# Patent

## Title
Methods for the early diagnosis of ovarian cancer

## Abstract

The present invention discloses the protease hepsin is specifically over-expressed in ovarian and other malignancies. A number of hepsin peptides can induce immune responses to hepsin, thereby demonstrating the potential of these peptides in monitoring and the development of immunotherapies for ovarian and other malignancies. There is also provided a hepsin protein variant that is useful as a marker for ovarian cancer cells, prostate cancer cells or kidney cancer cells.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a continuation-in-part application which claims the benefit of priority under 35 U.S.C. §120 of U.S. Ser. No. 10/135,795, filed Apr. 30, 2002, which is a continuation-in-part application which claims the benefit of priority under 35 U.S.C. §120 of U.S. Ser. No. 10/102,283, filed Mar. 20, 2002, now U.S. Pat. No. 6,875,609, which is a continuation-in-part application of U.S. Ser. No. 09/919,048, filed Jul. 30, 2001, now U.S. Pat. No. 6,787,354, which is a continuation-in-part application of Ser. No. 09/861,966 filed May 21, 2001, now U.S. Pat. No. 6,518,028, which is a divisional application of Ser. No. 09/510,738 filed Feb. 22, 2000, now 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
1. Field of the Invention
Generally, the present invention relates to the fields of molecular biology and medicine. More specifically, the present invention is in the field of cancer research, especially ovarian cancer diagnosis.
2. Background of the Invention
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.
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





uPA, Urokinase-type plasminogen activator;


tPA, Tissue-type plasminogen activator;


PAI-I, Plasminogen activator 0 inhibitors;


PAI-2, Plasminogen activator inhibitors;


NES-1, Normal epithelial cell-specific-1;


MMP, Matrix P metallo-protease.


*Overexpressed in gastrointestinal ulcers.






