0020]FIG. 3 shows amplification with serine protease redundant primers: histidine sense (S1) with aspartic acid antisense (AS1), using normal cDNA (Lane 1) and tumor cDNA (Lane 2); and histidine sense (S1) with serine antisense (AS2), using normal cDNA (Lane 3) and tumor cDNA (Lane 4).  
         [0021]    [0021]FIG. 4 shows amplification with cysteine protease redundant primers. Normal (Lane 1), low malignant potential (Lane 2), serious carcinoma (Lane 3), mucinous carcinoma (Lane 4), and clear cell carcinoma (Lane 5).  
         [0022]    [0022]FIG. 5 shows amplification with metallo-protease redundant primers. Normal (Lane 1), low malignant potential (Lane 2), serious carcinoma (Lane 3), mucinous carcinoma (Lane 4), and clear cell carcinoma (Lane 5).  
         [0023]    [0023]FIG. 6 shows amplification with specific primers directed towards the serine protease, hepsin. Expression in normal (Lanes 1-3), low malignant potential tumors (Lanes 4-8), and ovarian carcinomas (Lanes 9-12).  
         [0024]    [0024]FIG. 7 shows hepsin expression levels in normal, low malignant potential tumors, and ovarian carcinomas. S=serious, M=mucinous, LMP=low malignant potential.  
         [0025]    [0025]FIG. 8 shows serine protease stratum corneum chymotrypsin enzyme (SCCE) expression in normal, low malignant potential tumors, and ovarian carcinomas.  
         [0026]    [0026]FIG. 9 shows metallo-protease PUMP-1 (MMP-7) gene expression in normal (lanes 1-2) and ovarian carcinomas tissue (Lanes 3-10).  
         [0027]    [0027]FIG. 10A shows Northern blot analysis of hepsin expression in normal ovary and ovarian carcinomas. Lane 1, normal ovary (case 10); lane 2, serous carcinoma (case 35); lane 3, mucinous carcinoma (case 48); lane 4, endometrioid carcinoma (case 51); and lane 5, clear cell carcinoma (case 54). In cases 35, 51 and 54, more than a 10-fold increase in the hepsin 1.8 kb transcript abundance was observed. FIG. 10B shows Northern blot analysis of hepsin in normal human fetal. FIG. 10C shows Northern blot analysis of hepsin in adult tissues. Significant overexpression of the hepsin transcript is noted in both fetal liver and fetal kidney. Notably, hepsin overexpression is not observed in normal adult tissue. Slight expression above the background level is observed in the adult prostate.  
         [0028]    [0028]FIG. 11 A shows hepsin expression in normal (N), mucinous (M) and serous (S) low malignant potential (LMP) tumors and carcinomas (CA). β-tubulin was used as an internal control. FIG. 11B shows the ratio of hepsin:β-tubulin expression in normal ovary, LMP tumor, and ovarian carcinoma. Hepsin mRNA expression levels were significantly elevated in LMP tumors, (p&lt;0.005) and carcinomas (p&lt;0.0001) compared to levels in normal ovary. All 10 cases of normal ovaries showed a relatively low level of hepsin mRNA expression.  
         [0029]    [0029]FIG. 12A shows northern blot analysis of mRNA expression of the SCCE gene in fetal tissue. FIG. 12B shows northern blot analysis of mRNA expression of the SCCE gene in ovarian tissue.  
         [0030]    [0030]FIG. 13 A shows a comparison of quantitative PCR of SCCE cDNA from normal ovary and ovarian carcinomas. FIG. 13B shows a bar graph comparing the ratio of SCCE to -tubulin in 10 normal and 44 ovarian carcinoma tissues.  
         [0031]    [0031]FIG. 14 shows a comparison by quantitative PCR of normal and ovarian carcinoma expression of mRNA for protease M.  
         [0032]    [0032]FIG. 15 shows the TADG-12 catalytic domain including an insert near the His 5′-end.  
         [0033]    [0033]FIG. 16A shows northern blot analysis comparing TADG-14 expression in normal and ovarian carcinoma tissues.  
         [0034]    [0034]FIG. 16B shows preliminary quantitative PCR amplification of normal and carcinoma cDNAs using specific primers for TADG-14.  
         [0035]    [0035]FIG. 17A shows northern blot analysis of the PUMP-1 gene in human fetal tissue. FIG. 17B shows northern blot analysis of the PUMP-1 gene in normal ovary and ovarian carcinomas.  
         [0036]    [0036]FIG. 18A shows a comparison of PUMP-1 expression in normal and carcinoma tissues using quantitative PCR with an internal β-tubulin control. FIG. 18B shows the ratio of mRNA expression of PUMP-1 compared to the internal control β-tubulin in 10 normal and 44 ovarian carcinomas.  
         [0037]    [0037]FIG. 19 shows a comparison of PCR amplified products for the hepsin, SCCE, protease M, PUMP-1 and Cathepsin L genes.  
         [0038]    [0038]FIG. 20 shows CD8 + CTL recognition of hepsin 170-178 peptide in a 5 hour  51 Cr release assay. Targets were LCL loaded with hepsin 170-178 (closed circles) and control LCL (open circles).  
         [0039]    [0039]FIG. 21 shows CD8 + CTL recognition of hepsin 172-180 peptide in a 5 hr  51 Cr release assay. Targets were LCL loaded with hepsin 172-180 (closed circles) and control LCL (open circles). 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0040]    This invention identifies hepsin protease as a marker for ovarian tumor cells. In various combinations with other proteases, hepsin expression is characteristic of individual tumor types. Such information can provide the basis for diagnostic tests (assays or immunohistochemistry) and prognostic evaluation (depending on the display pattern). Long-term treatment of tumor growth, invasion and metastasis has not succeeded with existing chemotherapeutic agents. Most tumors become resistant to drugs after multiple cycles of chemotherapy. The present invention identifies hepsin as a new therapeutic intervention target utilizing either antibodies directed at the protease, antisense vehicles for downregulation or protease inhibitors both from established inhibition data and/or for the design of new drugs.  
         [0041]    A primary object of the present invention is a method for detecting the presence of malignant hyperplasia in a tissue sample. The cancer is detected by analyzing a biological sample for the presence of markers to proteases that are specific indicators of certain types of cancer cells. This object may be accomplished by isolating mRNA from a sample or by detection of proteins by polyclonal or preferably monoclonal antibodies. When using mRNA detection, the method may be carried out by converting the isolated mRNA to cDNA according to standard methods; treating the converted cDNA with amplification reaction reagents (such as cDNA PCR reaction reagents) in a container along with an appropriate mixture of nucleic acid primers selected from the list in Table 2; reacting the contents of the container to produce amplification products; and analyzing the amplification products to detect the presence of malignant hyperplasia markers in the sample. The analyzing step may be accomplished using Northern Blot analysis to detect the presence of malignant hyperplasia markers in the amplification product. Northern Blot analysis is known in the art. The analysis step may be further accomplished by quantitatively detecting the presence of malignant hyperplasia marker in the amplification products, and comparing the quantity of marker detected against a panel of expected values for known presence or absence in normal and malignant tissue derived using similar primers.  
         [0042]    The present invention also provides various nucleic acid sequences that are useful in the methods disclosed herein. These nucleic acid sequences are listed in Table 2. It is anticipated that these nucleic acid sequences be used in mixtures to accomplish the utility of this invention. Features of such mixtures include: SEQ ID No. 1 with SEQ ID No. 2; SEQ ID No. 1 with SEQ ID No. 3; SEQ ID No. 4 with SEQ ID No. 5; SEQ ID No. 6 with SEQ ID No. 7; SEQ ID No. 8 with SEQ ID No. 9; and SEQ ID No. 10 with SEQ ID No. 11. The skilled artisan may be able to develop other nucleic acid sequences and mixtures thereof to accomplish the benefit of this invention, but it is advantageous to have the sequences listed in Table 2 available without undue experimentation.  
         [0043]    The present invention provides a method for detecting malignant hyperplasia in a biological sample, comprising the steps of isolating mRNA from the sample; and detecting hepsin mRNA in the sample. The presence of the hepsin mRNA in the sample is indicative of the presence of malignant hyperplasia, wherein the absense of the hepsin mRNA in the sample is indicative of the absence of malignant hyperplasia. This method may further comprise the step of comparing the hepsin mRNA to reference information, wherein the comparison provides a diagnosis and/or determines a treatment of the malignant hyperplasia. A typical means of detection of hepsin mRNA is by PCR amplification, which, preferably, uses primers shown in SEQ ID No. 8 and SEQ ID No. 9. Representative biological samples are blood, urine, saliva, tears, interstitial fluid, ascites fluid, tumor tissue biopsy and circulating tumor cells.  
         [0044]    The present invention is further directed toward a method of inhibiting expression of hepsin in a cell, comprising the step of introducing into a cell a vector comprises a hepsin gene operably linked in opposite orientation to elements necessary for expression, wherein expression of the vector produces hepsin antisense mRNA in the cell. The hepsin antisense mRNA hybridizes to endogenous hepsin mRNA, thereby inhibiting expression of hepsin in the cell.  
         [0045]    The present invention is still further directed toward a method of inhibiting a hepsin protein in a cell, comprising the step of introducing an antibody into a cell, wherein the antibody is specific for a hepsin protein or a fragment thereof. Binding of the antibody to hepsin inhibits the hepsin protein. Preferably, the hepsin fragment is a 9-residue fragment up to a 20-residue fragment, and more preferably, the 9-residue fragment is SEQ ID Nos. 28, 29, 30, 31, 88, 89, 108, 109, 128, 129, 148, 149, 150, 151, 152, 153 and 154.  
         [0046]    The present invention is also directed toward a method of targeted therapy to an individual, comprising the step of administering a compound to an individual, wherein the compound has a targeting moiety and a therapeutic moiety, and wherein the targeting moiety is specific for hepsin. Preferably, the targeting moiety is an antibody specific for hepsin or a ligand or ligand binding domain that binds hepsin. Likewise, the therapeutic moiety is preferably a radioisotope, a toxin, a chemotherapeutic agent, an immune stimulant or cytotoxic agent. Generally, the individual suffers from a disease such as ovarian cancer, lung cancer, prostate cancer, colon cancer or another cancer in which hepsin is overexpressed.  
         [0047]    The present invention is additionally directed toward a method of vaccinating an individual against hepsin, comprising the steps of inoculating an individual with a hepsin protein or fragment thereof, wherein the hepsin protein or fragment thereof lack hepsin protease activity. Inoculation with the hepsin protein, or fragment thereof, elicits an immune response in the individual, thereby vaccinating the individual against hepsin. Generally, this method is applicable when the individual has cancer, is suspected of having cancer or is at risk of getting cancer. Sequences of preferred hepsin proteins or fragment thereof are shown in SEQ ID Nos. 28, 29, 30, 31, 88, 89, 108, 109, 128, 129, 148, 149, 150, 151, 152, 153 and 154.  
         [0048]    The present invention is yet directed toward a method of producing immune-activated cells directed toward hepsin, comprising the steps of exposing immune cells to hepsin protein or fragment thereof that lacks hepsin protease activity. Typically, exposure to hepsin protein or fragment thereof activates the immune cells, thereby producing immune-activated cells directed toward hepsin. Generally, the immune-activated cells are B-cells, T-cells and/or dendritic cells. Preferably, the hepsin fragment is a 9-residue fragment up to a 20-residue fragment, and more preferably, the 9-residue fragment is SEQ ID Nos. 28, 29, 30, 31, 88, 89, 108, 109, 128, 129, 148, 149, 150, 151, 152, 153 or 154. Oftentimes, the dendritic cells are isolated from an individual prior to exposure and then reintroduced into the individual subsequent to the exposure. Typically, the individual has cancer, is suspected of having cancer or is at risk of getting cancer.  
         [0049]    The present invention is further directed toward a n immunogenic composition, comprising an immunogenic fragment of hepsin protein and an appropriate adjuvant. Preferably, the fragment is a 9-residue fragment up to a 20-residue fragment, and more preferably, the 9-residue fragment is SEQ ID Nos. 28, 29, 30, 31, 88, 89, 108, 109, 128, 129, 148, 149, 150, 151, 152, 153 or 154.  
         [0050]    The present invention is further directed toward a n oligonucleotide having a sequence complementary to SEQ ID No. 188 or a frgament thereof. The present invention further provides a composition comprising the above-described oligonucleotide and a physiologically acceptable carrier, and a method of treating a neoplastic state in an individual in need of such treatment, comprising the step of administering to the individual an effective dose of the above-described oligonucleotide. Typically, the neoplastic state may be ovarian cancer, breast cancer, lung cancer, colon cancer, prostate cancer or another cancer in which hepsin is overexpressed.  
         [0051]    The present invention is still further directed toward a method of screening for compounds that inhibit hepsin activity, comprising the steps of contacting a sample with a compound, wherein the sample comprises hepsin protein; and assaying for hepsin protease activity. A decrease in the hepsin protease activity in the presence of the compound relative to hepsin protease activity in the absence of the compound is indicative of a compound that inhibits hepsin activity.  
         [0052]    It will be apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.  
         [0053]    In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch &amp; Sambrook, “Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning: A Practical Approach,” Volumes I and II (D. N. Glover ed. 1985); “Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic Acid Hybridization” (B. D. Hames &amp; S. J. Higgins eds. 1985); “Transcription and Translation” (B. D. Hames &amp; S. J. Higgins eds. 1984); “Animal Cell Culture” (R. I. Freshney, ed. 1986); “Immobilized Cells And Enzymes” (IRL Press, 1986); B. Perbal, “A Practical Guide To Molecular Cloning” (1984). Therefore, if appearing herein, the following terms shall have the definitions set out below.  
         [0054]    As used herein, the term “cDNA” shall refer to the DNA copy of the mRNA transcript of a gene.  
         [0055]    As used herein, the term “PCR” refers to the polymerase chain reaction that is the subject of U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis, as well as other improvements now known in the art.  
         [0056]    The present invention comprises a vector comprising a DNA sequence which encodes a hepsin protein, wherein said vector is capable of replication in a host, and comprises, in operable linkage: a) an origin of replication; b) a promoter; and c) a DNA sequence coding for said hepsin protein. Preferably, the vector of the present invention contains a portion of the DNA sequence shown in SEQ ID No. 188. Vectors may be used to amplify and/or express nucleic acid encoding a hepsin protein, a fragment of hepsin protein, or an antisense hepsin mRNA.  
         [0057]    An expression vector is a replicable construct in which a nucleic acid sequence encoding a polypeptide is operably linked to suitable control sequences capable of effecting expression of the polypeptide in a cell. The need for such control sequences will vary depending upon the cell selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter and/or enhancer, suitable mRNA ribosomal binding sites and sequences which control the termination of transcription and translation. Methods which are well known to those skilled in the art can be used to construct expression vectors containing appropriate transcriptional and translational control signals. See, for example, techniques described in Sambrook et al., 1989 , Molecular Cloning: A Laboratory Manual  (2nd Ed.), Cold Spring Harbor Press, N.Y. A gene and its transcription control sequences are defined as being “operably linked” if the transcription control sequences effectively control transcription of the gene. Vectors of the invention include, but are not limited to, plasmid vectors and viral vectors. Preferred viral vectors of the invention are those derived from retroviruses, adenovirus, adeno-associated virus, SV40 virus, or herpes viruses.  
         [0058]    As used herein, the term “host” is meant to include not only prokaryotes but also eukaryotes such as yeast, plant and animal cells. A recombinant DNA molecule or gene which encodes a human hepsin protein of the present invention can be used to transform a host using any of the techniques commonly known to those of ordinary skill in the art. Especially preferred is the use of a vector containing coding sequences for the gene which encodes a human hepsin protein of the present invention for purposes of prokaryote transformation. Prokaryotic hosts may include  E. coli, S. tymphimurium, Serratia marcescens  and  Bacillus subtilis . Eukaryotic hosts include yeasts such as  Pichia pastoris , mammalian cells and insect cells.  
         [0059]    The term “oligonucleotide”, as used herein, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors, which, in turn, depend upon the ultimate function and use of the oligonucleotide. The term “primer”, as used herein, refers to a n oligonucleotide, whether occurring naturally (as in a purified restriction digest) or produced synthetically, and which is capable of initiating synthesis of a strand complementary to a nucleic acid when placed under appropriate conditions, i.e., in the presence of nucleotides and an inducing agent, such as a DNA polymerase, and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, sequence and/or homology of primer and the method used. For example, in diagnostic applications, the oligonucleotide primer typically contains 15-25 or more nucleotides, depending upon the complexity of the target sequence, although it may contain fewer nucleotides.  
         [0060]    The primers herein are selected to be “substantially” complementary to particular target DNA sequences. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment (i.e., containing a restriction site) may be attached to the 5′ end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementary with the sequence to hybridize therewith and form the template for synthesis of the extension product.  
         [0061]    The probe to which the DNA of the invention hybridizes preferably consists of a sequence of at least 20 consecutive nucleotides, more preferably 40 nucleotides, even more preferably 50 nucleotides, and most preferably 100 nucleotides or more (up to 100%) of the coding sequence of the nucleotides listed in SEQ ID No. 188 or the complement thereof. Such a probe is useful for detecting expression of hepsin in a cell by a method including the steps of (a) contacting mRNA obtained from the cell with a labeled hepsin hybridization probe; and (b) detecting hybridization of the probe with the mRNA.  
         [0062]    As used herein, “substantially pure DNA” means DNA that is not part of a milieu in which the DNA naturally occurs, by virtue of separation (partial or total purification) of some or all of the molecules of that milieu, or by virtue of alteration of sequences that flank the claimed DNA. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into a n autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence, e.g., a fusion protein. Also included is a recombinant DNA which includes a portion of the nucleotides listed in SEQ ID No. 188 and which encodes an alternative splice variant of hepsin.  
         [0063]    The DNA may have at least about 70% sequence identity to the coding sequence of the nucleotides listed in SEQ ID No. 188, preferably at least 75% (e.g., at least 80%); and most preferably at least 90%. The identity between two sequences is a direct function of the number of matching or identical positions. When a position in both of the two sequences is occupied by the same monomeric subunit, e.g., if a given position is occupied by an adenine in each of two DNA molecules, then they are identical at that position. For example, if 7 positions in a sequence 10 nucleotides in length are identical to the corresponding positions in a second 10-nucleotide sequence, then the two sequences have 70% sequence identity. The length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705).  
         [0064]    Further included in this invention are hepsin proteins which are encoded, at least in part, by portions of SEQ ID No. 188, e.g., products of alternative mRNA splicing or alternative protein processing events, or in which a section of hepsin sequence has been deleted. The fragment, or the intact hepsin polypeptide, may be covalently linked to another polypeptide, e.g., one which acts as a label, a ligand or a means to increase antigenicity.  
         [0065]    A substantially pure hepsin protein may be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding a hepsin polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, such a s immunoaffinity chromatography using an antibody specific for hepsin, polyacrylamide gel electrophoresis, or HPLC analysis. A protein is substantially free of naturally associated components when it is separated from at least some of those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be, by definition, substantially free from its naturally associated components. Accordingly, substantially pure proteins include eukaryotic proteins synthesized in  E. coli , other prokaryotes, or any other organism in which they do not naturally occur.  
         [0066]    In addition to substantially full-length proteins, the invention also includes fragments (e.g., antigenic fragments) of the hepsin protein. As used herein, “fragment,” as applied to a polypeptide, will ordinarily be at least 10 residues, more typically at least 20 residues, and preferably at least 30 (e.g., 50) residues in length, but less than the entire, intact sequence. Fragments of the hepsin protein can be generated by methods known to those skilled in the art, e.g., by enzymatic digestion of naturally occurring or recombinant hepsin protein, by recombinant DNA techniques using an expression vector that encodes a defined fragment of hepsin, or by chemical synthesis. The ability of a candidate fragment to exhibit a characteristic of hepsin (e.g., binding to an antibody specific for hepsin) can be assessed by methods known in the art. Purified hepsin or antigenic fragments of hepsin can be used to generate new antibodies or to test existing antibodies (e.g., as positive controls in a diagnostic assay) by employing standard protocols known to those skilled in the art. Included in this invention is polyclonal antisera generated by using hepsin or a fragment of hepsin as the immunogen in, e.g., rabbits. Standard protocols for monoclonal and polyclonal antibody production known to those skilled in this art are employed. The monoclonal antibodies generated by this procedure can be screened for the ability to identify recombinant hepsin cDNA clones, and to distinguish them from other cDNA clones.  
         [0067]    The invention encompasses not only an intact anti-hepsin monoclonal antibody, but also an immunologically-active antibody fragment, e.g., a Fab or (Fab) 2  fragment; an engineered single chain Fv molecule; or a chimeric molecule, e.g., an antibody which contains the binding specificity of one antibody, e.g., of murine origin, and the remaining portions of another antibody, e.g., of human origin.  
         [0068]    In one embodiment, the antibody, or a fragment thereof, may be linked to a toxin or to a detectable label, e.g., a radioactive label, non-radioactive isotopic label, fluorescent label, chemiluminescent label, paramagnetic label, enzyme label, or calorimetric label. Examples of suitable toxins include diphtheria toxin, Pseudomonas exotoxin A, ricin, and cholera toxin. Examples of suitable enzyme labels include malate hydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholinesterase, etc. Examples of suitable radioisotopic labels include  3 H,  125 I,  131 I,  32 P,  35 S,  14 C, etc.  
         [0069]    Paramagnetic isotopes for purposes of in vivo diagnosis can also be used according to the methods of this invention. There are numerous examples of elements that are useful in magnetic resonance imaging. For discussions on in vivo nuclear magnetic resonance imaging, see, for example, Schaefer et al., (1989)  JACC  14, 472-480; Shreve et al., (1986)  Magn. Reson. Med.  3, 336-340; Wolf, G. L., (1984)  Physiol. Chem. Phys. Med. NMR  16, 93-95; Wesbey et al., (1984)  Physiol. Chem. Phys. Med. NMR  16, 145-155; Runge et al., (1984)  Invest. Radiol.  19, 408-415. Examples of suitable fluorescent labels include a fluorescein label, an isothiocyalate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an ophthaldehyde label, a fluorescamine label, etc. Examples of chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, an aequorin label, etc.  
         [0070]    Those of ordinary skill in the art will know of other suitable labels which may be employed in accordance with the present invention. The binding of these labels to antibodies or fragments thereof can be accomplished using standard techniques commonly known and used by those of ordinary skill in the art. Typical techniques are described by Kennedy et al., (1976)  Clin. Chim. Acta  70, 1-31; and Schurs et al., (1977)  Clin. Chim. Acta  81, 1-40. Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide ester method. All of these methods are incorporated by reference herein.  
         [0071]    Also within the invention is a method of detecting hepsin protein in a biological sample, which includes the steps of contacting the sample with the labeled antibody, e.g., radioactively tagged antibody specific for hepsin, and determining whether the antibody binds to a component of the sample. Antibodies to the hepsin protein can be used in an immunoassay to detect increased levels of hepsin protein expression in tissues suspected of neoplastic transformation. These same uses can be achieved with Northern blot assays and analyses.  
         [0072]    As described herein, the invention provides a number of diagnostic advantages and uses. For example, the hepsin protein is useful in diagnosing cancer in different tissues since this protein is highly overexpressed in tumor cells. Antibodies (or antigen-binding fragments thereof) which bind to an epitope specific for hepsin are useful in a method of detecting hepsin protein in a biological sample for diagnosis of cancerous or neoplastic transformation. This method includes the steps of obtaining a biological sample (e.g., cells, blood, plasma, tissue, etc.) from a patient suspected of having cancer, contacting the sample with a labeled antibody (e.g., radioactively tagged antibody) specific for hepsin, and detecting the hepsin protein using standard immunoassay techniques such as an ELISA. Antibody binding to the biological sample indicates that the sample contains a component which specifically binds to an epitope within hepsin.  
         [0073]    Likewise, a standard Northern blot assay can be used to ascertain the relative amounts of hepsin mRNA in a cell or tissue obtained from a patient suspected of having cancer, in accordance with conventional Northern hybridization techniques known to those of ordinary skill in the art. This Northern assay uses a hybridization probe, e.g., radiolabelled hepsin cDNA, either containing the full-length, single stranded DNA having a sequence complementary to SEQ ID No. 188, or a fragment of that DNA sequence at least 20 (preferably at least 30, more preferably at least 50, and most preferably at least 100 consecutive nucleotides in length). The DNA hybridization probe can be labeled by any of the many different methods known to those skilled in this art.  
         [0074]    The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion:  
       EXAMPLE 1  
       [0075]    Amplification Of Serine Proteases Using Redundant And Specific Primers  
         [0076]    Only cDNA preparations deemed free of genomic DNA were used for gene expression analysis. Redundant primers were prepared for serine proteases, metallo-proteases and cysteine protease. The primers were synthesized to consensus sequences of amino acid surrounding the catalytic triad for serine proteases, viz. histidine . . . aspartate . . . and serine. The sequences of both sense (histidine &amp; aspartate) and antisense (aspartate and serine) redundant primers are shown in Table 2.  
                                               TABLE 2                                   SEQ ID             PCR Primers   5′→3′   No.                    No.               Redundant Primers:       Serine Protease (histidine) = S1   tgggtigtiacigcigcica(ct)tg   1       Serine Protease (aspartic acid) = AS1   a(ag)ia(ag)igciatitcitticc   2       Serine Protease (serine) = AS11   a(ag)iggiccicci(cg)(ta)(ag)tcicc   3       Cysteine Protease - sense   ca(ag)ggica(ag)tg(ct)ggi(ta)(cg)itg(ct)tgg   4       Cysteine Protease - antisense   taiccicc(ag)tt(ag)caicc(ct)tc   5       Metallo Protease - sense   cci(ac)gitg(tc)ggi(ga)(ta)icciga   6       Metallo Protease - antisense   tt(ag)tgicciai(ct)tc(ag)tg   7       Specific Primers:       Serine Protease (hepsin) = sense   tgtcccgatggcgagtgttt   8       Serine Protease (hepsin) = antisense   cctgttggccatagtactgc       Serine Protease (SCCE) = sense   agatgaatgagtacaccgtg   10       Serine Protease (SCCE) = antisense   ccagtaagtccttgtaaacc   11       Serine Protease (Comp B) = sense   aagggacacgagagctgtat   12       Serine Protease (Comp B) = antisense   aagtggtagttggaggaagc   13       Serine Protease (Protease M) = sense   ctgtgatccaccctgactat   20       Serine Protease (Protease M) = antisense   caggtggatgtatgcacact   21       Serine Protease (TADG12) = sense (Ser10-s)   gcgcactgtgtttatgagat   22       Serine Protease (TADG12) = antisense (Ser10-as)   ctctttggcttgtacttgct   23       Serine Protease (TADG13) = sense   tgagggacatcattatgcac   24       Serine Protease (TADG13) = antisense   caagttttccccataattgg   25       Serine Protease (TADG14) = sense   acagtacgcctgggagacca   26       Serine Protease (TADG14) = antisense   ctgagacggtgcaattctgg   27       Cysteine Protease (Cath-L) = sense   attggagagagaaaggctac   14       Cysteine Protease (Cath-L) = antisense   cttgggattgtacttacagg   15       Metallo Protease (PUMP1) = sense   cttccaaagtggtcacctac   16       Metallo Protease (PUMP1) = antisense   ctagactgctaccatccgtc   17                  
 
