AU737659B2 - Method for detection of metastatic prostate cancer - Google Patents

Description

WO 98/21365 PCTfUS97/20948 METHOD FOR DETECTION OF METASTATIC PROSTATE CANCER Statement of Government Rights 5 This invention was made with the support of the U.S.

Government via grants from the National Institutes of Health (Grant Nos.

CA70893 and DK41995). The Government may have certain rights in this invention.

Background of the Invention The glandular kallikreins are a subgroup of serine proteases which are involved in the post-translational processing of specific polypeptide precursors to their biologically active forms. In humans, three members of this family have been identified, and some of their properties characterized (Clements, Endoc. Rev., 10, 343 (1989); Clements, Mol, Cell Endo., 99, 1 (1994); Jones et al., Acta Endoc., 122, 481 (1992)). The hKLK1 gene encodes the tissue kallikrein protein, hK1, the hKLK2 gene encodes the prostate-specific glandular kallikrein protein, hK2, and the hKLK3 gene encodes the prostatespecific antigen protein, hK3 (PSA). Northern blot analysis ofmRNA shows that both hK2 and PSA are expressed mainly in the human prostate, while expression of hK1 is found in the pancreas, submandibular gland, kidney, and other nonprostate tissues (Chapdelaine et al., FEBS Lett., 236, 205 (1988); Young et al., Biochem. 31, 818 (1992)).

The nucleotide sequence homology between the exons of hKLK2 and hKLK3 is 80%, whereas the nucleotide sequence homology between the exons of hKLK2 and hKLK1 is 65%. The deduced amino acid sequence homology of hK2 to PSA is 78%, whereas the deduced amino acid sequence homology of hK2 to hK1 is 57%. Moreover, the deduced amino acid sequence of hK2 suggests that hK2 may be a trypsin-like protease, whereas PSA is a chymotrypsin-like protease.

PSA levels are widely used as a prognostic indicator of prostate carcinoma. However, since the concentration of PSA in serum is elevated in patients with either prostatic cancer (pCa) or benign prostatic hyperplasia (BPH), WO 98/21365 PCTIUS97/20948 2 the detection of elevated levels of PSA does not distinguish between these diseases. Moreover, the high degree of homology of hK2 to PSA raises some question as to the specificity of antibodies currently used to detect the levels of PSA. If the levels of circulating hK2 are unrelated to pCa or BPH, then antibodies raised to preparations of PSA which are contaminated with hK2, or to regions of PSA with homology to hK2, can result in false positive results.

Although it is now generally accepted that serum PSA testing, combined with the digital rectal exam (DRE), is the most effective means to detect clinically significant and organ-confined prostate cancer, combinations of PSA, DRE and ultrasonic prostate examination can detect only some prostate tumors. For example, up to 40% of surgically treated patients with prostate cancer are subsequently found to be clinically understaged. Moreover, the actual incidence of histological cancers based on autopsy data relative to the incidence of clinically significant prostate cancer is high. Furthermore, approximately 30% of patients with alleged localized prostate cancer may have occult (distant) metastatic disease (Moreno et al., Cancer Res., 52, 6110 (1992)). Of these patients, 80% experience relapse biochemically, elevated PSA levels, or by the recurrence of local, or occurrence of frank systemic, disease, after therapy.

Operative therapy is not the appropriate treatment modality for patients having established metastasis. Screening modalities to assess early metastases often fail to identify a significant subset of patients with locally invasive disease involving penetration of the prostate capsule or seminal vesicle.

While immunohistochemical techniques have been employed to identify micrometastatic, or circulating, prostate tumor cells when no obvious metastatic deposit was evidenced by conventional means, immunohistochemical methods are laborious and lack the sensitivity needed for the early detection of metastatic or locally invasive prostate cancer.