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. (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. (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.
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.
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
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 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.
The present invention provides methods of vaccinating an individual against hepsin or produce immune-activated cells directed toward hepsin by inoculating an individual with an expression vector encoding a hepsin protein or a fragment thereof.
The present invention also provides methods of immunotherapy targeted toward hepsin in an individual, involving the steps of generating dendritic cells in vitro from peripheral blood drawn from an individual, loading these dendritic cells with hepsin protein or a fragment thereof, then transferring these dendritic cells back to the individual in single or multiple doses. Hepsin-loaded or hepsin-expressing dendritic cells can also be used to stimulate hepsin-specific T cell responses in vitro, followed by adoptive immunotherapy in which the individual is given autologous hepsin-specific T cells.
There is also provided a method of monitoring the efficacy of vaccinating an individual with hepsin or hepsin peptide. The method comprises measuring immune responses in response to said hepsin or hepsin peptide, wherein induction of immune responses to said hepsin or hepsin peptide indicates that said individual has been vaccinated by said hepsin or hepsin peptide.
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.
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.
In still yet 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.
In another aspect of the present invention, there is provided a hepsin protein variant or a fragment thereof that is useful as a marker for ovarian cancer cells, prostate cancer cells or kidney cancer cells.
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
FIG. 1 shows agarose gel comparison of PCR products derived from normal and carcinoma cDNA.
FIG. 2 shows Northern blot analysis of ovarian tumors using hepsin, SCCE, PUMP-1, TADG-14 and β-tubulin probes.
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).
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).
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).
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).
FIG. 7 shows hepsin expression levels in normal, low malignant potential tumors, and ovarian carcinomas. S=serious, M=mucinous, LMP=low malignant potential.
FIG. 8 shows serine protease stratum corneum chymotrypsin enzyme (SCCE) expression in normal, low malignant potential tumors, and ovarian carcinomas.
FIG. 9 shows metallo-protease PUMP-1 (MMP-7) gene expression in normal (lanes 1-2) and ovarian carcinomas tissue (Lanes 3-10).
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.
FIG. 11A 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<0.005) and carcinomas (p<0.0001) compared to levels in normal ovary. All 10 cases of normal ovaries showed a relatively low level of hepsin mRNA expression.
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.
FIG. 13A 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 β-tubulin in 10 normal and 44 ovarian carcinoma tissues.
FIG. 14 shows a comparison by quantitative PCR of normal and ovarian carcinoma expression of mRNA for protease M.
FIG. 15 shows the TADG-12 catalytic domain including an insert near the His 5′-end.
FIG. 16A shows northern blot analysis comparing TADG-14 expression in normal and ovarian carcinoma tissues. FIG. 16B shows preliminary quantitative PCR amplification of normal and carcinoma cDNAs using specific primers for TADG-14.
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.
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.
FIG. 19 shows a comparison of PCR amplified products for the hepsin, SCCE, protease M, PUMP-1 and Cathepsin L genes.
FIG. 20 shows CD8+ CTL recognition of hepsin 170-178 peptide in a 5 hr 51Cr release assay. Targets were LCL loaded with hepsin 170-178 (closed circles) and control LCL (open circles).
FIG. 21 shows CD8+ CTL recognition of hepsin 172-180 peptide in a 5 hr 51Cr release assay. Targets were LCL loaded with hepsin 172-180 (closed circles) and control LCL (open circles).
FIG. 22 shows CD8+ CTL recognition of hepsin 42-51 peptide in a 5 hr 51Cr release assay. Targets were LCL loaded with hepsin 42-51 (squares), control LCL (triangles) and K562 cells (diamonds).
FIG. 23 shows CD8+ CTL recognition of hepsin 284-293 peptide in a 5 hr 51Cr release assay. Targets were LCL loaded with hepsin 284-293 (closed circles) and control LCL (open circles).
FIG. 24 shows CD8+ CTL recognition of hepsin 308-317 peptide in a 5 hr 51Cr release assay. Targets cells were LCL loaded with or without hepsin 308-317.
FIG. 25 shows hepsin-specific CD4+ T cell and CD8+ T cell proliferative responses stimulated by full length hepsin protein. Solid histograms represent the stimulation index for T cells stimulated with hepsin-loaded dendritic cells, and open histograms represent the stimulation index for T cells stimulated with control dendritic cells.
FIG. 26 shows peptide-specific CD8+ CTL recognition of endogenously processed and presented hepsin tumor antigen. CTL were derived by stimulation with dendritic cells pulsed with hepsin peptide 170-178. Cytotoxicity was tested in a standard 5 hours 51Cr-release assay against autologous macrophages infected with Ad-GFP/hepsin (diamonds), macrophages infected with Ad-GFP-SCCE (diamonds), macrophages pulsed with hepsin 170-178 peptide (squares), or control untreated macrophages (filled circles).
FIG. 27 shows hepsin and hepsin protein variant expression in ovarian carcinoma and prostate carcinoma.
FIG. 28 shows PCR analysis of hepsin protein variant in prostate carcinoma.
FIG. 29 shows a diagram of transcript and open reading frame of hepsin and the hepsin intron 12 variant.
FIG. 30 shows amino acid sequence comparison of hepsin and hepsin protein variant.