       EXAMPLE 2  
       [0077]    Carcinoma Tissue  
         [0078]    Several protease entities were identified and subcloned from PCR amplification of cDNA derived from serous cystadenocarcinomas. Therefore, the proteases described herein are reflective of surface activities for this type of carcinoma, the most common form of ovarian cancer. Applicant also shows PCR amplification bands of similar base pair size unique to the mucinous tumor type and the clear cell type. About 20-25% of ovarian cancers are classified as either mucinous, clear cell, or endometrioid.  
       EXAMPLE 3  
       [0079]    Ligation, Transformation And Sequencing  
         [0080]    To determine the identity of the PCR products, all the appropriate bands were ligated into Promega T-vector plasmid and the ligation product was used to transform JM109 cells (Promega) grown on selective media. After selection and culturing of individual colonies, plasmid DNA was isolated by means of the WIZARD MINIPREP™ DNA purification system (Promega). Inserts were sequenced using a Prism Ready Reaction Dydeoxy Terminators cycle sequencing kit (Applied Biosystems). Residual dye terminators were removed from the completed sequencing reaction using a CENTRISEP SPIN™ column (Princeton Separation), and samples were loaded into an Applied Biosystems Model 373A DNA sequencing system. The results of subcloning and sequencing for the serine protease primers are summarized in Table 3.  
                                 TABLE 3                           Serine protease candidates                Subclone   Primer Set   Gene Candidate                       1   His-Ser   Hepsin           2   His-Ser   SCCE           3   His-Ser   Compliment B           4   His-Asp   Cofactor 1           5   His-Asp   TADG-12*           6   His-Ser   TADG-13*           7   His-Ser   TADG-14*           8   His-Ser   Protease M           9   His-Ser   TADG-15*                                  
 