There is, therefore, a need for early detection of prostate cancer cells with metastatic potential. Moreover, there is a need to accurately stage prostate cancer prior to subjecting a patient to invasive procedures. In particular, 3 there is a need for a marker for prostate cancer that can function independently of, or in combination with, PSA.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

Summary of the Invention The invention provides a diagnostic method for detecting hK2 DNA wherein the presence of prostate cancer cells in a physiological sample can be correlated to the detection of hK2 RNA in the sample. Because expression of hK2 is prostate tissue specific, hK2 RNA should theoretically not be detectable in cells present in bodily fluids or non-prostate tissue if there is no locally invasive or metastatic disease, or if all prostate tissue 25 (benign and malignant) has been removed or destroyed. The method comprises contacting an amount of DNA obtained by reverse transcription (RT) of RNA from a human physiological sample with a plurality of oligonucleotide primers, preferably at least two oligonucleotide primers, 30 at least one of which an hK2-specific oligonucleotide, in an amplification reaction so as to yield an amount of amplified hK2 DNA. A preferred amplification reaction is a polymerase chain reaction (PCR). The presence of the amplified hK2 DNA is then detected. As described hereinbelow, the presence of amplified hK2 DNA in blood cells, after RT-PCR, is correlated with prostate cancer, sixty-seven percent of the prostate cancer \\melb Eiles\home$\cintae\Keep\speci\52062.98.doc 11/01/01 3a patients expressed hK2, 17% expressed PSA, and 17% expressed both hK2 and PSA. Preferably, the source of the sample to be tested is human tissue, prostate, prostate capsule, seminal vesicle, bone marrow or lymph node. Another preferred source of the sample to be tested is a human physiological fluid which comprises cells, blood, serum, or seminal fluid.

More preferably, the sample is not obtained from the prostate.

As used herein, "amplified hK2 DNA" is defined to mean hK2 DNA in a sample, which was subjected to an amplification reaction, that is present in an amount that is greater than e.g. 10, preferably 10 4 and more preferably 106, times greater than, the amount of hK2 DNA S" 15 which was present in the sample prior to amplification.

As used herein, the term "hK2-specific oligonucleotide" or "hK2-specific primer" means a DNA e sequence that has at least about 80%, more preferably at least about 90%, and more preferably at least about 20 sequence i g* o go oooo *o* *oo *ooo H:\cintae\Keep\speci\52602.98.doc 10/07/01 WO 98/21365 PCT/US97/20948 4 identity or homology to SEQ ID NO:4 in regions of SEQ ID NO:4 that are divergent from nucleotide sequences which encode hK3 (SEQ ID NO:23). An oligonucleotide or primer of the invention has at least about 7-50, preferably at least about 10-40, and more preferably at least about 15-35, nucleotides.

Preferably, the oligonucleotide primers of the invention comprise at least 7 nucleotides at the 3' of the oligonucleotide primer which have at least about more preferably at least about 85%, and more preferably at least about identity to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. The oligonucleotides of the invention may also include sequences which are unrelated to hK2 nucleic acid sequences, they may encode restriction endonuclease recognition sequences. A preferred hK2-specific oligonucleotide of the invention comprises SEQ ID NO:14. Another preferred hK2-specific oligonucleotide of the invention comprises SEQ ID NO:17. Yet another preferred hK2-specific oligonucleotide of the invention comprises SEQ ID NO:18.

A preferred diagnostic method of the invention combines RT- PCR detection of hK2 transcripts with RT-PCR detection of transcripts of other gene products associated with prostate cancer. Combined detection of two or more gene products may provide greater diagnostic certainty or yield more informative staging information. Combined detection may also be helpful in differentiating those cells with aggressive growth potential from those that are more indolent. In a particularly preferred embodiment of the method provided by the invention, RT-PCR detection of hK2 RNA is combined with RT-PCR detection of PSA RNA.

The invention further provides a diagnostic method for detecting hK2 RNA. The method comprises extracting RNA from a physiological sample obtained from a human. The extracted RNA is reverse transcribed to yield DNA.

The DNA is contacted with an amount of at least two oligonucleotides effective to amplify the DNA to yield an amount amplified hK2 DNA, wherein at least one oligonucleotide is an hK2-specific oligonucleotide. The presence of the WO 98/21365 PCT/US97/20948 amplified hK2 DNA is then detected. The presence of the amplified hK2 DNA is indicative of metastatic prostate cancer in the human.

The presence ofhK2 RNA, or a level ofhK2 RNA that rises over time, in bodily fluids or non-prostate tissue may be reasonably expected to indicate the presence of previously undiagnosed metastatic disease. Early detection of metastatic disease provides a "lead time" during which alternative therapeutic strategies, including those that may not exist at the time of surgery but are subsequently developed, can be evaluated. Thus, the present invention provides a method for monitoring the progression of prostate cancer.