DETAILED DESCRIPTION OF THE INVENTION
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 for the design of new drugs.
The present invention is directed toward a method of vaccinating an individual against hepsin, comprising the steps of inoculating an individual with an expression vector encoding a hepsin peptide or with peptide-loaded dendritic cells. Expression of the hepsin peptide elicits an immune response in the individual, thereby vaccinating the individual against hepsin. Generally, this method is applicable when the individual has cancer or is at risk of getting a cancer such as ovarian cancer, lung cancer, prostate cancer and colon cancer. Sequences of preferred hepsin peptides are shown in SEQ ID Nos. 28, 29, 30, 31, 88, 89, 108, 109, 128, 129, 148, 149, 150, 151, 152, 153, 154, 189, 190 and 191.
The present invention also provides a method of producing immune-activated cells directed toward hepsin, comprising the steps of exposing immune cells to hepsin protein or fragment thereof. 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 fragment is SEQ ID Nos. 28, 29, 30, 31, 88, 89, 108, 109, 128, 129, 148, 149, 150, 151, 152, 153, 154, 189, 190 or 191. 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 or is at risk of getting a cancer such as ovarian cancer, lung cancer, prostate cancer and colon cancer.
The present invention also provides methods of immunotherapy targeted toward hepsin in an individual. In one embodiment, the method involves generating dendritic cells in vitro from peripheral blood drawn from the individual, loading these dendritic cells with hepsin protein or a fragment thereof by lipofection or other means, then transferring these dendritic cells back to the individual in single or multiple doses. Hepsin may also be expressed in these dendritic cells following transduction with a recombinant DNA vector. Alternatively, hepsin-loaded or hepsin-expressing dendritic cells can be used to stimulate hepsin-specific T cell responses in vitro, followed by adoptive immunotherapy in which the individual is given autologous hepsin-specific T cells. Typically, the individual has cancer or is at risk of getting a cancer such as ovarian cancer, lung cancer, prostate cancer and colon cancer. In general, a full length or a fragment of hepsin protein is expressed in the isolated dendritic cells. Preferably, the fragment is a 9-residue fragment up to a 20-residue fragment, and more preferably, the fragment is SEQ ID Nos. 28, 29, 30, 31, 88, 89, 108, 109, 128, 129, 148, 149, 150, 151, 152, 153, 154, 189, 190 or 191.
There is also provided a method of monitoring the efficacy of vaccinating an individual with hepsin or hepsin peptide such as those shown in SEQ ID Nos. 28, 29, 30, 31, 88, 89, 108, 109, 128, 129, 148, 149, 150, 151, 152, 153, 154, 189, 190 or 191. The method comprises isolating T cells or CD8+ T cells from the vaccinated individual and measuring immune responses specific to said hepsin or hepsin peptide. An increased level of immune responses compared to those exhibited by cells from normal individual indicates that said individual has been vaccinated by said hepsin or hepsin peptide. In general, the individual is vaccinated to hepsin if there is an increased level of hepsin-specific T cells proliferation, an increased frequency of hepsin-specific T cells or an increased frequency of hepsin-specific cytokine-secreting T cells. Standard assays well-known in the art such as tetramer analysis and ELISPOT assay can be used to determine the frequency of hepsin-specific T cells and the frequency of hepsin-specific cytokine-secreting T cells respectively.
In another aspect of the present invention, there is provided a method of inhibiting expression of hepsin in a cell, comprising the step of introducing into a cell a vector comprises a sequence complementary to SEQ ID No. 188, wherein expression of the vector produces hepsin antisense RNA in the cell. The hepsin antisense RNA hybridizes to endogenous hepsin mRNA, thereby inhibiting expression of hepsin in the cell.
Expression of hepsin can also be inhibited by antibody. An antibody specific for a hepsin protein or a fragment thereof is introduced into a cell, and binding of the antibody to hepsin would inhibit the hepsin protein. Preferably, the hepsin fragment is a 9-residue fragment up to a 20-residue fragment, and more preferably, the fragment is SEQ ID Nos. 28, 29, 30, 31, 88, 89, 108, 109, 128, 129, 148, 149, 150, 151, 152, 153, 154, 189, 190 or 191.
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.
The present invention is further directed toward an immunogenic composition, comprising an appropriate adjuvant and an immunogenic full length hepsin protein or a fragment thereof. Preferably, the fragment is a 9-residue fragment up to a 20-residue fragment, and more preferably, the fragment is SEQ ID Nos. 28, 29, 30, 31, 88, 89, 108, 109, 128, 129, 148, 149, 150, 151, 152, 153, 154, 189, 190 or 191.
The present invention also provides an oligonucleotide having a sequence complementary to SEQ ID No. 188 or a fragment 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.
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.
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 & 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 & S. J. Higgins eds. 1985); “Transcription and Translation” (B. D. Hames & 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.
As used herein, the term “cDNA” shall refer to the DNA copy of the mRNA transcript of a gene.
The present invention comprises a vector comprising a DNA sequence which encodes a hepsin protein or a fragment thereof, 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 RNA. Furthermore, the vectors may express nucleic acid encoding a fusion protein comprising an immunologically active component and a hepsin protein or a fragment thereof. These vectors would be useful in methods of vaccination against hepsin in an individual.
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 that 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.
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 an 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.
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 an 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 or a fragment thereof.
The present invention is also directed to an isolated DNA encoding a hepsin variant, said hepsin variant comprises the amino acid sequence of SEQ ID NO. 195 or a fragment thereof. In a related embodiment, an isolated and purified hepsin variant protein in which the hepsin variant comprises the amino acid sequence of SEQ ID NO. 195 or a fragment thereof. The present invention is also directed to a method of detecting tumor cells in a sample, comprising the step of detecting the expression of a hepsin protein variant that comprises the amino acid sequence of SEQ ID NO. 195 or a fragment thereof, wherein the presence of said hepsin variant in said sample indicates that said sample contains tumor cells. Generally, the method of claim 1, wherein said detection of hepsin variant expression or said or a fragment thereof is performed at DNA or protein level. Representative tumor cells include ovarian cancer cells, prostate cancer cells and kidney cancer cells.
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).
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.
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 as 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 that 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.
In addition to substantially full-length proteins, the invention also includes fragments (e.g., antigenic fragments) of the hepsin or hepsin variant proteins. 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.
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.
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 well-known in the art. Examples of suitable toxins include diphtheria toxin, Pseudomonas exotoxin A, ricin, and cholera toxin. Examples of suitable enzyme labels include alkaline phosphatase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, etc. Examples of suitable radioisotopic labels include 3H, 125I, 131I, 32P, 35S, 14C, etc.
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.
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.
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. 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. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.
EXAMPLE 1
Amplification of Serine Proteases Using Redundant and Specific Primers
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 & aspartate) and antisense (aspartate and serine) redundant primers are shown in Table 2.
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.
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 WIZARD MINIPREP DNA purification system (Promega), a kit used to rapidly isolate plasmid DNA. Inserts were sequenced using Prism Ready Dydeoxy Terminators cycle sequencing kit (Applied Biosystems). Residual dye terminators were removed from the completed sequencing reaction using CENTRISEP SPIN column (Princeton Separation), a column used for separating large molecules from small molecules, 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 2