       EXAMPLE 4  
       [0081]    Cloning And Characterization  
         [0082]    Cloning and characterization of new gene candidates was undertaken to expand the panel representative of extracellular proteases specific for ovarian carcinoma subtypes. Sequencing of the PCR products derived from tumor cDNA confirms the potential candidacy of these genes. The three novel genes all have conserved residues within the catalytic triad sequence consistent with their membership in the serine protease family.  
         [0083]    Applicant compared the PCR products amplified from normal and carcinoma cDNAs using sense-histidine and antisense-aspartate as well as sense-histidine and antisense-serine. The anticipated PCR products of approximately 200 bp and 500 bp for those pairs of primers were observed (aspartate is approximately 50-70 amino acids downstream from histidine, and serine is about 100-150 amino acids toward the carboxy end from histidine).  
         [0084]    [0084]FIG. 1 shows a comparison of PCR products derived from normal and carcinoma cDNA as shown by staining in an agarose gel. Two distinct bands in Lane 2 were present in the primer pair sense-His/antisense ASP (AS1) and multiple bands of about 500 bp are noted in the carcinoma lane for the sense-His/antisense-Ser (AS2) primer pairs in Lane 4.  
       EXAMPLE 5  
       [0085]    Quantitative PCR  
         [0086]    The mRNA overexpression of hepsin was detected and determined using quantitative PCR. Quantitative PCR was performed generally according to the method of Noonan et al. [ Proc. Natl. Acad. Sci. USA,  87:7160-7164 (1990)]. The following oligonucleotide primers were used: hepsin:  
         [0087]    forward 5′-TGTCCCGATGGCGAGTGTTT-3′ (SEQ ID No. 8), and  
         [0088]    reverse 5′-CCTGTTGGCCATAGTACTGC-3′ (SEQ ID No. 9); and β-tubulin:  
         [0089]    forward 5′-TGCATTGACAACGAGGC -3′ (SEQ ID No. 18), and  
         [0090]    reverse 5′-CTGTCTTGA CATTGTTG -3′ (SEQ ID No. 19).  
         [0091]    β-tubulin was utilized as an internal control. The predicted sizes of the amplified genes were 282 bp for hepsin and 454 bp for β-tubulin. The primer sequences used in this study were designed according to the cDNA sequences described by Leytus et al. [ Biochemistry,  27, 1067-1074 (1988)] for hepsin, and Hall et al. [ Mol. Cell. Biol.,  3, 854-862 (1983)] for β-tubulin. The PCR reaction mixture consisted of cDNA derived from 50 ng of mRNA converted by conventional techniques, 5 pmol of sense and antisense primers for both the hepsin gene and the β-tubulin gene, 200 μmol of dNTPs, 5 μCi of α- 32 PdCTP and 0.25 units of Taq DNA polymerase with reaction buffer (Promega) in a final volume of 25 μl. The target sequences were amplified in parallel with the β-tubulin gene. Thirty cycles of PCR were carried out in a Thermal Cycler (Perkin-Elmer Cetus). Each cycle of PCR included 30 sec of denaturation at 95° C., 30 sec of annealing at 63° C. and 30 sec of extension at 72° C. The PCR products were separated on 2% agarose gels and the radioactivity of each PCR product was determined by using a Phosphorlmager™ (Molecular Dynamics). Student&#39;s t test was used for comparison of mean values.  
         [0092]    Experiments comparing PCR amplification in normal ovary and ovarian carcinoma suggested overexpression and/or alteration in mRNA transcript in tumor tissues. Northern blot analysis of TADG-14 confirms a transcript size of 1.4 kb and data indicate overexpression in ovarian carcinoma (FIG. 2). Isolation and purification using both PCR and a specific 250 bp PCR product to screen positive plaques yielded a 1.2 kb clone of TADG-14. Other proteases were amplified by the same method using the appropriate primers from Table 2.  
       EXAMPLE 6  
       [0093]    Tissue Bank  
         [0094]    A tumor tissue bank of fresh frozen tissue of ovarian carcinomas as shown in Table 4 was used for evaluation. Approximately 100 normal ovaries removed for medical reasons other than malignancy were obtained from surgery and were available as controls.  
                                                               TABLE 4                           Ovarian cancer tissue bank                Total   Stage I/11   Stage III/IV   No                            Stage Serous                           Malignant   166   15   140   8           LMP   16   9   7   0           Benign   12   0   0   12           Mucinous           Malignant   26   6   14   6           LMP   28   25   3   0           Benign   3   0   0   3           Endometrioid           Malignant   38   17   21   0           LMP   2   2   0   0           Benign   0   0   0   0           Other*           Malignant   61   23   29   9           LMP   0   0   0   0           Benign   5   0   0   5                                  
 
         [0095]    From the tumor bank, approximately 100 carcinomas were evaluated encompassing most histological sub-types of ovarian carcinoma, including borderline or low-malignant potential tumors and overt carcinomas. The approach included using mRNA prepared from fresh frozen tissue (both normal and malignant) to compare expression of genes in normal, low malignant potential tumors and overt carcinomas. The cDNA prepared from polyA+mRNA was deemed to be genomic DNA-free by checking all preparations with primers that encompassed a known intron-exon splice site using both β-tubulin and p53 primers.  
       EXAMPLE 7  
       [0096]    Northern Blots Analysis  
         [0097]    Significant information can be obtained by examining the expression of these candidate genes by Northern blot. Analysis of normal adult multi-tissue blots offers the opportunity to identify normal tissues which may express the protease. Ultimately, if strategies for inhibition of proteases for therapeutic intervention are to be developed, it is essential to appreciate the expression of these genes in normal tissues.  
         [0098]    Significant information is expected from Northern blot analysis of fetal tissue. Genes overexpressed in carcinomas are often highly expressed in organogenesis. As indicated, the hepsin gene cloned from hepatoma cells and overexpressed in ovarian carcinoma is overtly expressed in fetal liver. Hepsin gene expression was also detected in fetal kidney, and therefore, could be a candidate for expression in renal carcinomas.  
         [0099]    Northern panels for examining expression of genes in a multi-tissue normal adult as well as fetal tissue are commercially available (CLONTECH). Such evaluation tools are not only important to confirm the overexpression of individual transcripts in tumor versus normal tissues, but also provides the opportunity to confirm transcript size, and to determine if alternate splicing or other transcript alteration may occur in ovarian carcinoma.  
         [0100]    Northern blot analysis was performed as follows: 10 μg of mRNA was loaded onto a 1% formaldehyde-agarose gel, electrophoresed and blotted onto a HyBond-N + ™ nylon membrane (Amersham).  32 P-labeled cDNA probes were made using Prime-a-Gene Labeling System™ (Promega). The PCR products amplified by specific primers were used as probes. Blots were prehybridized for 30 min and then hybridized for 60 min at 68° C. with  32 P-labeled cDNA probe in ExpressHyb™ Hybridization Solution (CLONTECH). Control hybridization to determine relative gel loading was accomplished using the β-tubulin probe.  
         [0101]    Normal human tissues including spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocyte, heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas and normal human fetal tissues (Human Multiple Tissue Northern Blot; CLONTECH) were all examined using the same hybridization procedure.  
       EXAMPLE 8  
       [0102]    PCR Products Corresponding To Serine, Cysteine And Metallo-Proteases  
         [0103]    Based on their unique expression in either low malignant potential tumors or carcinomas, PCR-amplified cDNA products were cloned and sequenced and the appropriate gene identified based upon nucleotide and amino acid sequences stored in the GCG and EST databases. FIGS. 3, 4 &amp;  5  show the PCR product displays comparing normal and carcinomatous tissues using redundant primers for serine proteases (FIG. 3), for cysteine proteases (FIG. 4) and for metallo-proteases (FIG. 5). Note the differential expression in the carcinoma tissues versus the normal tissues. The proteases were identified using redundant cDNA primers (see Table 2) directed towards conserved sequences that are associated with intrinsic enzyme activity (for serine proteases, cysteine proteases and metallo-proteases) by comparing mRNA expression in normal, low malignant potential and overt ovarian carcinoma tissues according to Sakanari et al. [ Biochemistry  86, 4863-4867 (1989)].  
       EXAMPLE 9  
       [0104]    Serine Proteases  
         [0105]    For the serine protease group, using the histidine domain primer sense, S1, in combination with antisense primer AS2, the following proteases were identified:  
         [0106]    (a) Hepsin, a trypsin-like serine protease cloned from hepatoma cells shown to be a cell surface protease essential for the growth of hepatoma cells in culture and highly expressed in hepatoma tumor cells (FIG. 3, Lane 4);  
         [0107]    (b) Complement factor B protease (human factor IX), a protease involved in the coagulation cascade and associated with the production and accumulation of fibrin split products associated with tumor cells (FIG. 3, Lane 4). Compliment factor B belongs in the family of coagulation factors X (Christmas factor). As part of the intrinsic pathway, compliment factor B catalyzes the proteolytic activation of coagulation factor X in the presence of Ca 2+ phospholipid and factor VIIIa e5; and  
         [0108]    (c) A stratum corneum chymotryptic enzyme (SCCE) serine protease involved in desquarnation of skin cells from the human stratum corneum (FIG. 3, Lane 4). SCCE is expressed in keratinocytes of the epidermis and functions to degrade the cohesive structures in the cornified layer to allow continuous skin surface shedding.  
       EXAMPLE 10  
       [0109]    Cysteine Proteases  
         [0110]    In the cysteine protease group, using redundant sense and anti-sense primers for cysteine proteases, one unique PCR product was identified by overexpression in ovarian carcinoma when compared to normal ovarian tissue (FIG. 4, Lanes 3-5). Cloning and sequencing this PCR product identified a sequence of Cathepsin L, which is a lysomal cysteine protease whose expression and secretion is induced by malignant transformation, growth factors and tumor promoters. Many human tumors (including ovarian) express high levels of Cathepsin L. Cathepsin L cysteine protease belongs in the stromolysin family and has potent elastase and collagenase activities. Published data indicates increased levels in the serum of patients with mucinous cystadenocarcinoma of the ovary. It has not heretofore been shown to be expressed in other ovarian tumors.  
       EXAMPLE 11  
       [0111]    Metallo-Proteases  
         [0112]    Using redundant sense and anti-sense primers for the metallo-protease group, one unique PCR product was detected in the tumor tissue which was absent in normal ovarian tissue (FIG. 5, Lanes 2-5). Subcloning and sequencing this product indicates it has complete homology in the appropriate region with the so-called PUMP-1 (MMP-7) gene. This zinc-binding metallo-protease is expressed as a proenzyme with a signal sequence and is active in gelatin and collagenase digestion. PUMP-1 has also been shown to be induced and overexpressed in 9 of 10 colorectal carcinomas compared to normal colon tissue, suggesting a role for this substrate in the progression of this disease.  
       EXAMPLE 12  
       [0113]    Expression Of Hepsin  
         [0114]    The expression of the serine protease hepsin gene in 8 normal, 11 low malignant potential tumors, and 14 carcinoma (both mucinous and serous type) by quantitative PCR using hepsin-specific primers (see Table 2) was determined (primers directed toward the β-tubulin message were used as an internal standard) (Table 5). These data confirm the overexpression of the hepsin surface protease gene in ovarian carcinoma, including both low malignant potential tumors and overt carcinoma. Expression of hepsin is increased over normal levels in low malignant potential tumors, and high stage tumors (Stage III) of this group have higher expression of hepsin when compared to low stage tumors (Stage 1) (Table 6). In overt carcinoma, serous tumors exhibit the highest levels of hepsin expression, while mucinous tumors express levels of hepsin comparable with the high stage low malignant potential group (FIGS. 6 &amp; 7).  
                                                           TABLE 5                           Patient Characteristics and Expression of Hepsin Gene                            mRNA                       expression of       Case   Histological type a     Stage/Grade   LN b     hepsin c                      1   normal ovary           n       2   normal ovary           n       3   normal ovary           n       4   normal ovary           n       5   normal ovary           n       6   normal ovary           n       7   normal ovary           n       8   normal ovary           n       9   normal ovary           n       10   normal ovary           n       11   S adenoma (LMP)   1/1   N   4+       12   S adenoma (LMP)   1/1   NE   4+       13   S adenoma (LMP)   1/1   NE   n       14   S adenoma (LMP)   1/1   N   2+       15   S adenoma (LMP)   3/1   P   4+       16   S adenoma (LMP)   3/1   P   4+       17   S adenoma (LMP)   3/1   P   4+       18   M adenoma (LMP)   1/1   NE   4+       19   M adenoma (LMP)   1/1   N   n       20   M adenoma (LMP)   1/1   N   n       21   M adenoma (LMP)   1/1   N   n       22   M adenoma (LMP)   1/1   NE   n       23   S carcinoma   1/2   N   4+       24   S carcinoma   1/3   N   4+       25   S carcinoma   3/1   NE   2+       26   S carcinoma   3/2   NE   4+       27   S carcinoma   3/2   P   4+       28   S carcinoma   3/2   NE   2+       29   S carcinoma   3/3   NE   2+       30   S carcinoma   3/3   NE   4+       31   S carcinoma   3/3   NE   4+       32   S carcinoma   3/3   NE   4+       33   S carcinoma   3/3   N   4+       34   S carcinoma   3/3   NE   n       35   S carcinoma   3/3   NE   4+       36   S carcinoma   3/3   NE   4+       37   S carcinoma   3/3   NE   4+       38   S carcinoma   3/3   N   4+       39   S carcinoma   3/2   NE   2+       40   S carcinoma   3/3   NE   4+       41   S carcinoma   3/2   NE   4+       42   M carcinoma   1/2   N   n       43   M carcinoma   2/2   NE   4+       44   M carcinoma   2/2   N   4+       45   M carcinoma   3/1   NE   n       46   M carcinoma   3/2   NE   4+       47   M carcinoma   3/2   NE   n       48   M carcinoma   3/3   NE   n       49   E carcinoma   2/3   N   4+       50   E carcinoma   3/2   NE   4+       51   E carcinoma   3/3   NE   4+       52   C carcinoma   1/3   N   4+       53   C carcinoma   1/1   N   4+       54   C carcinoma   3/2   P   4+                                          
 