The method comprises contacting an amount of DNA obtained by reverse transcription of RNA from a physiological sample obtained from a human afflicted with prostate cancer with an amount of at least two oligonucleotides, at least one of which an hK2-specific oligonucleotide, effective to amplify the DNA to yield an amount of amplified hK2 DNA. The presence or amount of the amplified hK2 DNA is detected or determined. At least one point later in time, another sample is taken and the amount of amplified hK2 DNA detected or determined. Then the amounts of amplified hK2 DNA, obtained at least at two different time points, are compared.

Also provided is a method for pathologically staging prostate cancer in a human. The method comprises contacting an amount of DNA obtained by reverse transcription of RNA from a physiological sample obtained from the human afflicted with prostate cancer with an amount of at least two oligonucleotides, at least one of which is an hK2-specific oligonucleotide, effective to amplify the DNA to yield an amount of amplified hK2 DNA. The presence or amount of the amplified hK2 DNA is then detected or determined.

The presence or amount of amplified hK2 DNA is indicative of the pathological stage of the prostate cancer.

Another embodiment of the invention provides a method for monitoring therapeutic interventions involving hormone therapies. For example, because hK2 expression is androgen-dependent, hK2 RNA levels in peripheral blood or other bodily tissue or fluid may be used as a marker during intermittent WO 98/21365 PCT/US97/20948 6 androgen therapy, or during androgen provocative testing, wherein a patient is temporarily placed in a hyperandrogenized state to stimulate a level of hK2 production by any persistent prostate cancer cells sufficient to render them detectable. See T. K. Takayama et al., Sem. in Oncol., 21, 542-553 (1994), and references cited therein. hK2 levels are preferably monitored periodically during the course of hormone therapy. It may be advantageous to also determine hK2 levels before commencement of therapy, and periodically after the conclusion of a therapeutic regimen.

Also provided is a diagnostic kit for detecting hK2 RNA in a physiological sample suspected of containing hK2 RNA. The kit comprises packaging containing a known amount of a first hK2-specific oligonucleotide, wherein the oligonucleotide consists of at least about 7-50 nucleotides, and wherein the oligonucleotide has at least about 80% identity to SEQ ID NO: 4, and a known amount of a second hK2-specific oligonucleotide, wherein the oligonucleotide consists of at least about 7-50 nucleotides, and wherein the oligonucleotide has at least about 80% identity to a nucleotide sequence which is complementary to SEQ ID NO:4.

The invention further provides an isolated, purified peptide comprising SEQ ID NO:22, a biologically active subunit thereof, or a biologically active variant thereof. The invention further provides an isolated, purified peptide comprising SEQ ID NO:26, a biologically active subunit thereof, or a biologically active variant thereof. Also provided is an isolated purified antibody or antibody preparation that specifically reacts with a protein or polypeptide which comprises the peptides of the invention described above.

As used herein, the term biologically active subunit" of a peptide of the invention is preferably defined to mean a subunit of a peptide having SEQ ID NO:22, which has at least about 10%, preferably at least about and more preferably at least about 90%, the activity of a peptide having SEQ ID NO:22. The activity of a peptide of the invention can be measured by methods well known to the art including, but not limited to, the ability of the peptide to elicit a sequence-specific immunologic response when the peptide is administered to an organism, goat, rabbit, sheep or mice.

As used herein, the term "biologically active variant" of a peptide of the invention is preferably defined to mean a peptide which has at least about 80%, preferably at least about 90%, and more preferably at least about identity or homology to SEQ ID NO:22. Biologically active variants of the peptides of the invention have at least about-0%, preferably at least about and more preferably at least about 90%, the activity of a peptide having SEQ ID NO:22. The activity of a variant peptide of the invention can be measured by methods described hereinabove.

The invention further provides a method for detecting or determining the presence of metastatic prostate cancer in a human non-prostate tissue sample. The method comprises mixing an amount of an agent, which binds to an hK2 polypeptide and which does not bind to hK3, with the cells of the mammalian tissue sample so as to form a binary complex comprising the agent and the cells. The presence or amount of complex formation in the sample is then detected or determined. The presence or amount of the complex provides an indication of the presence of micrometastatic prostate cancer. As used herein, "micrometastatic" means locally invasive disease, which typically 20 involves penetration of the prostate capsule or seminal vesicle, or occult disease.