SEQ



PCR Primers
5′→3′
ID 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
 9


 


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
ctctttggcttgtacttgct
23


(Ser10-as)


 


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
















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*





*indicates novel proteases






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.
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).
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 2
Northern Blots Analysis
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.
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.
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.
Northern blot analysis was performed as follows: 10 μg of mRNA was loaded onto a 1% formaldehyde-agarose gel, electrophoresed and blotted onto HYBOND-N+ nylon membrane (Amersham), a positively charged nylon membrane. 32P-labeled cDNA probes were made using PRIME-A-GENE LABELING SYSTEM (Promega), a kit used for random-primed labeling of linear template DNA with radionucleotides. 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 32P-labeled cDNA probe in EXPRESSHYB Hybridization Solution (CLONTECH), a hybridization solution used to hybridize blots with the labeled cDNA probe. Control hybridization to determine relative gel loading was accomplished using the β-tubulin.
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.
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 3
PCR Products Corresponding to Serine, Cysteine and Metallo-Proteases
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 & 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., (1989).
EXAMPLE 4
Serine Proteases
For the serine protease group, using the histidine domain primer sense, S1, in combination with antisense primer AS2, the following proteases were identified:

    
    
        (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);
        (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 Ca2+ phospholipid and factor VIIIa e5; and
        (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 5
Cysteine Proteases
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 6
Metallo-Protease
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 7
Expression of Hepsin
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. (1990). The following oligonucleotide primers were used:

    
    
        hepsin forward 5′-TGTCCCGATGGCGAGTGTTT-3′ (SEQ ID No. 8), and hepsin reverse 5′-CCTGTTGGCCATAGTACTGC-3′ (SEQ ID No. 9); β-tubulin forward 5′-TGCATTGACAACGAGGC-3′ (SEQ ID No. 18), and β-tubulin reverse 5′-CTGTCTTGA CATTGTTG-3′ (SEQ ID No. 19).
    