         [0115]    [0115]                                                           TABLE 6                           Overexpression of hepsin in normal ovaries and ovarian tumors                            Ratio of                   Hepsin   Hepsin           Type   N   Overexpression   to β-tubulin                            Normal   10    0 (0%)   0.06 ± 0.05           LMP   12    7 (58.3%)   0.26 ± 0.19           Serous   7    6 (85.7%)   0.34 ± 0.20           Mucinous   5    1 (20.0%)   0.14 ± 0.12           Carcinomous   32   27 (84.4%)   0.46 ± 0.29           Serous   19   18 (94.7%)   0.56 ± 0.32           Mucinous   7    3 (42.9%)   0.26 ± 0.22           Endometrioid   3    3 (100%)   0.34 ± 0.01           Clear Cell   3    3 (100%)   0.45 ± 0.08                        
       EXAMPLE 13  
       [0116]    Expression of SCCE and PUMP-1  
         [0117]    Studies using both SCCE-specific primers (FIG. 8) and PUMP-specific primers (FIG. 9) indicate overexpression of these proteases in ovarian carcinomas.  
       EXAMPLE 14  
       [0118]    Summary Of Proteases Detected Herein  
         [0119]    Most of the proteases described herein were identified from the sense-His/antisense-Ser primer pair, yielding a 500 bp PCR product (FIG. 1, Lane 4). Some of the enzymes are familiar, a short summary of each follows.  
         [0120]    Hepsin  
         [0121]    Hepsin is a trypsin-like serine protease cloned from hepatoma cells. Hepsin is an extracellular protease (the enzyme includes a secretion signal sequence) which is anchored in the plasma membrane by its amino terminal domain, thereby exposing its catalytic domain to the extracellular matrix. Hepsin has also been shown to be expressed in breast cancer cell lines and peripheral nerve cells. Hepsin has never before been associated with ovarian carcinoma. Specific primers for the hepsin gene were synthesized and the expression of hepsin examined using Northern blots of fetal tissue and ovarian tissue (both normal and ovarian carcinoma).  
         [0122]    [0122]FIG. 10A shows that hepsin was expressed in ovarian carcinomas of different histologic types, but not in normal ovary. FIG. 10B shows that hepsin was expressed in fetal liver and fetal kidney as anticipated, but at very low levels or not at all in fetal brain and lung. FIG. 10C shows that hepsin overexpression is not observed in normal adult tissue. Slight expression above the background level is observed in the adult prostate. The mRNA identified in both Northern blots was the appropriate size for the hepsin transcript. The expression of hepsin was examined in 10 normal ovaries and 44 ovarian tumors using specific primers to β-tubulin and hepsin in a quantitative PCR assay, and found it to be linear over 35 cycles. Expression is presented as the ratio of  32 p-hepsin band to the internal control, the  32 P-β-tubulin band.  
         [0123]    Hepsin expression was investigated in normal (N), mucinous (M) and serous (S) low malignant potential (LMP) tumors and carcinomas (CA). FIG. 11A shows quantitative PCR of hepsin and internal control β-tubulin. FIG. 11B shows the ratio of hepsin:β-tubulin expression in normal ovary, LMP tumor, and ovarian carcinoma. It was observed that Hepsin mRNA expression levels were significantly elevated in LMP tumors, (p&lt;0.005) and carcinomas (p&lt;0.0001) compared to levels in normal ovary. All 10 cases of normal ovaries showed a relatively low level of hepsin mRNA expression.  
         [0124]    Hepsin mRNA is highly overexpressed in most histopathologic types of ovarian carcinomas including some low malignant potential tumors (see FIGS. 11A &amp; 11B). Most noticeably, hepsin is highly expressed in serous, endometrioid and clear cell tumors tested. It is highly expressed in some mucinous tumors, but it is not overexpressed in the majority of such tumors.  
         [0125]    Stratum Corneum Chymotrypsin Enzyme (SCCE)  
         [0126]    The PCR product identified was the catalytic domain of the sense-His/antisense-Ser of the stratum corneum chymotrypsin enzyme. This extracellular protease was cloned, sequenced and shown to be expressed on the surface of keratinocytes in the epidermis. Stratum corneum chymotrypsin enzyme is a chymotrypsin-like serine protease whose function is suggested to be in the catalytic degradation of intercellular cohesive structures in the stratum corneum layer of the skin. This degradation allows continuous shedding (desquamation) of cells from the skin surface. The subcellular localization of stratum corneum chymotrypsin enzyme is in the upper granular layer in the stratum corneum of normal non-palmoplantar skin and in the cohesive parts of hypertrophic plantar stratum corneum. Stratum corneum chymotrypsin enzyme is exclusively associated with the stratum corneum and has not so far been shown to be expressed in any carcinomatous tissues.  
         [0127]    Northern blots were probed with the PCR product to determine expression of stratum corneum chymotrypsin enzyme in fetal tissue and ovarian carcinoma (FIGS. 12A &amp; 12B). Noticeably, detection of stratum corneum chymotrypsin enzyme messenger RNA on the fetal Northern was almost non-existent (a problem with the probe or the blot was excluded by performing the proper controls). A faint band appeared in fetal kidney. On the other hand, stratum corneum chymotrypsin enzyme mRNA is abundant in the ovarian carcinoma mRNA (FIG. 12B). Two transcripts of the correct size are observed for stratum corneum chymotrypsin enzyme. The same panel of cDNA used for hepsin analysis was used for stratum corneum chymotrypsin enzyme expression.  
         [0128]    No stratum corneum chymotrypsin enzyme expression was detected in the normal ovary lane of the Northern blot. A comparison of all candidate genes, including a loading marker (β-tubulin), was shown to confirm that this observation was not a result of a loading bias. Quantitative PCR using stratum corneum chymotrypsin enzyme primers, along with β-tubulin internal control primers, confirmed the overexpression of stratum corneum chymotrypsin enzyme mRNA in carcinoma of the ovary with no expression in normal ovarian tissue (FIG. 13).  
         [0129]    [0129]FIG. 13A shows a comparison using quantitative PCR of stratum corneum chymotrypsin enzyme cDNA from normal ovary and ovarian carcinomas. FIG. 13B shows the ratio of stratum corneum chymotrypsin enzyme to the β-tubulin internal standard in 10 normal and 44 ovarian carcinoma tissues. Again, it is observed that stratum corneum chymotrypsin enzyme is highly overexpressed in ovarian carcinoma cells. It is also noted that some mucinous tumors overexpress stratum corneum chymotrypsin enzyme, but the majority do not.  
         [0130]    Protease M  
         [0131]    Protease M was identified from subclones of the His—ser primer pair. This protease was first cloned by Anisowicz, et al., [ Molecular Medicine,  2, 624-636 (1996)] and shown to be overexpressed in carcinomas. A preliminary evaluation indicates that this enzyme is overexpressed in ovarian carcinoma (FIG. 14).  
         [0132]    Cofactor I and Complement Factor B  
         [0133]    Several serine proteases associated with the coagulation pathway were also subcloned. Examination of normal and ovarian carcinomas by quantitative PCR for expression of these enzymes, it was noticeable that this mRNA was not clearly overexpressed in ovarian carcinomas when compared to normal ovarian tissue. It should be noted that the same panel of tumors was used for the evaluation of each candidate protease.  
       EXAMPLE 15  
       [0134]    Summary Of Previously Unknown Proteases Detected Herein TADG-12  
         [0135]    TADG-12 was identified from the primer pairs, sense-His/antisense-Asp (see FIG. 1, Lanes 1 &amp; 2). Upon subcloning both PCR products in lane 2, the 200 bp product had a unique protease-like sequence not included in GenBank. This 200 bp product contains many of the conserved amino acids common for the His-Asp domain of the family of serine proteins. The second and larger PCR product (300 bp) was shown to have a high degree of homology with TADG-12 (His-Asp sequence), but also contained approximately 100 bp of unique sequence. Synthesis of specific primers and the sequencing of the subsequent PCR products from three different tumors demonstrated that the larger PCR product (present in about 50% of ovarian carcinomas) includes an insert of about 100 bp near the 5′ end (and near the histidine) of the sequence. This insert may be a retained genomic intron because of the appropriate position of splice sites and the fact that the insert does not contain an open reading frame (see FIG. 15). This suggests the possibility of a splice site mutation which gives rise to retention of the intron, or a translocation of a sequence into the TADG-12 gene in as many as half of all ovarian carcinomas.  
         [0136]    TADG-13 and TADG-14  
         [0137]    Specific primers were synthesized for TADG-13 and TADG-14 to evaluate expression of genes in normal and ovarian carcinoma tissue. Northern blot analysis of ovarian tissues indicates the transcript for the TADG-14 gene is approximately 1.4 kb and is expressed in ovarian carcinoma tissues (FIG. 16A) with no noticeable transcript presence in normal tissue. In quantitative PCR studies using specific primers, increased expression of TADG-14 in ovarian carcinoma tissues was noted compared to a normal ovary (FIG. 16B). The presence of a specific PCR product for TADG-14 in both an HeLa library and an ovarian carcinoma library was also confirmed. Several candidate sequences corresponding to TADG-14 have been screened and isolated from the HeLa library.  
         [0138]    Clearly from sequence homology, these genes fit into the family of serine proteases. TADG-13 and -14 are, however, heretofore undocumented genes which the specific primers of the invention allow to be evaluated in normal and tumor cells, and with which the presence or absence of expression of these genes is useful in the diagnosis or treatment selection for specific tumor types.  
         [0139]    PUMP-1  
         [0140]    In a similar strategy using redundant primers to metal binding domains and conserved histidine domains, a differentially expressed PCR product identical to matrix metallo-protease 7 (MMP-7) was identified, herein called PUMP-1. Using specific primers for PUMP-1, PCR produced a 250 bp product for Northern blot analysis.  
         [0141]    PUMP-1 is differentially expressed in fetal lung and kidney tissues. FIG. 17A shows the expression of PUMP-1 in human fetal tissue, while no transcript could be detected in either fetal brain or fetal liver. FIG. 17B compares PUMP-1 expression in normal ovary and carcinoma subtypes using Northern blot analysis. Notably, PUMP-1 is expressed in ovarian carcinoma tissues, and again, the presence of a transcript in normal tissue was not detected. Quantitative PCR comparing normal versus ovarian carcinoma expression of the PUMP-1 mRNA indicates that this gene is highly expressed in serous carcinomas, including most low malignant serous tumors, and is, again, expressed to a lesser extent in mucinous tumors (see FIGS. 18A &amp; 18B). PUMP-1, however, is so far the protease most frequently found overexpressed in mucinous tumors (See Table 7).  
         [0142]    Cathepsin-L  
         [0143]    Using redundant cysteine protease primers to conserved domains surrounding individual cysteine and histidine residues, the cathepsin-L protease was identified in several serous carcinomas. An initial examination of the expression of cathepsin L in normal and ovarian tumor tissue indicates that transcripts for the cathepsin-L protease are present in both normal and tumor tissues (FIG. 19). However, its presence or absence in combination with other proteases of the present invention permits identification of specific tumor types and treatment choices.  
         [0144]    Discussion  
         [0145]    Redundant primers to conserved domains of serine, metallo-, and cysteine proteases have yielded a set of genes whose mRNAs are overexpressed in ovarian carcinoma. The genes which are clearly overexpressed include the serine proteases hepsin, stratum corneum chymotrypsin enzyme, protease M TADG12, TADG14 and the metallo-protease PUMP-1 (see FIG. 19 and Table 7). Northern blot analysis of normal and ovarian carcinoma tissues, summarized in FIG. 14, indicated overexpression of hepsin, stratum corneum chymotrypsin enzyme, PUMP-1 and TADG-14. A β-tubulin probe to control for loading levels was included.  
                                                                                   TABLE 7                           Overexpression of Proteases in Ovarian Tumors            Type   N   Hepsin   SCCE   Pump-1   Protease M                    Normal   10   0%   (0/10)   0%   (0/10)   0%   (0/10)   0%   (0/10)       LMP   12   58.3%   (7/12)   66.7%   (8/12)   75.0%   (9/12)   75%   (9/12)       serous   7   85.7%   (6/7)   85.7%   (6/7)   85.7%   (6/7)   100%   (7/7)       mucinous   5   20.0%   (1/5)   40.0%   (2/5)   60%   (3/5)   40.0%   (2/5)       Carcinoma   32   84.4%   (27/32)   78.1%   (25/32)   81.3%   (26/32)   90.6%   (29/32)       serous   19   94.7%   (18/19)   89.5%   (17/19)   78.9%   (15/19)   94.7%   (18/19)       mucinous   7   42.9%   (3/7)   28.6%   (2/7)   71.4%   (5/7)   85.7%   (6/7)       endometr.   3   100%   (3/3)   100%   (3/3)   100%   (3/3)   100%   (3/3)       clear cell   3   100%   (3/3)   100%   (3/3)   100%   (3/3)   67.7%   (2/3)                  
 