A preferred agent for use in the method is an antibody. The term "antibody" includes human and animal mAbs, and preparations of polyclonal antibodies, as well as antibody fragments, synthetic antibodies, including recombinant antibodies, chimeric antibodies, including humanized antibodies, anti-idiotopic *oo 25 antibodies and derivatives thereof. To prepare antibodies which bind to hK2 and not to hK3, isolated hK2 polypeptides, isolated hK2 peptides, as well as variants and subunits thereof, can be used to prepare populations of antibodies. These antibodies in turn can be used as the basis for direct or competitive assays to detect and quantify hK2 polypeptides (or "protein") in samples derived from Atissues such as bone marrow and lymph nodes, and samples of cells such as from p~hysiological fluids which comprise cells.

7a For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

\\melb files\home$\cintae\Keep\speci\52062.98.doc 11/01/01 WO 98/21365 PCT/US97/20948 8 Brief Description of the Figures Figure 1 depicts the amino acid sequences of wild type mature hK2 (SEQ ID NO:1) and hK3 (SEQ ID NO:7).

Figure 2 depicts the amino acid sequence, and corresponding nucleic acid sequence, of wild type pphK2 (SEQ ID NO:3 and SEQ ID NO:4, respectively), phK2 (SEQ ID NO:5 and SEQ ID NO:6) and hK2 (SEQ ID NO:1 and SEQ ID NO:2). Codon 217 (GCT, Ala) is shown in bold and underlined.

Figure 3 is a schematic diagram of the pGT expression vectors pGThK2 and pGThK2v 21 7 Figure 4 depicts chromatographic profiles from the purification of phK2 217 A DEAE chromatogram of 7 day spent medium from AV12 cells transfected with a vector encoding pphK2v 2 7 A sample of the spent medium was applied in bicarbonate buffer, pH 8 and eluted with a salt gradient. The A 280 elution profile is represented by a solid line. The dotted line represents the results of an ELISA assay of a portion of individual column fractions which was dried onto microtiter plates and developed with a rabbit anti-pphK2 antibody.

The hydrophobic interaction profile of pooled DEAE fractions. Fractions 24 to 30 from the DEAE chromatographic eluates of were pooled, concentrated and applied to an HIC (hydrophobic interaction column) column in 1.2 M sodium sulfate, and eluted with a decreasing salt gradient. The elution profile

(A

28 0 is represented by a solid line. The dotted line represents the results of an ELISA assay of a portion of individual column fractions which was dried onto microtiter plates and developed with a rabbit anti-hK2 antibody. hK2containing fractions from the 22 minute peak from were concentrated and applied to a Pharmacia S12 size exclusion column. Fractions were collected and analyzed by SDS/PAGE. The 19.4 minute peak appeared homogeneous by

SDS-PAGE.

Figure 5 represents an SDS/PAGE analysis of purified hK2 and PSA. A 1.5 mg sample of purified phK2v 217 or PSA was boiled in sample buffer with or without 1% beta-mercaptoethanol. Samples were subjected to WO 98/21365 PCT/US97/20948 9 SDS/PAGE on a 4-20% gel. The protein bands were visualized by staining the gel with silver.

Figure 6 depicts Conconavalin A staining ofphK2v 21 7 The predicted position ofphK2 is designated by an arrow. ZCE (an anti-CEA mAb) and BSA were included as examples of glycosylated and non-glycosylated proteins, respectively. The presence of a band at the predicted position in the phK2 lane demonstrates that this protein is glycosylated.

Figure 7 represents the conversion of pro to mature hK2v 2 7 by trypsin cleavage. Trypsin w/w) was incubated with phK2v 21 7 for 10 minutes at 37°C in 100 mM borate buffer pH 8, and then subjected to HIC-HPLC. The dashed line represents the profile of the phK2v 2 1 7 prior to incubation with trypsin.

The solid line represents the profile ofphK2 after trypsin digestion. The profiles have been superimposed for comparison. The identity of the two forms was confirmed by N-terminal sequencing of the protein.

Figure 8 depicts Western blot analysis of seminal fluid using monoclonal antibody (mAb) hK1G 586.1. Processed seminal fluid was diluted 1:1 in PBS and centrifuged at 10,000 X g for 20 minutes. The supernatant was subjected to SDS/PAGE on a 8-25% gel using the PhastSystem (Pharmacia).