    


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. (1988) for hepsin, and Hall et al. (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-32PdCTP 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 PhosphorImagerTM (Molecular Dynamics). Student's t test was used for comparison of mean values.
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).
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 32P-hepsin band to the internal control, the 32P-β-tubulin band.
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<0.005) and carcinomas (p<0.0001) compared to levels in normal ovary. All 10 cases of normal ovaries showed a relatively low level of hepsin mRNA expression.
Hepsin mRNA is highly overexpressed in most histopathologic types of ovarian carcinomas including some low malignant potential tumors (see FIGS. 11A & 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.
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.
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.







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





*Other category includes the following tumor types: Brenner's tumor, thecoma, teratoma, fibrothecoma, fibroma, granulosa cell, clear cell, germ cell, mixed mullerian, stromal, undifferentiated, and dysgerminoma.






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 & 7).







TABLE 5







Patient Characteristics and Expression of Hepsin Gene















mRNA expression


Case
Histological typea
Stage/Grade
INb
of hepsinc














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+





aS, serous; M, mucinous; E, endometrioid; C, clear cell;


bLN, lymph node metastasis; P, positive; N, negative; NE, not examined;


cn, normal range = mean ± 2SD; 2+, mean ± 2SD to ± 4SD; 4+, mean ± 4SD or greater.













TABLE 6







Overexpression of hepsin in normal ovaries and ovarian tumors














Hepsin
Ratio of 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 8
Expression of SCCE and PUMP-1
Studies using both SCCE-specific primers (FIG. 8) and PUMP-specific primers (FIG. 9) indicate overexpression of these proteases in ovarian carcinomas.
EXAMPLE 9
Summary of Proteases Detected Herein
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.
Stratum Corneum Chymotrypsin Enzyme (SCCE)
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.
Northern blots were probed with the PCR product to determine expression of stratum corneum chymotrypsin enzyme in fetal tissue and ovarian carcinoma (FIGS. 12A & 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.
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).
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.
Protease M
Protease M was identified from subclones of the His—ser primer pair. This protease was first cloned by Anisowicz et al. (1996) and shown to be overexpressed in carcinomas. A preliminary evaluation indicates that this enzyme is overexpressed in ovarian carcinoma (FIG. 14).
Cofactor I and Complement Factor B
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.
TADG-12
TADG-12 was identified from the primer pairs, sense-His/antisense-Asp (see FIG. 1, Lanes 1 & 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.
TADG-13 and TADG-14
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.
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.
PUMP-1
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.
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 & 18B). PUMP-1, however, is so far the protease most frequently found overexpressed in mucinous tumors (See Table 7).
Cathepsin-L
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.
SUMMARY
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.
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.
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'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.







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)









EXAMPLE 10
Hepsin Peptide Ranking
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'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


& Ranking
Start
Peptide
Dissociation1/2
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 1
34.154
 31


 