         [0146]    For the most part, these proteins previously have not been associated with the extracellular matrix of ovarian carcinoma cells. No panel of proteases which might contribute to the growth, shedding, invasion and colony development of metastatic carcinoma has been previously described, including the three new candidate serine proteases which are herein disclosed. The establishment of an extracellular protease panel associated with either malignant growth or malignant potential offers the opportunity for the identification of diagnostic or prognostic markers and for therapeutic intervention through inhibition or down regulation of these proteases.  
         [0147]    The availability of the instant gene-specific primers coding for the appropriate region of tumor specific proteases allows for the amplification of a specific cDNA probe using Northern and Southern analysis, and their use as markers to detect the presence of the cancer in tissue. The probes also allow more extensive evaluation of the expression of the gene in normal ovary versus low malignant potential tumor, as well as both high- and low-stage carcinomas. The evaluation of a panel of fresh frozen tissue from all the carcinoma subtypes (Table 4) allowed the determination of whether a protease is expressed predominantly in early stage disease or within specific carcinoma subtypes. It was also determined whether each gene&#39;s expression is confined to a particular stage in tumor progression and/or is associated with metastatic lesions. Detection of specific combinations of proteases is an identifying characteristic of the specific tumor types and yields valuable information for diagnoses and treatment selection. Particular tumor types may be more accurately diagnosed by the characteristic expression pattern of each specific tumor.  
       EXAMPLE 16  
       [0148]    Hepsin Peptide Ranking  
         [0149]    For vaccine or immune stimulation, individual 9-mers to 11-mers of the hepsin protein were examined to rank the binding of individual peptides to the top 8 haplotypes in the general population (Parker et al., (1994)). The computer program used for this analyses can be found on the web site of National Institutes of Health. Table 8 shows the peptide ranking based upon the predicted half-life of each peptide&#39;s binding to a particular HLA allele. A larger half-life indicates a stronger association with that peptide and the particular HLA molecule. The hepsin peptides that strongly bind to an HLA allele are putative immunogens, and are used to innoculate an individual against hepsin.  
                                                           TABLE 8                           Hepsin peptide ranking            HLA Type           Predicted   SEQ       &amp; Ranking   Start   Peptide   Dissociation ½     ID No.                    HLA A0201                        1   170   SLGRWPWQV   521.640   28        2   191   SLLSGDWVL   243.051   29        3   229   GLQLGVQAV   159.970   30        4   392   KVSDFREWI   134.154   31        5   308   VLQEARVPI   72.717   32        6   130   RLLEVISVC   71.069   33        7   98   ALTHSELDV   69.552   34        8   211   VLSRWRVFA   46.451   35        9   26   LLLLTAIGA   31.249   36       10   284   ALVDGKICT   30.553   37       11   145   FLAAICQDC   22.853   38       12   192   LLSGDWVLT   21.536   39       13   20   ALTAGTLLL   21.362   40       14   259   ALVHLSSPL   21.362   41       15   277   CLPAAGQAL   21.362   42       16   230   LQLGVQAVV   18.186   43       17   268   PLTEYIQPV   14.429   44       18   31   AIGAASWAI   10.759   45       19   285   LVDGKICTV   9.518   46       20   27   LLLTAIGAA   9.343   47       HLA A0205        1   191   SLLSGDWVL   25.200   48        2   163   IVGGRDTSL   23.800   49        3   392   KVSDFREWI   18.000   50        4   64   MVFDKTEGT   15.300   51        5   236   AVVYHGGYL   14.000   52        6   55   QVSSADARL   14.000   53        7   130   RLLEVISVC   9.000   54        8   230   LQLGVQAVV   8.160   55        9   20   ALTAGTLLL   7.000   56       10   259   ALVHLSSPL   7.000   57       11   277   CLPAAGQAL   7.000   58       12   17   KVAALTAGT   6.000   59       13   285   LVDGKICTV   5.440   60       14   308   VLQEARVPI   5.100   61       15   27   LLLTAIGAA   5.100   62       16   229   GLQLGVQAV   4.000   63       17   313   RVPIISNDV   4.000   64       18   88   LSCEEMGFL   3.570   65       19   192   LLSGDWVLT   3.400   66       20   284   ALVDGKICT   3.000   67       HLA A1        1   89   SCEEMGFLR   45.000   68        2   58   SADARLMVF   25.000   69        3   393   VSDFREWIF   7.500   70        4   407   HSEASGMVT   6.750   71        5   137   VCDCPRGRF   5.000   72        6   269   LTEYIQPVC   4.500   73        7   47   DQEPLYPVQ   2.700   74        8   119   CVDEGRLPH   2.500   75        9   68   KTEGTWRLL   2.250   76       10   101   HSELDVRTA   1.350   77       11   250   NSEENSNDI   1.350   78       12   293   VTGWGNTQY   1.250   79       13   231   QLGVQAVVY   1.000   80       14   103   ELDVRTAGA   1.000   81       15   378   GTGCALAQK   1.000   82       16   358   VCEDSISRT   0.900   83       17   264   SSPLPLTEY   0.750   84       18   87   GLSCEEMGF   0.500   85       19   272   YIQPVCLPA   0.500   86       20   345   GIDACQGDS   0.500   87       HLA A24        1   301   YYGQQAGVL   200.000   88        2   238   VYHGGYLPF   100.000   89        3   204   CFPERNRVL   36.000   90        4   117   FFCVDEGRL   20.000   91        5   124   RLPHTQRLL   12.000   92        6   80   RSNARVAGL   12.000   93        7   68   KTEGTWRLL   12.000   94        8   340   GYPEGGIDA   9.000   95        9   242   GYLPFRDPN   9.000   96       10   51   LYPVQVSSA   7.500   97       11   259   ALVHLSSPL   7.200   98       12   277   CLPAAGQAL   7.200   99       13   191   SLLSGDWVL   6.000   100       14   210   RVLSRWRVF   6.000   101       15   222   VAQASPHGL   6.000   102       16   236   AVVYHGGYL   6.000   103       17   19   AALTAGTLL   6.000   104       18   36   SWAIVAVLL   5.600   105       19   35   ASWAIVAVL   5.600   106       20   300   QYYGQQAGV   5.600   107       HLA B7        1   363   ISRTPRWRL   90.000   108        2   366   TPRWRLCGI   80.000   109        3   236   AVVYHGGYL   60.000   110        4   13   CSRPKVAAL   40.000   111        5   179   SLRYDGAHL   40.000   112        6   43   LLRSDQEPL   40.000   113        7   19   AALTAGTLL   36.000   114        8   55   QVSSADARL   20.000   115        9   163   IVGGRDTSL   20.000   116       10   140   CPRGRFLAA   20.000   117       11   20   ALTAGTLLL   12.000   118       12   409   EASGMVTQL   12.000   119       13   259   ALVHLSSPL   12.000   120       14   35   ASWAIVAVL   12.000   121       15   184   GAHLCGGSL   12.000   122       16   18   VAALTAGTL   12.000   123       17   222   VAQASPHGL   12.000   124       18   224   QASPHGLQL   12.000   125       19   265   SPLPLTEYI   8.000   126       20   355   GPFVCEDSI   8.00   127       HLA B8        1   13   CSRPKVAAL   80.000   128        2   366   TPRWRLCGI   80.000   129        3   140   CPRGRFLAA   16.000   130        4   152   DCGRRKLPV   4.800   131        5   363   ISRTPRWRL   4.000   132        6   163   IVGGRDTSL   4.000   133        7   331   QJKPKMFCA   4.000   134        8   80   RSNARVAGL   2.000   135        9   179   SLRYDGAHL   1.600   136       10   43   LLRSDQEPL   1.600   137       11   409   EASGMVTQL   1.600   138       12   311   EARVPIISN   0.800   139       13   222   VAQASPHGL   0.800   140       14   19   AALTAGTLL   0.800   141       15   18   VAALTAGTL   0.800   142       16   184   GAHLCGGSL   0.800   143       17   224   QASPHGLQL   0.800   144       18   82   NARVAGLSC   0.800   145       19   204   CFPERNRVL   0.600   146       20   212   LSRWRVFAG   0.400   147       HLA B2702        1   172   GRWPWQVSL   300.000   148        2   44   LRSDQEPLY   200.00   149        3   155   RRKLPVDRI   180.000   150        4   213   SRWRVFAGA   100.000   151        5   166   GRDTSLGRW   100.000   152        6   369   WRLCGIVSW   100.000   153        7   180   LRYDGAHLC   100.000   154        8   96   LRALTHSEL   60.000   155        9   396   FREWIFQAI   60.000   156       10   123   GRLPHTQRL   60.000   157       11   207   ERNRVLSRW   30.000   158       12   209   NRVLSRWRV   20.000   159       13   14   SRPKVAALT   20.000   160       14   106   VRTAGANGT   20.000   161       15   129   QRLLEVISV   20.000   162       16   349   CQGDSGGPF   20.000   163       17   61   ARLMVFDKT   20.000   164       18   215   WRYFAGAVA   20.000   165       19   143   GRFLAAICQ   10.000   166       20   246   FRDPNSEEN   10.000   167       HLA B4403        1   132   LEVISVCDC   36.000   168        2   91   EEMGFLRAL   18.000   169        3   264   SSPLPLTEY   13.500   170        4   310   QEARVPIIS   12.000   171        5   319   NDVCNGADF   10.000   172        6   4   KEGGRTVPC   9.000   173        7   251   SEENSNDIA   8.000   174        8   256   NDIALVHLS   7.500   175        9   294   TGWGNTQYY   6.750   176       10   361   DSISRTPRW   6.750   177       11   235   QAVVYHGGY   6.000   178       12   109   AGANGTSGF   6.000   179       13   270   TEYIQPVCL   6.000   180       14   174   WPWQVSLRY   4.500   181       15   293   VTGWGNTQY   4.500   182       16   69   TEGTWRLLC   4.000   183       17   90   CEEMGFLRA   4.000   184       18   252   EENSNDIAL   4.000   185       19   48   QEPLYPVQV   4.000   186       20   102   SELDVRTAG   3.600   187                  
 
       EXAMPLE 17  
       [0150]    Hepsin Peptides as Target Epitopes For Human CD8 + Cytotoxic T Cells  
         [0151]    Two computer programs were used to identify 9-mer peptides containing binding motifs for HLA class I molecules. The first, based on a scheme devised by Parker et al (1994), was developed by the Bioinformatics and Molecular Analysis Section (BIMAS) of the Center for Information Technology, NIH, and the second, known as SYFPEITHI, was formulated by Rammensee and colleagues at the University of Tubingen, Germany.  
         [0152]    Peptides that possessed HLA A2.1 binding motifs were synthesized and tested directly for their ability to bind HLA A2.1. This technique employs T2 cells which are peptide transporter-deficient and thus express low endogenous HLA class I levels due to inability to load peptide and stabilize HLA class I folding for surface expression. It has been showed that addition of exogenous peptides capable of binding HLA A2.1 (A*0201) could increase the number of properly folded HLA A2.1 molecules on the cell surface, as revealed by flow cytometry (Nijman et al, 1993).  
         [0153]    Peptides that possessed binding motifs for HLA class I molecules other than A2.1 were tested directly for their ability to induce specific CD8 + CTL responses from normal adult donors as described below.  
         [0154]    Monocyte-derived dendritic cells were generated from peripheral blood drawn from normal adult donors of the appropriate HLA type. Adherent monocytes were cultured in AIM-V (Gibco-BRL) supplemented with GM-CSF and IL-4 according to standard techniques (Santin et al, 2000, 2001). After 5-6 days, dendritic cell maturation was induced by addition of PGE 2 , IL-1β and TNFα for a further 48 h.  
         [0155]    Mature dendritic cells were loaded with peptide (2×10 6  dendritic cells with 50 μg/ml peptide in 1 ml serum-free AIM-V medium for 2 h at 37° C.) and washed once prior to culture with 1×10 6 /ml peripheral blood mononuclear cells (PBMC) in AIM-V or AIM-V plus 5% human AB serum. The PBMC:DC ratio was between 20:1 and 30:1. After 7 days, responder T cells were restimulated with peptide-loaded, irradiated autologous dendritic cells or peripheral blood mononuclear cells at responder:stimulator ratios between 10:1 and 20:1 or 1:1 and 1:10 respectively. At this point, cultures were supplemented with recombinant human IL-2 (10-100 U/ml), and fed with 50-75% changes of fresh medium plus IL-2 every 2-4 days. T cell lines were established and maintained by peptide restimulation every 14-21 days. Responder CD8 + T cells were purified by positive selection with anti-CD8-coupled magnetic beads (Dynal, Inc.) after the 2 nd  or 3 rd  antigen stimulation.  
         [0156]    Peptide-specific cytotoxicity was tested in standard 5-6 h microwell  51 Cr-release assays (Nazaruk et al, 1998). Autologous EBV-transformed lymphoblastoid cell lines (LCL) were loaded with peptide (50 μg/ml, 1 h at 37° C.) and subsequently  51 Cr-labeled (50 μCi in 200-300 μl, 1 h at 37° C.). Peptide-loaded  51 Cr-labeled LCL were incubated with CD8 + T cells at effector-target ration between 5:1 and 1.25:1. Cytotoxicity was recorded as percentage  51 Cr released into culture supernatants.  
         [0157]    Hepsin peptide 170-178 (SEQ ID No. 28) is an HLA A2.1-binding peptide, as revealed by upregulation of A2.1 expression in T2 cells (data not shown). CD8 + CTL specific for hepsin 170-178 killed peptide-loaded autologous lymphoblastoid cell lines, but did not kill control, peptide-free lymphoblastoid cell lines (FIG. 20). Heterologous HLA A2.1-expressing peptide-loaded lymphoblastoid cell lines were efficiently killed, but targets lacking HLA A2.1 were not killed. Natural killer-sensitive K562 cells were not lysed. Cytotoxicity against hepsin 170-178 loaded lymphoblastoid cell lines could be blocked with a monoclonal antibody specific for a non-polymorphic HLA class I determinant, confirming that lysis was HLA class I-restricted. Cytotoxicity was also blocked by MAb specific for HLA A2.1.  
         [0158]    Hepsin peptide 172-180 (SEQ ID No. 148) was predicted by computer analysis to bind HLA B27. While this could not be demonstrated directly, cytotoxicity assays showed that CD8 + CIL specific for hepsin 172-180 could kill peptide-loaded, HLA B27-expressing autologous and heterologous lymphoblastoid cell lines, but failed to recognize heterologous peptide-loaded lymphoblastoid cell lines that did not express HLA B27.  
         [0159]    CD8 + CTL specific for hepsin 172-180 killed peptide-loaded autologous lymphoblastoid cell lines, but did not kill peptide-free control lymphoblastoid cell lines (FIG. 21). Natural killer-sensitive K562 cells were not lysed. Cytotoxicity against hepsin 172-180 loaded lymphoblastoid cell lines could be blocked with MAb specific for a non-polymorphic HLA class I determinant, confirming that lysis was HLA class I-restricted.  
         [0160]    Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. Further, these patents and publications are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.  
         [0161]    One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present examples, along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.  
     