Protein was transferred to nitrocellulose and incubated with protein-G purified HK1G 586.1 (1 pg/ml) followed by goat anti-mouse IgG-HRP (1:1000). The blot was developed using the ECL detection system (Amersham).

Figure 9 represents a time course study of hK2 expression in AV12 cells. AV12-hK2 clone #27 was grown to -60-70% confluency, then cells were washed with HBSS and serum free HH4 media was added. Spent medium was withdrawn each day, concentrated and subjected to SDS/PAGE on a 12% gel. Proteins were electroblotted and probed with monoclonal antibody HK1D 106.4, which detects both phK2 and hK2 (1:1000) or HK1G 464.3, which detects phK2 (1:1000), followed by goat anti-mouse IgG-HRP (1:500). The blot was developed with ECL (Amersham) according to the manufacturer's instructions. Purified phK2v 2 17 and hK2v 217 were used as controls. The position ofhK2 is indicated by the arrow.

WO 98/21365 PCT/US97/20948 Figure 10 represents a time course study of expression of the variant form of hK2 in transfected AV12 cells. At -60-70% confluency, AV12hK2v 2 17 cells were washed with HBSS and serum free HH4 media was added.

Spent media was withdrawn each day, concentrated and subjected to SDS/PAGE on a 12% gel. Proteins were electroblotted and probed with monoclonal antibodies HK1D 106.4 and HK1G 464.3. Goat anti-mouse IgG-HRP (1:500) was used as a secondary antibody and the blot was developed with ECL (Amersham) according to the manufacturer's instructions. Purified phK2v 21 7 and hK2v 2 17 were used as controls. The position of hK2 is indicated by the arrow.

Figure 11 is a plot of hK2 expression and cell viability over time.

AV 12-hK2 clone #27 was grown to 60-70% confluency, washed with HBSS and serum free HH4 media was added. Spent media was withdrawn each day and the hK2 concentration was measured by ELISA using HK1D 106.4 or HK1G 464.3 as a primary antibody, and goat anti-mouse IgG-HRP as a secondary antibody.

The reaction was developed with OPD (Sigma, St. Louis, MO). Viable cells were enumerated daily using trypan blue dye exclusion.

Figure 12 depicts the expression of hK2 in PC3 and DU145 cells.

PC3 and DU145 cells transfected with pGThK2 were grown to -60-70% confluency, washed and resuspended in serum free HH4 media.. The spent medium of pGThK2 transfected DU145 cells was collected 3 days after resuspension and the spent medium of pGThK2 transfected PC3 cells was collected 5 days after resuspension. Spent media were concentrated and subjected to SDS/PAGE on 12% gels. Proteins were electroblotted and probed with HK1D 106.4 or HKIG 464.3 as described above. Purified phK2v 2 1 7 and phK2v 2 17 were used as controls. The position of hK2 is indicated by an arrow.

Figure 13 depicts the expression of hK2 by selected hK2containing AV12 clones. Cells from hK2 containing AV12 clone numbers 27, 31 and 32 were grown to -60-70% confluency and washed with HBSS, then serum free HH4 media was added. Spent media was withdrawn 7 days after the addition of serum free media, concentrated and subjected to SDS/PAGE on a 12% gel. Proteins were electroblotted and probed with HK1D 106.4 or HK1G WO 98/21365 PCTIUS97/20948 11 464.3. Goat anti-mouse IgG-HRP (1:500) was used as a secondary antibody and the blot was developed with ECL (Amersham) according to manufacturer's instructions. Purified phK2v 2 17 and hK2 v 21 7 were used as controls. The position of hK2 is indicated by an arrow.

Figure 14 depicts the expression of phK2v 21 7 in selected AV12hK2v 2 17 clones. Cells from AV12 clone numbers 2, 3, 4, 45 and 48 were grown to approximately 60-70% confluency and washed with HBSS, and serum free HH4 media was added. Spent media was withdrawn 7 days after the addition of serum free media, concentrated and subjected to SDS/PAGE on a 12% gel.

Proteins were electroblotted and probed with HKlD 106.4 or HK1G 464.3.