 5
308
VLQEARVPI 7
2.717
 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
QIKPKMFCA
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
WRVFAGAVA
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 11
Hepsin Peptides as Target Epitopes for Human CD8+ Cytotoxic T Cells
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.
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).
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.
Monocyte-derived DC 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). After 5-6 days, DC maturation was induced by addition of PGE2, IL-1β and TNFα for a further 48 h.
Mature DC were loaded with peptide (2×106 DC 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 1×106/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 DC or PBMC 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 2nd or 3rd antigen stimulation.
Peptide-specific cytotoxicity was tested in standard 5-6 h microwell 51Cr-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 51Cr-labeled (50 μCi in 200-300 μl, 1 h at 37° C.). Peptide-loaded 51Cr-labeled LCL were incubated with CD8+ T cells at effector-target ration between 10:1 and 1.25:1. Cytotoxicity was recorded as percentage 51Cr released into culture supernatants.
Hepsin Peptide 170-178
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 LCL, but did not kill control, peptide-free LCL (FIG. 20). Heterologous HLA A2.1-expressing peptide-loaded LCL 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 LCL could be blocked with MAb 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.
Hepsin Peptide 172-180
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+ CTL specific for hepsin 172-180 could kill peptide-loaded, HLA B27-expressing autologous and heterologous LCL, but failed to recognize heterologous peptide-loaded LCL that did not express HLA B27, or peptide-free control LCL (FIG. 21). Natural killer-sensitive K562 cells were not lysed. Cytotoxicity against hepsin 172-180 loaded LCL could be blocked with MAb specific for a non-polymorphic HLA class I determinant, confirming that lysis was HLA class I-restricted.
Hepsin Peptide 42-51
Hepsin peptide 42-51 (SEQ ID No. 189) was predicted by computer analysis to bind HLA A*0201. CD8+ CTL specific for hepsin 42-51 killed peptide-loaded autologous LCL, but did not kill control, peptide-free LCL (FIG. 22). Heterologous HLA A*0201-expressing peptide-loaded LCL were efficiently killed, but targets lacking HLA A*0201 were not killed. Natural killer-sensitive K562 cells were not lysed. Cytotoxicity against hepsin 42-51 loaded LCL could be blocked with MAb 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.
Hepsin Peptide 284-293
Hepsin peptide 284-293 (SEQ ID No. 190) was predicted by computer analysis to bind HLA A*0201. CD8+CTL specific for hepsin 284-293 killed peptide-loaded autologous LCL, but did not kill control, peptide-free LCL (FIG. 23). Heterologous HLA A*0201-expressing peptide-loaded LCL were efficiently killed, but targets lacking HLA A*0201 were not killed. Natural killer-sensitive K562 cells were not lysed. Cytotoxicity against hepsin 284-293 loaded LCL could be blocked with MAb specific for a non-polymorphic HLA class I determinant, confirming that lysis was HLA class I-restricted.
Hepsin Peptide 308-317
Hepsin peptide 308-317 (SEQ ID No. 191) was predicted by computer analysis to bind HLA A*0201. CD8+ CTL specific for hepsin 308-317 killed peptide-loaded autologous LCL, but did not kill control, peptide-free LCL (FIG. 24). Heterologous HLA A*0201-expressing peptide-loaded LCL were efficiently killed, but targets lacking HLA A*0201 were not killed. Cytotoxicity against hepsin 308-317 loaded LCL could be blocked with MAb specific for a non-polymorphic HLA class I determinant, confirming that lysis was HLA class I-restricted.
EXAMPLE 12
Recombinant Full Length Hepsin Induces CD4+ and CD8+ T Cell Proliferative Responses
The following example shows that dendritic cells loaded with full-length recombinant hepsin are capable of inducing both CD4+ T cell and CD8+ T cell proliferative responses to hepsin.
Results disclosed above that show dendritic cells (DC) loaded with hepsin-derived peptides can efficiently stimulate HLA A2.1-restricted and HLA B27-restricted CD8+ CTL responses in normal adults suggest that hepsin may be a leading candidate as a target for dendritic cell-based immunotherapy of ovarian cancer. Furthermore, the utility of hepsin as a target antigen for immunotherapeutic purposes may not be confined to ovarian cancer. A recent series of gene expression profiling studies identified hepsin as a major tumor marker for prostate cancer. Hepsin was consistently highly expressed in prostate cancer, but not in benign prostatic hyperplasia (Luo et al., (2001); Magee et al., (2001); Welsh et al., (2001); Dhanasekaran et al., (2001) and Stamey et al., (2001)). These reports strongly support the proposal that hepsin may also be a leading target for dendritic cell-based immunotherapy of prostate cancer.