       
       
         1 
         
           
             188  
           
           
             1  
             23  
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               primer_bind  
               6, 9, 12, 15, 18  
               sense oligonucleotide primer for amplifying 
      serine proteases, n = Inosine  
             
           
            1 

tgggtngtna cngcngcnca ytg                                             23 

 
           
             2  
             20  
             DNA  
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               primer_bind  
               3, 6, 9, 12, 15, 18  
               antisense oligonucleotide primer for amplifying 
      serine proteases, n = Inosine  
             
           
            2 

arnarngcna tntcnttncc                                                 20 

 
           
             3  
             20  
             DNA  
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               primer_bind  
               3, 6, 9, 12, 18  
               antisense oligonucleotide primer for amplifying 
      serine proteases, n = Inosine  
             
           
            3 

arnggnccnc cnswrtcncc                                                 20 

 
           
             4  
             24  
             DNA  
             Artificial sequence  
             
               primer_bind  
               6, 15, 18  
               sense oligonucleotide primer for amplifying 
      cysteine proteases, n = Inosine  
             
           
            4 

carggncart gyggnwsntg ytgg                                            24 

 
           
             5  
             20  
             DNA  
             Artificial sequence  
             
               primer_bind  
               3, 6, 15  
               antisense oligonucleotide primer for amplifying 
      cysteine proteases, n = Inosine  
             
           
            5 

tanccnccrt trcanccytc                                                 20 

 
           
             6  
             20  
             DNA  
             Artificial sequence  
             
               primer_bind  
               3, 6, 12, 15, 18  
               sense oligonucleotide primer for amplifying 
      metallo-proteases, n = Inosine  
             
           
            6 

ccnmgntgyg gnrwnccnga                                                 20 

 
           
             7  
             17  
             DNA  
             Artificial sequence  
             
               primer_bind  
               6, 9, 11  
               antisense oligonucleotide primer for amplifying 
      metallo-proteases, n = Inosine  
             
           
            7 

ttrtgnccna nytcrtg                                                    17 

 
           
             8  
             20  
             DNA  
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               sense oligonucleotide primer specific for 
      hepsin  
             
           
            8 

tgtcccgatg gcgagtgttt                                                 20 

 
           
             9  
             20  
             DNA  
             Artificial sequence  
             
               antisense oligonucleotide primer specific for 
      hepsin  
             
           
            9 

cctgttggcc atagtactgc                                                 20 

 
           
             10  
             20  
             DNA  
             Artificial sequence  
             
               sense oligonucleotide primer specific for SCCE  
             
           
            10 

agatgaatga gtacaccgtg                                                 20 

 
           
             11  
             20  
             DNA  
             Artificial sequence  
             
               antisense oligonucleotide primer specific for 
      SCCE  
             
           
            11 

ccagtaagtc cttgtaaacc                                                 20 

 
           
             12  
             20  
             DNA  
             Artificial sequence  
             
               sense oligonucleotide primer specific for CompB  
             
           
            12 

aagggacacg agagctgtat                                                 20 

 
           
             13  
             20  
             DNA  
             Artificial sequence  
             
               antisense oligonucleotide primer specific for 
      CompB  
             
           
            13 

aagtggtagt tggaggaagc                                                 20 

 
           
             14  
             20  
             DNA  
             Artificial sequence  
             
               sense oligonucleotide primer specific for 
      Cath-L  
             
           
            14 

attggagaga gaaaggctac                                                 20 

 
           
             15  
             20  
             DNA  
             Artificial sequence  
             
               antisense oligonucleotide primer specific for 
      Cath-L  
             
           
            15 

cttgggattg tacttacagg                                                 20 

 
           
             16  
             20  
             DNA  
             Artificial sequence  
             
               sense oligonucleotide primer specific for 
      PUMP-1  
             
           
            16 

cttccaaagt ggtcacctac                                                 20 

 
           
             17  
             20  
             DNA  
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               antisense oligonucleotide primer specific for 
      PUMP-1  
             
           
            17 

ctagactgct accatccgtc                                                 20 

 
           
             18  
             17  
             DNA  
             Artificial sequence  
             
               sense oligonucleotide primer specific for 
      (-tubulin  
             
           
            18 

tgcattgaca acgaggc                                                    17 

 
           
             19  
             17  
             DNA  
             Artificial sequence  
             
               antisense oligonucleotide primer specific for 
      (-tubulin  
             
           
            19 

ctgtcttgac attgttg                                                    17 

 
           
             20  
             20  
             DNA  
             Artificial sequence  
             
               sense oligonucleotide primer specific for 
      Protease M  
             
           
            20 

ctgtgatcca ccctgactat                                                 20 

 
           
             21  
             20  
             DNA  
             Artificial sequence  
             
               antisense oligonucleotide primer specific for 
      Protease M  
             
           
            21 

caggtggatg tatgcacact                                                 20 

 
           
             22  
             20  
             DNA  
             Artificial sequence  
             
               sense oligonucleotide primer specific for 
      TADG-12  
             
           
            22 

gcgcactgtg tttatgagat                                                 20 

 
           
             23  
             20  
             DNA  
             Artificial sequence  
             
               antisense oligonucleotide primer specific for 
      TADG-12  
             
           
            23 

ctctttggct tgtacttgct                                                 20 

 
           
             24  
             20  
             DNA  
             Artificial sequence  
             
               sense oligonucleotide primer specific for 
      TADG-13  
             
           
            24 

tgagggacat cattatgcac                                                 20 

 
           
             25  
             20  
             DNA  
             Artificial sequence  
             
               antisense oligonucleotide primer specific for 
      TADG-13  
             
           
            25 

caagttttcc ccataattgg                                                 20 

 
           
             26  
             20  
             DNA  
             Artificial sequence  
             
               sense oligonucleotide primer specific for 
      TADG-14  
             
           
            26 

acagtacgcc tgggagacca                                                 20 

 
           
             27  
             20  
             DNA  
             Artificial sequence  
             
               antisense oligonucleotide primer specific for 
      TADG-14  
             
           
            27 

ctgagacggt gcaattctgg                                                 20 

 
           
             28  
             9  
             PRT  
             Homo sapiens  
             
               Residues 170-178 of the hepsin protein  
             
           
            28 

Ser Leu Gly Arg Trp Pro Trp Gln Val 
                 5 

 
           
             29  
             9  
             PRT  
             Homo sapiens  
             
               Residues 191-199 of the hepsin protein  
             
           
            29 

Ser Leu Leu Ser Gly Asp Trp Val Leu 
                 5 

 
           
             30  
             9  
             PRT  
             Homo sapiens  
             
               Residues 229-237 of the hepsin protein  
             
           
            30 

Gly Leu Gln Leu Gly Val Gln Ala Val 
                 5 

 
           
             31  
             9  
             PRT  
             Homo sapiens  
             
               Residues 392-400 of the hepsin protein  
             
           
            31 

Lys Val Ser Asp Phe Arg Glu Trp Ile 
                 5 

 
           
             32  
             9  
             PRT  
             Homo sapiens  
             
               Residues 308-316 of the hepsin protein  
             
           
            32 

Val Leu Gln Glu Ala Arg Val Pro Ile 
                 5 

 
           
             33  
             9  
             PRT  
             Homo sapiens  
             
               Residues 130-138 of the hepsin protein  
             
           
            33 

Arg Leu Leu Glu Val Ile Ser Val Cys 
                 5 

 
           
             34  
             9  
             PRT  
             Homo sapiens  
             
               Residues 98-106 of the hepsin protein  
             
           
            34 

Ala Leu Thr His Ser Glu Leu Asp Val 
                 5 

 
           
             35  
             9  
             PRT  
             Homo sapiens  
             
               Residues 211-219 of the hepsin protein  
             
           
            35 

Val Leu Ser Arg Trp Arg Val Phe Ala 
                 5 

 
           
             36  
             9  
             PRT  
             Homo sapiens  
             
               Residues 26-34 of the hepsin protein  
             
           
            36 

Leu Leu Leu Leu Thr Ala Ile Gly Ala 
                 5 

 
           
             37  
             9  
             PRT  
             Homo sapiens  
             
               Residues 284-292 of the hepsin protein  
             
           
            37 

Ala Leu Val Asp Gly Lys Ile Cys Thr 
                 5 

 
           
             38  
             9  
             PRT  
             Homo sapiens  
             
               Residues 145-153 of the hepsin protein  
             
           
            38 

Phe Leu Ala Ala Ile Cys Gln Asp Cys 
                 5 

 
           
             39  
             9  
             PRT  
             Homo sapiens  
             
               Residues 192-200 of the hepsin protein  
             
           
            39 

Leu Leu Ser Gly Asp Trp Val Leu Thr 
                 5 

 
           
             40  
             9  
             PRT  
             Homo sapiens  
             
               Residues 20-28 of the hepsin protein  
             
           
            40 

Ala Leu Thr Ala Gly Thr Leu Leu Leu 
                 5 

 
           
             41  
             9  
             PRT  
             Homo sapiens  
             
               Residues 259-267 of the hepsin protein  
             
           
            41 

Ala Leu Val His Leu Ser Ser Pro Leu 
                 5 

 
           
             42  
             9  
             PRT  
             Homo sapiens  
             
               Residues 277-285 of the hepsin protein  
             
           
            42 

Cys Leu Pro Ala Ala Gly Gln Ala Leu 
                 5 

 
           
             43  
             9  
             PRT  
             Homo sapiens  
             
               Residues 230-238 of the hepsin protein  
             
           
            43 

Leu Gln Leu Gly Val Gln Ala Val Val 
                 5 

 
           
             44  
             9  
             PRT  
             Homo sapiens  
             
               Residues 268-276 of the hepsin protein  
             
           
            44 

Pro Leu Thr Glu Tyr Ile Gln Pro Val 
                 5 

 
           
             45  
             9  
             PRT  
             Homo sapiens  
             
               Residues 31-39 of the hepsin protein  
             
           
            45 

Ala Ile Gly Ala Ala Ser Trp Ala Ile 
                 5 

 
           
             46  
             9  
             PRT  
             Homo sapiens  
             
               Residues 285-293 of the hepsin protein  
             
           
            46 

Leu Val Asp Gly Lys Ile Cys Thr Val 
                 5 

 
           
             47  
             9  
             PRT  
             Homo sapiens  
             
               Residues 27-35 of the hepsin protein  
             
           
            47 

Leu Leu Leu Thr Ala Ile Gly Ala Ala 
                 5 

 
           
             48  
             9  
             PRT  
             Homo sapiens  
             
               Residues 191-199 of the hepsin protein  
             
           
            48 

Ser Leu Leu Ser Gly Asp Trp Val Leu 
                 5 

 
           
             49  
             9  
             PRT  
             Homo sapiens  
             
               Residues 163-171 of the hepsin protein  
             
           
            49 

Ile Val Gly Gly Arg Asp Thr Ser Leu 
                 5 

 
           
             50  
             9  
             PRT  
             Homo sapiens  
             
               Residues 392-400 of the hepsin protein  
             
           
            50 

Lys Val Ser Asp Phe Arg Glu Trp Ile 
                 5 

 
           
             51  
             9  
             PRT  
             Homo sapiens  
             
               Residues 64-72 of the hepsin protein  
             
           
            51 

Met Val Phe Asp Lys Thr Glu Gly Thr 
                 5 

 
           
             52  
             9  
             PRT  
             Homo sapiens  
             
               Residues 236-244 of the hepsin protein  
             
           
            52 

Ala Val Val Tyr His Gly Gly Tyr Leu 
                 5 

 
           
             53  
             9  
             PRT  
             Homo sapiens  
             
               Residues 55-63 of the hepsin protein  
             
           
            53 

Gln Val Ser Ser Ala Asp Ala Arg Leu 
                 5 

 
           
             54  
             9  
             PRT  
             Homo sapiens  
             
               Residues 130-138 of the hepsin protein  
             
           
            54 

Arg Leu Leu Glu Val Ile Ser Val Cys 
                 5 

 
           
             55  
             9  
             PRT  
             Homo sapiens  
             
               Residues 230-238 of the hepsin protein  
             
           
            55 

Leu Gln Leu Gly Val Gln Ala Val Val 
                 5 

 
           
             56  
             9  
             PRT  
             Homo sapiens  
             
               Residues 20-28 of the hepsin protein  
             
           
            56 

Ala Leu Thr Ala Gly Thr Leu Leu Leu 
                 5 

 
           
             57  
             9  
             PRT  
             Homo sapiens  
             
               Residues 259-267 of the hepsin protein  
             
           
            57 

Ala Leu Val His Leu Ser Ser Pro Leu 
                 5 

 
           
             58  
             9  
             PRT  
             Homo sapiens  
             
               Residues 277-285 of the hepsin protein  
             
           
            58 

Cys Leu Pro Ala Ala Gly Gln Ala Leu 
                 5 

 
           
             59  
             9  
             PRT  
             Homo sapiens  
             
               Residues 17-25 of the hepsin protein  
             
           
            59 

Lys Val Ala Ala Leu Thr Ala Gly Thr 
                 5 

 
           
             60  
             9  
             PRT  
             Homo sapiens  
             
               Residues 285-293 of the hepsin protein  
             
           
            60 

Leu Val Asp Gly Lys Ile Cys Thr Val 
                 5 

 
           
             61  
             9  
             PRT  
             Homo sapiens  
             
               Residues 308-316 of the hepsin protein  
             
           
            61 

Val Leu Gln Glu Ala Arg Val Pro Ile 
                 5 

 
           
             62  
             9  
             PRT  
             Homo sapiens  
             
               Residues 27-35 of the hepsin protein  
             
           
            62 

Leu Leu Leu Thr Ala Ile Gly Ala Ala 
                 5 

 
           
             63  
             9  
             PRT  
             Homo sapiens  
             
               Residues 229-237 of the hepsin protein  
             
           
            63 

Gly Leu Gln Leu Gly Val Gln Ala Val 
                 5 

 
           
             64  
             9  
             PRT  
             Homo sapiens  
             
               Residues 313-321 of the hepsin protein  
             
           
            64 

Arg Val Pro Ile Ile Ser Asn Asp Val 
                 5 

 
           
             65  
             9  
             PRT  
             Homo sapiens  
             
               Residues 88-96 of the hepsin protein  
             
           
            65 

Leu Ser Cys Glu Glu Met Gly Phe Leu 
                 5 

 
           
             66  
             9  
             PRT  
             Homo sapiens  
             
               Residues 192-200 of the hepsin protein  
             
           
            66 

Leu Leu Ser Gly Asp Trp Val Leu Thr 
                 5 

 
           
             67  
             9  
             PRT  
             Homo sapiens  
             
               Residues 284-292 of the hepsin protein  
             
           
            67 

Ala Leu Val Asp Gly Lys Ile Cys Thr 
                 5 

 
           
             68  
             9  
             PRT  
             Homo sapiens  
             
               Residues 89-97 of the hepsin protein  
             
           
            68 

Ser Cys Glu Glu Met Gly Phe Leu Arg 
                 5 

 
           
             69  
             9  
             PRT  
             Homo sapiens  
             
               Residues 58-66 of the hepsin protein  
             
           
            69 

Ser Ala Asp Ala Arg Leu Met Val Phe 
                 5 

 
           
             70  
             9  
             PRT  
             Homo sapiens  
             
               Residues 393-401 of the hepsin protein  
             
           
            70 

Val Ser Asp Phe Arg Glu Trp Ile Phe 
                 5 

 
           
             71  
             9  
             PRT  
             Homo sapiens  
             
               Residues 407-415 of the hepsin protein  
             
           
            71 

His Ser Glu Ala Ser Gly Met Val Thr 
                 5 

 
           