Goat anti-mouse IgG-HRP (1:500) was used as a secondary antibody and the blot was developed with ECL (Amersham) according to manufacturer's instructions. Purified phK2v 2 17 and hK2v 2 17 were used as controls. The position of hK2 is indicated by an arrow.

Figure 15 depicts the amidolytic specificity of hK2v 21 7 hK2, and PSA for residues 210-236 of hK2. The synthetic peptide (0.63 mM) was digested overnight at 37 0 C with 1 Ig/ml hK2, 40 ig/ml hK2v21 7 or 100 ig/ml PSA, and the digestion products separated by RP-HPLC. Peaks were normalized to compare the qualitative aspects of cleavage.

Figure 16 depicts the specificity of hK2 and PSA for different peptide substrates. Open arrows denote peptide bonds cleaved by PSA; solid arrows denote bonds cleaved by hK2. Peptide #1 represents amino acid residues 210-236 of hK2. Peptide #2 represents amino acid residues 1-14 of angiotensinogen, the renin substrate tetradecapeptide. Peptide #3 represents amino acid residues -7 to +7 of phK2. Peptide #4 represents amino acid residues 41-56 of hK2. Peptide #5 represents the amino acid sequence of the oxidized beta chain of insulin. Peptide #6 represents amino acid residues 196-213 of

PMSA.

Figure 17 depicts the activation of phK2v 21 7 by hK2 but not hK2v 2 17 phK2 v 2 1 7 contains the pro leader peptide sequence VPLIQSR, a sequence not present in hK2 v21 7 Panel A shows phK2 v21 7 incubated with 1% WO 98/21365 PCT/US97/20948 12 w/w hK2. Panel B is a control with 40% w/w hK2v 2 17 incubated with phK2v 217 for 6 hours.

Figure 18 depicts Western blot analysis of hK2 incubated with protease inhibitors. Each sample was separated on a 8-25% gradient SDS- PAGE, blotted and probed with HK1G586.1. hK2 was incubated for 4 hours at 37 0 C with the following inhibitors: Lane 1, antichymotrypsin (ACT); lane 2, alpha 2-antiplasmin; lane 3, anti-thrombin III; lane 4, alpha 1-protease inhibitor (anti-trypsin); lane 5, alpha 2-macroglobulin; lanes 1 and 2 show a covalent complex of the predicted Mr of 90-100 kD. Serpin inhibitors were employed at 20 pM, macroglobulin at 2.8 utM, and hK2 at 0.175 uM. Lane 5 shows the higher Mr complexes representing covalent complex formation of hK2 with alpha 2-macroglobulin.

Figure 19 depicts complex formation of hK2 in human serum.

Western blots of hK2 and PSA were incubated with human serum. hK2 samples were probed with HK1G586.1 and PSA samples with PSM773 anti-PSA mAb Lanes 1-6 contain hK2 samples and lanes 7 and 8 are PSA samples. Lane 1 represents an hK2 control. Lane 2 contains hK2 incubated with ACT for 4 hours. Lane 3 represents a serum control with no added protease. Lane 4 contains hK2 incubated for 15 minutes with serum. Lane 5 contains hK2 incubated with serum for 4 hours. Lane 6 contains hK2 incubated with purified alpha-2 macroglobulin for 4 hours. Lane 7 contains PSA incubated with serum for 4 hours. Lane 8 contains PSA incubated with purified alpha-2 macroglobulin for 4 hours.

Figure 20 depicts immunoreactivity of monoclonal antibody HK G 586 in untreated human prostate (n=257).

Figure 21 depicts immunoreactivity of monoclonal antibody HK1G 586 in androgen deprivation therapy-treated human prostate Figure 22 depicts RT-PCR detection of PSA and hK2 mRNAs in RNA extracted from LNCaP cells diluted in whole human blood, and RT-PCR detection of PSA and hK2 mRNAs in RNA extracted from whole human blood of the following patients: patient 17, age 31, male control; patient WO 98/21365 PCT/US97/20948 13 21, age 58, clinical stage B; patient 24, age 83, known metastatic disease (D2); patient 26, age 75, pathological stage C (+margins); patient 28, age 57, pathological stage C (+margins); patient 49, age 64, pathological stage C (+seminal vesicles); patient 59, age 31, male control; patient 60, age 73, known metastatic disease.