However, the use of peptides restricts the response to predetermined HLA class I types, which imposes limitations on patient selection. The use of dendritic cells loaded with full-length recombinant tumor antigens circumvents this problem, and also offers the prospect of being able to induce both CD8+ T cell responses and helper CD4+ T cell responses, the latter of which may play a critical role in the generation and maintenance of effective anti-tumor immunity. A further, potentially critical, advantage of using full-length tumor antigen is that CD8+ T cell responses are induced against naturally processed epitopes, which markedly increases the likelihood that CD8+ T cells will recognize endogenously synthesized antigens that are also naturally processed and presented by the target ovarian tumor cell.
Hepsin cDNA was cloned into the IPTG-inducible pQE-30 vector (Qiagen) and expressed in E. coli. Addition of a 6×-histidine tag on the amino terminus facilitates affinity purification with Ni-NTA resin. Dendritic cells were derived from peripheral blood monocyte precursors as described above. Mature dendritic cells express high levels of HLA class I and class II molecules, costimulatory molecules (e.g., CD86 and CD40), and CD83 (expressed on mature, but not immature, monocyte-derived dendritic cells), but do not express CD14 (a macrophage/monocyte marker).
To stimulate hepsin-specific T cell proliferation, mature dendritic cells were loaded with purified recombinant hepsin by DOTAP lipofection. Briefly 25 μg hepsin was combined with 15 μg DOTAP (Roche Applied Science, Indianapolis, Ind.) in 500 μl AIM-V medium (Invitrogen, Grand Island, N.Y.). This mixture was incubated with 1-2×106 dendritic cells for up to 2 hours at 37° C. Hepsin-loaded dendritic cells were cocultured with autologous peripheral blood lymphocytes from a normal male donor at a responder:stimulator ratio of 30:1 in AIM-V medium plus 5% human AB serum. After 7-10 days, responder T cells were restimulated with hepsin-loaded dendritic cells at a responder:stimulator ratio of 10:1. T cell cultures were supplemented with recombinant IL-2 (10-100 U/ml), and fed every 2-4 days with 50-75% changes of medium plus IL-2. T cell lines were subsequently maintained by restimulation with hepsin-loaded DC every 14 days. Before the 3rd restimulation, CD4+ T cells and CD8+ T cells were purified by positive selection with anti-CD4 or anti-CD8-conjugated magnetic beads, as appropriate. Resultant populations were >98% pure by flow cytometry.
CD4+ T cells and CD8+ T cells were tested in microwell lymphoproliferation assays after the 4th and 5th passages, respectively. T cells (2×104/well) were incubated with dendritic cells loaded with hepsin by DOTAP lipofection (5×103/well) or control dendritic cells treated with DOTAP only (5×103/well). The assay was incubated for 72 hours. Proliferation was determined by the addition of 3H-thymidine (1 μCi/well) to each microwell culture for the final 24 hours. Results are presented as the mean of triplicate microwells, calculated as a stimulation index (ratio of 3H-thymidine uptake by T cells cultured with dendritic cells versus 3H-thymidine uptake by T cells cultured alone).
Although some background proliferation in response to stimulation with control dendritic cells was seen, this assay clearly shows that hepsin-loaded dendritic cells are capable of inducing a significant antigen-specific lymphoproliferative response by both CD4+ T cells and CD8+ T cells (FIG. 25). These results underline the potential for dendritic cell-based immunotherapy using hepsin as a tumor target antigen.
In summary, the present invention provides immunotherapeutic applications specially targeted at hepsin, and applied to the treatment of tumors that express hepsin. Target diseases will include ovarian cancer and prostate cancer, but will also include any other malignancy for which hepsin expression can be demonstrated. Immunotherapeutic applications will include, but are not limited to: Immunotherapy may take the form of hepsin-loaded dendritic cell vaccination, in which dendritic cells are generated in vitro from peripheral blood drawn from the patient, loaded with hepsin by lipofection or other means, and then given back to the patient as an autologous cellular vaccine, either in single doses or multiple doses. Hepsin may also be expressed in dendritic cells following transduction with a recombinant DNA vector, and such hepsin-transduced dendritic cells may then be used as a cellular vaccine.
Recombinant DNA vectors that express hepsin, either alone or as a fusion protein with other immunologically active components, may be used as a DNA vaccine for treatment of tumors that express hepsin. Hepsin-loaded or hepsin-expressing dendritic cells may also be used to stimulate tumor antigen-specific T cell responses in vitro, followed by adoptive immunotherapy, in which the patient will be given autologous hepsin-specific T cells.