             72  
             9  
             PRT  
             Homo sapiens  
             
               Residues 137-145 of the hepsin protein  
             
           
            72 

Val Cys Asp Cys Pro Arg Gly Arg Phe 
                 5 

 
           
             73  
             9  
             PRT  
             Homo sapiens  
             
               Residues 269-277 of the hepsin protein  
             
           
            73 

Leu Thr Glu Tyr Ile Gln Pro Val Cys 
                 5 

 
           
             74  
             9  
             PRT  
             Homo sapiens  
             
               Residues 47-55 of the hepsin protein  
             
           
            74 

Asp Gln Glu Pro Leu Tyr Pro Val Gln 
                 5 

 
           
             75  
             9  
             PRT  
             Homo sapiens  
             
               Residues 119-127 of the hepsin protein  
             
           
            75 

Cys Val Asp Glu Gly Arg Leu Pro His 
                 5 

 
           
             76  
             9  
             PRT  
             Homo sapiens  
             
               Residues 68-76 of the hepsin protein  
             
           
            76 

Lys Thr Glu Gly Thr Trp Arg Leu Leu 
                 5 

 
           
             77  
             9  
             PRT  
             Homo sapiens  
             
               Residues 101-109 of the hepsin protein  
             
           
            77 

His Ser Glu Leu Asp Val Arg Thr Ala 
                 5 

 
           
             78  
             9  
             PRT  
             Homo sapiens  
             
               Residues 250-258 of the hepsin protein  
             
           
            78 

Asn Ser Glu Glu Asn Ser Asn Asp Ile 
                 5 

 
           
             79  
             9  
             PRT  
             Homo sapiens  
             
               Residues 293-301 of the hepsin protein  
             
           
            79 

Val Thr Gly Trp Gly Asn Thr Gln Tyr 
                 5 

 
           
             80  
             9  
             PRT  
             Homo sapiens  
             
               Residues 231-239 of the hepsin protein  
             
           
            80 

Gln Leu Gly Val Gln Ala Val Val Tyr 
                 5 

 
           
             81  
             9  
             PRT  
             Homo sapiens  
             
               Residues 103-111 of the hepsin protein  
             
           
            81 

Glu Leu Asp Val Arg Thr Ala Gly Ala 
                 5 

 
           
             82  
             9  
             PRT  
             Homo sapiens  
             
               Residues 378-386 of the hepsin protein  
             
           
            82 

Gly Thr Gly Cys Ala Leu Ala Gln Lys 
                 5 

 
           
             83  
             9  
             PRT  
             Homo sapiens  
             
               Residues 358-366 of the hepsin protein  
             
           
            83 

Val Cys Glu Asp Ser Ile Ser Arg Thr 
                 5 

 
           
             84  
             9  
             PRT  
             Homo sapiens  
             
               Residues 264-272 of the hepsin protein  
             
           
            84 

Ser Ser Pro Leu Pro Leu Thr Glu Tyr 
                 5 

 
           
             85  
             9  
             PRT  
             Homo sapiens  
             
               Residues 87-95 of the hepsin protein  
             
           
            85 

Gly Leu Ser Cys Glu Glu Met Gly Phe 
                 5 

 
           
             86  
             9  
             PRT  
             Homo sapiens  
             
               Residues 272-280 of the hepsin protein  
             
           
            86 

Tyr Ile Gln Pro Val Cys Leu Pro Ala 
                 5 

 
           
             87  
             9  
             PRT  
             Homo sapiens  
             
               Residues 345-353 of the hepsin protein  
             
           
            87 

Gly Ile Asp Ala Cys Gln Gly Asp Ser 
                 5 

 
           
             88  
             9  
             PRT  
             Homo sapiens  
             
               Residues 301-309 of the hepsin protein  
             
           
            88 

Tyr Tyr Gly Gln Gln Ala Gly Val Leu 
                 5 

 
           
             89  
             9  
             PRT  
             Homo sapiens  
             
               Residues 238-246 of the hepsin protein  
             
           
            89 

Val Tyr His Gly Gly Tyr Leu Pro Phe 
                 5 

 
           
             90  
             9  
             PRT  
             Homo sapiens  
             
               Residues 204-212 of the hepsin protein  
             
           
            90 

Cys Phe Pro Glu Arg Asn Arg Val Leu 
                 5 

 
           
             91  
             9  
             PRT  
             Homo sapiens  
             
               Residues 117-125 of the hepsin protein  
             
           
            91 

Phe Phe Cys Val Asp Glu Gly Arg Leu 
                 5 

 
           
             92  
             9  
             PRT  
             Homo sapiens  
             
               Residues 124-132 of the hepsin protein  
             
           
            92 

Arg Leu Pro His Thr Gln Arg Leu Leu 
                 5 

 
           
             93  
             9  
             PRT  
             Homo sapiens  
             
               Residues 80-88 of the hepsin protein  
             
           
            93 

Arg Ser Asn Ala Arg Val Ala Gly Leu 
                 5 

 
           
             94  
             9  
             PRT  
             Homo sapiens  
             
               Residues 68-76 of the hepsin protein  
             
           
            94 

Lys Thr Glu Gly Thr Trp Arg Leu Leu 
                 5 

 
           
             95  
             9  
             PRT  
             Homo sapiens  
             
               Residues 340-348 of the hepsin protein  
             
           
            95 

Gly Tyr Pro Glu Gly Gly Ile Asp Ala 
                 5 

 
           
             96  
             9  
             PRT  
             Homo sapiens  
             
               Residues 242-250 of the hepsin protein  
             
           
            96 

Gly Tyr Leu Pro Phe Arg Asp Pro Asn 
                 5 

 
           
             97  
             9  
             PRT  
             Homo sapiens  
             
               Residues 51-59 of the hepsin protein  
             
           
            97 

Leu Tyr Pro Val Gln Val Ser Ser Ala 
                 5 

 
           
             98  
             9  
             PRT  
             Homo sapiens  
             
               Residues 259-267 of the hepsin protein  
             
           
            98 

Ala Leu Val His Leu Ser Ser Pro Leu 
                 5 

 
           
             99  
             9  
             PRT  
             Homo sapiens  
             
               Residues 277-285 of the hepsin protein  
             
           
            99 

Cys Leu Pro Ala Ala Gly Gln Ala Leu 
                 5 

 
           
             100  
             9  
             PRT  
             Homo sapiens  
             
               Residues 191-199 of the hepsin protein  
             
           
            100 

Ser Leu Leu Ser Gly Asp Trp Val Leu 
                 5 

 
           
             101  
             9  
             PRT  
             Homo sapiens  
             
               Residues 210-218 of the hepsin protein  
             
           
            101 

Arg Val Leu Ser Arg Trp Arg Val Phe 
                 5 

 
           
             102  
             9  
             PRT  
             Homo sapiens  
             
               Residues 222-230 of the hepsin protein  
             
           
            102 

Val Ala Gln Ala Ser Pro His Gly Leu 
                 5 

 
           
             103  
             9  
             PRT  
             Homo sapiens  
             
               Residues 236-244 of the hepsin protein  
             
           
            103 

Ala Val Val Tyr His Gly Gly Tyr Leu 
                 5 

 
           
             104  
             9  
             PRT  
             Homo sapiens  
             
               Residues 19-27 of the hepsin protein  
             
           
            104 

Ala Ala Leu Thr Ala Gly Thr Leu Leu 
                 5 

 
           
             105  
             9  
             PRT  
             Homo sapiens  
             
               Residues 36-44 of the hepsin protein  
             
           
            105 

Ser Trp Ala Ile Val Ala Val Leu Leu 
                 5 

 
           
             106  
             9  
             PRT  
             Homo sapiens  
             
               Residues 35-43 of the hepsin protein  
             
           
            106 

Ala Ser Trp Ala Ile Val Ala Val Leu 
                 5 

 
           
             107  
             9  
             PRT  
             Homo sapiens  
             
               Residues 300-308 of the hepsin protein  
             
           
            107 

Gln Tyr Tyr Gly Gln Gln Ala Gly Val 
                 5 

 
           
             108  
             9  
             PRT  
             Homo sapiens  
             
               Residues 363-371 of the hepsin protein  
             
           
            108 

Ile Ser Arg Thr Pro Arg Trp Arg Leu 
                 5 

 
           
             109  
             9  
             PRT  
             Homo sapiens  
             
               Residues 366-374 of the hepsin protein  
             
           
            109 

Thr Pro Arg Trp Arg Leu Cys Gly Ile 
                 5 

 
           
             110  
             9  
             PRT  
             Homo sapiens  
             
               Residues 236-244 of the hepsin protein  
             
           
            110 

Ala Val Val Tyr His Gly Gly Tyr Leu 
                 5 

 
           
             111  
             9  
             PRT  
             Homo sapiens  
             
               Residues 13-21 of the hepsin protein  
             
           
            111 

Cys Ser Arg Pro Lys Val Ala Ala Leu 
                 5 

 
           
             112  
             9  
             PRT  
             Homo sapiens  
             
               Residues 179-187 of the hepsin protein  
             
           
            112 

Ser Leu Arg Tyr Asp Gly Ala His Leu 
                 5 

 
           
             113  
             9  
             PRT  
             Homo sapiens  
             
               Residues 43-51 of the hepsin protein  
             
           
            113 

Leu Leu Arg Ser Asp Gln Glu Pro Leu 
                 5 

 
           
             114  
             9  
             PRT  
             Homo sapiens  
             
               Residues 19-27 of the hepsin protein  
             
           
            114 

Ala Ala Leu Thr Ala Gly Thr Leu Leu 
                 5 

 
           
             115  
             9  
             PRT  
             Homo sapiens  
             
               Residues 55-63 of the hepsin protein  
             
           
            115 

Gln Val Ser Ser Ala Asp Ala Arg Leu 
                 5 

 
           
             116  
             9  
             PRT  
             Homo sapiens  
             
               Residues 163-171 of the hepsin protein  
             
           
            116 

Ile Val Gly Gly Arg Asp Thr Ser Leu 
                 5 

 
           
             117  
             9  
             PRT  
             Homo sapiens  
             
               Residues 140-148 of the hepsin protein  
             
           
            117 

Cys Pro Arg Gly Arg Phe Leu Ala Ala 
                 5 

 
           
             118  
             9  
             PRT  
             Homo sapiens  
             
               Residues 20-28 of the hepsin protein  
             
           
            118 

Ala Leu Thr Ala Gly Thr Leu Leu Leu 
                 5 

 
           
             119  
             9  
             PRT  
             Homo sapiens  
             
               Residues 409-417 of the hepsin protein  
             
           
            119 

Glu Ala Ser Gly Met Val Thr Gln Leu 
                 5 

 
           
             120  
             9  
             PRT  
             Homo sapiens  
             
               Residues 259-267 of the hepsin protein  
             
           
            120 

Ala Leu Val His Leu Ser Ser Pro Leu 
                 5 

 
           
             121  
             9  
             PRT  
             Homo sapiens  
             
               Residues 35-43 of the hepsin protein  
             
           
            121 

Ala Ser Trp Ala Ile Val Ala Val Leu 
                 5 

 
           
             122  
             9  
             PRT  
             Homo sapiens  
             
               Residues 184-192 of the hepsin protein  
             
           
            122 

Gly Ala His Leu Cys Gly Gly Ser Leu 
                 5 

 
           
             123  
             9  
             PRT  
             Homo sapiens  
             
               Residues 18-26 of the hepsin protein  
             
           
            123 

Val Ala Ala Leu Thr Ala Gly Thr Leu 
                 5 

 
           
             124  
             9  
             PRT  
             Homo sapiens  
             
               Residues 222-230 of the hepsin protein  
             
           
            124 

Val Ala Gln Ala Ser Pro His Gly Leu 
                 5 

 
           
             125  
             9  
             PRT  
             Homo sapiens  
             
               Residues 224-232 of the hepsin protein  
             
           
            125 

Gln Ala Ser Pro His Gly Leu Gln Leu 
                 5 

 
           
             126  
             9  
             PRT  
             Homo sapiens  
             
               Residues 265-273 of the hepsin protein  
             
           
            126 

Ser Pro Leu Pro Leu Thr Glu Tyr Ile 
                 5 

 
           
             127  
             9  
             PRT  
             Homo sapiens  
             
               Residues 355-363 of the hepsin protein  
             
           
            127 

Gly Pro Phe Val Cys Glu Asp Ser Ile 
                 5 

 
           
             128  
             9  
             PRT  
             Homo sapiens  
             
               Residues 13-21 of the hepsin protein  
             
           
            128 

Cys Ser Arg Pro Lys Val Ala Ala Leu 
                 5 

 
           
             129  
             9  
             PRT  
             Homo sapiens  
             
               Residues 366-374 of the hepsin protein  
             
           
            129 

Thr Pro Arg Trp Arg Leu Cys Gly Ile 
                 5 

 
           
             130  
             9  
             PRT  
             Homo sapiens  
             
               Residues 140-148 of the hepsin protein  
             
           
            130 

Cys Pro Arg Gly Arg Phe Leu Ala Ala 
                 5 

 
           
             131  
             9  
             PRT  
             Homo sapiens  
             
               Residues 152-160 of the hepsin protein  
             
           
            131 

Asp Cys Gly Arg Arg Lys Leu Pro Val 
                 5 

 
           
             132  
             9  
             PRT  
             Homo sapiens  
             
               Residues 363-371 of the hepsin protein  
             
           
            132 

Ile Ser Arg Thr Pro Arg Trp Arg Leu 
                 5 

 
           
             133  
             9  
             PRT  
             Homo sapiens  
             
               Residues 133-141 of the hepsin protein  
             
           
            133 

Ile Val Gly Gly Arg Asp Thr Ser Leu 
                 5 

 
           
             134  
             9  
             PRT  
             Homo sapiens  
             
               Residues 331-339 of the hepsin protein  
             
           
            134 

Gln Ile Lys Pro Lys Met Phe Cys Ala 
                 5 

 
           
             135  
             9  
             PRT  
             Homo sapiens  
             
               Residues 80-88 of the hepsin protein  
             
           
            135 

Arg Ser Asn Ala Arg Val Ala Gly Leu 
                 5 

 
           
             136  
             9  
             PRT  
             Homo sapiens  
             
               Residues 179-187 of the hepsin protein  
             
           
            136 

Ser Leu Arg Tyr Asp Gly Ala His Leu 
                 5 

 
           
             137  
             9  
             PRT  
             Homo sapiens  
             
               Residues 43-51 of the hepsin protein  
             
           
            137 

Leu Leu Arg Ser Asp Gln Glu Pro Leu 
                 5 

 
           