Detailed Description of the Invention The high degree of amino acid sequence homology of hK2 to PSA, and the fact that the expression of both hK2 and PSA is essentially limited to prostate cells, suggests that measuring the amount or presence of hK2 and PSA in tissue samples, or measuring the levels of hK2 transcripts in physiological fluids comprising cells, blood, or in tissue samples, e.g., lymph node, can be useful in the diagnosis and monitoring of prostatic cancer (pCa).

Definitions As used herein, the term "hK2 polypeptide" includes recombinant pre-pro, pro and mature hK2 polypeptides. A mature hK2 polypeptide having the amino acid sequence shown in Figure 1 (SEQ ID NO:1), as well as "variant" polypeptides which share at least 90% homology with SEQ ID NO:1 in the regions which are substantially homologous with hK3, which regions are not identified by bars as shown in Figure 1. The variant hK2 polypeptides of the invention have at least one amino acid substitution relative to the corresponding wild type hK2 polypeptide. A preferred variant hK2 polypeptide comprises SEQ ID NO:8, a mature hK2 polypeptide having an alanine to valine substitution at amino acid position 217. hK2 polypeptides possess antigenic function in common with the mature hK2 molecule of Figure 1, in that said polypeptides are also definable by antibodies which bind specifically thereto, but which do not cross-react with hK3 (or hKl). Preferably, said antibodies react with antigenic sites or epitopes that are also present on the mature hK2 molecule of Figure 1.

Antibodies useful to define common antigenic function are described in detail in Serial No. 08/096,946, now U.S. Patent No. 5,516,639, i.e., polyclonal antisera prepared in vivo against hK2 subunit 41-56.

WO 98/21365 PCT/US97/20948 14 "Isolated hK2 nucleic acid" is RNA or DNA containing greater than 7, preferably 15, and more preferably 20 or more, sequential nucleotide bases that are complementary to the non-coding or coding strand of the native hK2 polypeptide RNA or DNA, or hybridizes to said RNA or DNA and remains stably bound under stringent conditions. Preferably, the isolated nucleic acid encodes a biologically active hK2 polypeptide, a variant thereof, or a subunit thereof. The biological activity of an hK2 polypeptide can be detected by methods well known to the art including, but not limited to, the ability to react with antibodies specific for hK2 polypeptides, the ability to cleave hK2-specific substrates (see Example or the ability to bind to serum proteins (see Example A variant hK2 polypeptide or subunit thereof, or a subunit of an hK2 polypeptide, has at least about 10%, preferably at least about 50%, and more preferably at least about 90%, the biological activity of an hK2 polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

Thus, the RNA or DNA is isolated in that it is free from at least one contaminating nucleic acid with which it is normally associated in the natural source of the nucleic acid and is preferably substantially free of any other mammalian RNA or DNA. The phrase "free from at least one contaminating source nucleic acid with which it is normally associated" includes the case where the nucleic acid is reintroduced into the source or natural cell but is in a different chromosomal location or is otherwise flanked by nucleic acid sequences not normally found in the source cell. An example of isolated hK2 nucleic acid is RNA or DNA that encodes a biologically active hK2 polypeptide sharing at least sequence identity with the hK3-homologous regions of the hK2 peptide of Figure 1, as described above. The term "isolated, purified" as used with respect to an hK2 polypeptide is defined in terms of the methodologies discussed hereinbelow.

As used herein, the term "recombinant nucleic acid," i.e., "recombinant DNA" refers to a nucleic acid, to DNA that has been derived or isolated from any appropriate tissue source, that may be subsequently chemically altered in vitro, and later introduced into target host cells, such as WO 98/21365 PCTIUS97/20948 cells derived from animal, plant, insect, yeast, fungal or bacterial sources. An example of recombinant DNA "derived" from a source, would be a DNA sequence that is identified as a useful fragment encoding hK2, or a fragment or variant thereof, and which is then chemically synthesized in essentially pure form. An example of such DNA "isolated" from a source would be a useful DNA sequence that is excised or removed from said source by chemical means, e.g, by the use of restriction endonucleases, so that it can be further manipulated, amplified, for use in the invention, by the methodology of genetic engineering.

Therefore, "recombinant DNA" includes completely synthetic DNA sequences, semi-synthetic DNA sequences, DNA sequences isolated from biological sources, and DNA sequences derived from introduced RNA, as well as mixtures thereof. Generally, the recombinant DNA sequence is not originally resident in the genome of the host target cell which is the recipient of the DNA, or it is resident in the genome but is not expressed, or not highly expressed.