Monoclonal antibody therapy based on hepsin are also apparent. Hepsin is expressed as a transmembrane protein on the surface of tumor cells. Construction of human monoclonal antibodies, or chimeric humanized monoclonal antibodies specific for hepsin offers an attractive option for immunotherapy of hepsin-expressing malignancies.
EXAMPLE 13
CD8+ CTL Specific for Hepsin Peptide 170-178 Recognize Endogenously Expressed Hepsin Tumor Antigen
To determine whether peptide-specific CD8+ CTL are capable of recognizing targets that process and present endogenously expressed hepsin tumor antigens, recombinant adenoviruses expressing hepsin and SCCE, both in conjunction with green fluorescent protein (GFP) as a means of directly monitoring expression levels by flow cytometric techniques were constructed. It was found that CD8+ CTL specific for hepsin 170-178 recognize and kill autologous targets infected with recombinant adenoviruses expressing the full-length hepsin antigen (Ad-GFP/hepsin) but did not recognize targets infected with Ad-GFP/SCCE (FIG. 26). These results show that the hepsin 170-178 peptide is a naturally processed and presented CTL epitope for hepsin-specific CD8+ CTL.
EXAMPLE 14
Hepsin Variant
Because members of the serine protease family are highly expressed and secreted by tumors, they offer potential targets for both diagnosis and therapy. While many of these enzymes are predominantly tumor produced, there is often some level of expression in a limited number of normal tissues.
To further enhance the potential for more specific tumor diagnosis and targeting, it would be helpful to provide unique sequences which might be included in the enzyme families. The present example discloses a transcription variant of the hepsin enzyme. The hepsin variant includes a unique intron sequence which could provide potential specificity to the recognition of hepsin in tumor.
When the complete transcript of the hepsin gene was examined for potential variants, one variant was detected which included intron sequence between exon 12 and exon 13. PCR analysis of normal tissue as well as tissue from carcinomas of the ovary and prostate confirmed the expression of a PCR band of greater size and appropriate length for the expression of a hepsin variant which included the complete sequence of intron 12.
FIG. 27 shows the expression of the hepsin variant in ovarian and prostate carcinomas. Examining a more extensive group of prostate carcinomas demonstrated the presence of this variant hepsin V12 in all the carcinomas examined (FIG. 28). Thus far hepsin V12 was shown to be expressed in 4/6 ovarian carcinomas and 10/10 prostate carcinomas.
Sense and antisense primers were made to exons 12 and 13 of hepsin (HepsinV Sense, 5′-GCG GTG GTC CCT TTG TGT GT-3′, SEQ ID NO. 192; HepsinV Antisense, 5′-AAG AGC ATC CCG TCA TCA GG-3′, SEQ ID NO. 193). All PCR was run in 20 ul reactions consisting of ovarian tumor cDNA derived from 50 ng of mRNA, 5 pmol each of sense and antisense primers, 0.2 mmol of dNTPs, 2.5 mmol of MgCl2 and 1 U of Taq polymerase in 1× buffer. This mixture was subjected to 1.5 minutes of denaturation at 94° C. followed by 35 cycles of PCR consisting of the following: denaturation for 30 seconds at 94° C., 30 seconds of annealing at the appropriate temperature for each primer set, and 1 minute of extension at 72° C. with an additional 7 minutes of extension on the last cycle.
The inclusion of the intron 12 sequence resulted in an extended transcript size (FIG. 29) and a modified amino acid sequence because of the presence of a stop codon toward the middle of the added intron sequence (FIG. 30). Analysis of the EST data for expression of intron 12 of hepsin indicates its presence in a clear cell carcinoma of the kidney and the Jurkat cell line. The presence of the truncated amino acid sequence derived from intron 12 of hepsin can provide the potential for more specific diagnosis and targeting of tumor which express this variant. These tumors include, but are not limited to, carcinomas of the ovary, prostate and kidney.
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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.

## Claims

1. A method of detecting the presence of one or more of ovarian cancer, prostate cancer or kidney cancer in a subject, comprising:
obtaining a ovarian, prostate, or kidney tumor sample from said subject and a corresponding ovarian, prostate, or kidney tissue control sample from a normal tissue obtained from a healthy individual;
detecting a mRNA encoding hepsin protein variant consisting of the amino acid sequence shown in SEQ ID NO. 195 in said subject sample and in said control sample; and
comparing a level of the subject sample mRNA with a level of corresponding mRNA in the control sample, wherein a higher level of said mRNA in said subject sample as compared to said control sample indicates presence of one or more of ovarian cancer, prostate cancer or kidney cancer in said subject.