             138  
             9  
             PRT  
             Homo sapiens  
             
               Residues 409-417 of the hepsin protein  
             
           
            138 

Glu Ala Ser Gly Met Val Thr Gln Leu 
                 5 

 
           
             139  
             9  
             PRT  
             Homo sapiens  
             
               Residues 311-319 of the hepsin protein  
             
           
            139 

Glu Ala Arg Val Pro Ile Ile Ser Asn 
                 5 

 
           
             140  
             9  
             PRT  
             Homo sapiens  
             
               Residues 222-230 of the hepsin protein  
             
           
            140 

Val Ala Gln Ala Ser Pro His Gly Leu 
                 5 

 
           
             141  
             9  
             PRT  
             Homo sapiens  
             
               Residues 19-27 of the hepsin protein  
             
           
            141 

Ala Ala Leu Thr Ala Gly Thr Leu Leu 
                 5 

 
           
             142  
             9  
             PRT  
             Homo sapiens  
             
               Residues 18-26 of the hepsin protein  
             
           
            142 

Val Ala Ala Leu Thr Ala Gly Thr Leu 
                 5 

 
           
             143  
             9  
             PRT  
             Homo sapiens  
             
               Residues 184-192 of the hepsin protein  
             
           
            143 

Gly Ala His Leu Cys Gly Gly Ser Leu 
                 5 

 
           
             144  
             9  
             PRT  
             Homo sapiens  
             
               Residues 224-232 of the hepsin protein  
             
           
            144 

Gln Ala Ser Pro His Gly Leu Gln Leu 
                 5 

 
           
             145  
             9  
             PRT  
             Homo sapiens  
             
               Residues 82-90 of the hepsin protein  
             
           
            145 

Asn Ala Arg Val Ala Gly Leu Ser Cys 
                 5 

 
           
             146  
             9  
             PRT  
             Homo sapiens  
             
               Residues 204-212 of the hepsin protein  
             
           
            146 

Cys Phe Pro Glu Arg Asn Arg Val Leu 
                 5 

 
           
             147  
             9  
             PRT  
             Homo sapiens  
             
               Residues 212-220 of the hepsin protein  
             
           
            147 

Leu Ser Arg Trp Arg Val Phe Ala Gly 
                 5 

 
           
             148  
             9  
             PRT  
             Homo sapiens  
             
               Residues 172-180 of the hepsin protein  
             
           
            148 

Gly Arg Trp Pro Trp Gln Val Ser Leu 
                 5 

 
           
             149  
             9  
             PRT  
             Homo sapiens  
             
               Residues 44-52 of the hepsin protein  
             
           
            149 

Leu Arg Ser Asp Gln Glu Pro Leu Tyr 
                 5 

 
           
             150  
             9  
             PRT  
             Homo sapiens  
             
               Residues 155-163 of the hepsin protein  
             
           
            150 

Arg Arg Lys Leu Pro Val Asp Arg Ile 
                 5 

 
           
             151  
             9  
             PRT  
             Homo sapiens  
             
               Residues 213-221 of the hepsin protein  
             
           
            151 

Ser Arg Trp Arg Val Phe Ala Gly Ala 
                 5 

 
           
             152  
             9  
             PRT  
             Homo sapiens  
             
               Residues 166-174 of the hepsin protein  
             
           
            152 

Gly Arg Asp Thr Ser Leu Gly Arg Trp 
                 5 

 
           
             153  
             9  
             PRT  
             Homo sapiens  
             
               Residues 369-377 of the hepsin protein  
             
           
            153 

Trp Arg Leu Cys Gly Ile Val Ser Trp 
                 5 

 
           
             154  
             9  
             PRT  
             Homo sapiens  
             
               Residues 180-188 of the hepsin protein  
             
           
            154 

Leu Arg Tyr Asp Gly Ala His Leu Cys 
                 5 

 
           
             155  
             9  
             PRT  
             Homo sapiens  
             
               Residues 96-104 of the hepsin protein  
             
           
            155 

Leu Arg Ala Leu Thr His Ser Glu Leu 
                 5 

 
           
             156  
             9  
             PRT  
             Homo sapiens  
             
               Residues 396-404 of the hepsin protein  
             
           
            156 

Phe Arg Glu Trp Ile Phe Gln Ala Ile 
                 5 

 
           
             157  
             9  
             PRT  
             Homo sapiens  
             
               Residues 123-131 of the hepsin protein  
             
           
            157 

Gly Arg Leu Pro His Thr Gln Arg Leu 
                 5 

 
           
             158  
             9  
             PRT  
             Homo sapiens  
             
               Residues 207-215 of the hepsin protein  
             
           
            158 

Glu Arg Asn Arg Val Leu Ser Arg Trp 
                 5 

 
           
             159  
             9  
             PRT  
             Homo sapiens  
             
               Residues 209-217 of the hepsin protein  
             
           
            159 

Asn Arg Val Leu Ser Arg Trp Arg Val 
                 5 

 
           
             160  
             9  
             PRT  
             Homo sapiens  
             
               Residues 14-22 of the hepsin protein  
             
           
            160 

Ser Arg Pro Lys Val Ala Ala Leu Thr 
                 5 

 
           
             161  
             9  
             PRT  
             Homo sapiens  
             
               Residues 106-114 of the hepsin protein  
             
           
            161 

Val Arg Thr Ala Gly Ala Asn Gly Thr 
                 5 

 
           
             162  
             9  
             PRT  
             Homo sapiens  
             
               Residues 129-137 of the hepsin protein  
             
           
            162 

Gln Arg Leu Leu Glu Val Ile Ser Val 
                 5 

 
           
             163  
             9  
             PRT  
             Homo sapiens  
             
               Residues 349-357 of the hepsin protein  
             
           
            163 

Cys Gln Gly Asp Ser Gly Gly Pro Phe 
                 5 

 
           
             164  
             9  
             PRT  
             Homo sapiens  
             
               Residues 61-69 of the hepsin protein  
             
           
            164 

Ala Arg Leu Met Val Phe Asp Lys Thr 
                 5 

 
           
             165  
             9  
             PRT  
             Homo sapiens  
             
               Residues 215-223 of the hepsin protein  
             
           
            165 

Trp Arg Val Phe Ala Gly Ala Val Ala 
                 5 

 
           
             166  
             9  
             PRT  
             Homo sapiens  
             
               Residues 143-151 of the hepsin protein  
             
           
            166 

Gly Arg Phe Leu Ala Ala Ile Cys Gln 
                 5 

 
           
             167  
             9  
             PRT  
             Homo sapiens  
             
               Residues 246-254 of the hepsin protein  
             
           
            167 

Phe Arg Asp Pro Asn Ser Glu Glu Asn 
                 5 

 
           
             168  
             9  
             PRT  
             Homo sapiens  
             
               Residues 132-140 of the hepsin protein  
             
           
            168 

Leu Glu Val Ile Ser Val Cys Asp Cys 
                 5 

 
           
             169  
             9  
             PRT  
             Homo sapiens  
             
               Residues 91-99 of the hepsin protein  
             
           
            169 

Glu Glu Met Gly Phe Leu Arg Ala Leu 
                 5 

 
           
             170  
             9  
             PRT  
             Homo sapiens  
             
               Residues 264-272 of the hepsin protein  
             
           
            170 

Ser Ser Pro Leu Pro Leu Thr Glu Tyr 
                 5 

 
           
             171  
             9  
             PRT  
             Homo sapiens  
             
               Residues 310-318 of the hepsin protein  
             
           
            171 

Gln Glu Ala Arg Val Pro Ile Ile Ser 
                 5 

 
           
             172  
             9  
             PRT  
             Homo sapiens  
             
               Residues 319-327 of the hepsin protein  
             
           
            172 

Asn Asp Val Cys Asn Gly Ala Asp Phe 
                 5 

 
           
             173  
             9  
             PRT  
             Homo sapiens  
             
               Residues 4-12 of the hepsin protein  
             
           
            173 

Lys Glu Gly Gly Arg Thr Val Pro Cys 
                 5 

 
           
             174  
             9  
             PRT  
             Homo sapiens  
             
               Residues 251-259 of the hepsin protein  
             
           
            174 

Ser Glu Glu Asn Ser Asn Asp Ile Ala 
                 5 

 
           
             175  
             9  
             PRT  
             Homo sapiens  
             
               Residues 256-264 of the hepsin protein  
             
           
            175 

Asn Asp Ile Ala Leu Val His Leu Ser 
                 5 

 
           
             176  
             9  
             PRT  
             Homo sapiens  
             
               Residues 294-302 of the hepsin protein  
             
           
            176 

Thr Gly Trp Gly Asn Thr Gln Tyr Tyr 
                 5 

 
           
             177  
             9  
             PRT  
             Homo sapiens  
             
               Residues 361-369 of the hepsin protein  
             
           
            177 

Asp Ser Ile Ser Arg Thr Pro Arg Trp 
                 5 

 
           
             178  
             9  
             PRT  
             Homo sapiens  
             
               Residues 235-243 of the hepsin protein  
             
           
            178 

Gln Ala Val Val Tyr His Gly Gly Tyr 
                 5 

 
           
             179  
             9  
             PRT  
             Homo sapiens  
             
               Residues 109-117 of the hepsin protein  
             
           
            179 

Ala Gly Ala Asn Gly Thr Ser Gly Phe 
                 5 

 
           
             180  
             9  
             PRT  
             Homo sapiens  
             
               Residues 270-278 of the hepsin protein  
             
           
            180 

Thr Glu Tyr Ile Gln Pro Val Cys Leu 
                 5 

 
           
             181  
             9  
             PRT  
             Homo sapiens  
             
               Residues 174-182 of the hepsin protein  
             
           
            181 

Trp Pro Trp Gln Val Ser Leu Arg Tyr 

 
           
             182  
             9  
             PRT  
             Homo sapiens  
             
               Residues 293-301 of the hepsin protein  
             
           
            182 

Val Thr Gly Trp Gly Asn Thr Gln Tyr 
                 5 

 
           
             183  
             9  
             PRT  
             Homo sapiens  
             
               Residues 69-77 of the hepsin protein  
             
           
            183 

Thr Glu Gly Thr Trp Arg Leu Leu Cys 
                 5 

 
           
             184  
             9  
             PRT  
             Homo sapiens  
             
               Residues 90-98 of the hepsin protein  
             
           
            184 

Cys Glu Glu Met Gly Phe Leu Arg Ala 
                 5 

 
           
             185  
             9  
             PRT  
             Homo sapiens  
             
               Residues 252-260 of the hepsin protein  
             
           
            185 

Glu Glu Asn Ser Asn Asp Ile Ala Leu 
                 5 

 
           
             186  
             9  
             PRT  
             Homo sapiens  
             
               Residues 48-56 of the hepsin protein  
             
           
            186 

Gln Glu Pro Leu Tyr Pro Val Gln Val 
                 5 

 
           
             187  
             9  
             PRT  
             Homo sapiens  
             
               Residues 102-110 of the hepsin protein  
             
           
            187 

Ser Glu Leu Asp Val Arg Thr Ala Gly 
                 5 

 
           
             188  
             1783  
             DNA  
             Homo sapiens  
             
               full length cDNA of hepsin  
             
           
            188 

tcgagcccgc tttccaggga ccctacctga gggcccacag gtgaggcagc                50 

ctggcctagc aggccccacg ccaccgcctc tgcctccagg ccgcccgctg               100 

ctgcggggcc accatgctcc tgcccaggcc tggagactga cccgaccccg               150 

gcactacctc gaggctccgc ccccacctgc tggaccccag ggtcccaccc               200 

tggcccagga ggtcagccag ggaatcatta acaagaggca gtgacatggc               250 

gcagaaggag ggtggccgga ctgtgccatg ctgctccaga cccaaggtgg               300 

cagctctcac tgcggggacc ctgctacttc tgacagccat cggggcggca               350 

tcctgggcca ttgtggctgt tctcctcagg agtgaccagg agccgctgta               400 

cccagtgcag gtcagctctg cggacgctcg gctcatggtc tttgacaaga               450 

cggaagggac gtggcggctg ctgtgctcct cgcgctccaa cgccagggta               500 

gccggactca gctgcgagga gatgggcttc ctcagggcac tgacccactc               550 

cgagctggac gtgcgaacgg cgggcgccaa tggcacgtcg ggcttcttct               600 

gtgtggacga ggggaggctg ccccacaccc agaggctgct ggaggtcatc               650 

tccgtgtgtg attgccccag aggccgtttc ttggccgcca tctgccaaga               700 

ctgtggccgc aggaagctgc ccgtggaccg catcgtggga ggccgggaca               750 

ccagcttggg ccggtggccg tggcaagtca gccttcgcta tgatggagca               800 

cacctctgtg ggggatccct gctctccggg gactgggtgc tgacagccgc               850 

ccactgcttc ccggagcgga accgggtcct gtcccgatgg cgagtgtttg               900 

ccggtgccgt ggcccaggcc tctccccacg gtctgcagct gggggtgcag               950 

gctgtggtct accacggggg ctatcttccc tttcgggacc ccaacagcga              1000 

ggagaacagc aacgatattg ccctggtcca cctctccagt cccctgcccc              1050 

tcacagaata catccagcct gtgtgcctcc cagctgccgg ccaggccctg              1100 

gtggatggca agatctgtac cgtgacgggc tggggcaaca cgcagtacta              1150 

tggccaacag gccggggtac tccaggaggc tcgagtcccc ataatcagca              1200 

atgatgtctg caatggcgct gacttctatg gaaaccagat caagcccaag              1250 

atgttctgtg ctggctaccc cgagggtggc attgatgcct gccagggcga              1300 

cagcggtggt ccctttgtgt gtgaggacag catctctcgg acgccacgtt              1350 

ggcggctgtg tggcattgtg agttggggca ctggctgtgc cctggcccag              1400 

aagccaggcg tctacaccaa agtcagtgac ttccgggagt ggatcttcca              1450 

ggccataaag actcactccg aagccagcgg catggtgacc cagctctgac              1500 

cggtggcttc tcgctgcgca gcctccaggg cccgaggtga tcccggtggt              1550 

gggatccacg ctgggccgag gatgggacgt ttttcttctt gggcccggtc              1600 

cacaggtcca aggacaccct ccctccaggg tcctctcttc cacagtggcg              1650 

ggcccactca gccccgagac cacccaacct caccctcctg acccccatgt              1700 

aaatattgtt ctgctgtctg ggactcctgt ctaggtgccc ctgatgatgg              1750 

gatgctcttt aaataataaa gatggttttg att                                1783