As used herein, "chimeric" means that a vector comprises DNA from at least two different species, or comprises DNA from the same species, which is linked or associated in a manner which does not occur in the "native" or wild type of the species.

"Control sequences" is defined to mean DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotic cells, for example, include a promoter, and optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

"Operably linked" is defined to mean that the nucleic acids are placed in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome WO 98/21365 PCT/US97/20948 16 binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites.

If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accord with conventional practice.

"Southern analysis" or "Southern blotting" is a method by which the presence of DNA sequences in a restriction endonuclease digest of DNA or DNA-containing composition is confirmed by hybridization to a known, labeled oligonucleotide or DNA fragment. Southern analysis typically involves electrophoretic separation of DNA digests on agarose gels, denaturation of the DNA after electrophoretic separation, and transfer of the DNA to nitrocellulose, nylon, or another suitable membrane support for analysis with a radiolabeled, biotinylated, or enzyme-labeled probe as described in sections 9.37-9.52 of Sambrook et al., supra.

"Northern analysis" or "Northern blotting" is a method used to identify RNA sequences that hybridize to a known probe such as an oligonucleotide, DNA fragment, cDNA or fragment thereof, or RNA fragment.

The probe is labeled with a radioisotope such as 32 P, by biotinylation or with an enzyme. The RNA to be analyzed can be usually electrophoretically separated on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon, or other suitable membrane, and hybridized with the probe, using standard techniques well known in the art such as those described in sections 7.39-7.52 of Sambrook et al., supra.

"Stringent conditions" are those that employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate (SSC); 0.1% sodium lauryl sulfate (SDS) at 50 0 C, or (2) employ a denaturing agent such as formamide during hybridization, formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% mM sodium phosphate buffer at pH 6.5 with 750 mM WO 98/21365 PCT/US97/20948 17 NaC1, 75 mM sodium citrate at 42 0 C. Another example is use of formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 jg/ml), 0.1% SDS, and 10% dextran sulfate at 42 0 C, with washes at 42 0 C in 0.2 x SSC and 0.1% SDS. See Sambrook et al., supra, for other examples of stringent conditions.

Expression Cassettes or Expression Vectors An expression cassette or vector comprising a recombinant DNA sequence encoding hK2 which is operably linked to a promoter functional in a host cell, may be circular or linear, double-stranded or single-stranded.

Generally, the expression cassette or vector is in the form of chimeric DNA, such as plasmid DNA, that can also contain coding regions flanked by control sequences which promote the expression of the recombinant DNA present in the resultant cell line. For example, the expression cassette may itself comprise a promoter that is active in mammalian cells, or may utilize a promoter already present in the genome that is the transformation target. Such promoters include the CMV promoter, as well as the SV40 late promoter and retroviral LTRs (long terminal repeat elements). Aside from recombinant DNA sequences that serve as transcription units for hK2 or portions thereof, a portion of the.recombinant DNA may be untranscribed, serving a regulatory or a structural function.

The expression cassettes or expression vectors to be introduced into the cells further will generally contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of transformed cells from the population of cells sought to be transformed. Alternatively, the selectable marker may be carried on a separate piece of DNA and used in a cotransformation procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are well known in the art and include, for example, antibiotic and herbicide-resistance genes, such as neo, hpt, dhfr, bar, aroA and the like. See also Table 1 of Lundquist et al. Patent No.

5,554,798).

WO 98/21365 PCTfUS9720948 18 Reporter genes are used for identifying potentially transformed cells and for evaluating the functionality of regulatory sequences. Reporter genes which encode for easily assayable proteins are well known in the art. In general, a reporter gene is a gene which is not present in or expressed by the recipient organism or tissue and which encodes a protein whose expression is manifested by some easily detectable property, enzymatic activity.

Preferred genes include the chloramphenicol acetyl transferase gene (cat) from Tn9 ofE. coli, the beta-glucuronidase gene (gus) of the uidA locus ofE. coli, and the luciferase gene from firefly Photinuspyralis. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.

Other elements functional in the host cells, such as introns, enhancers, polyadenylation sequences and the like, may also be a part of the recombinant DNA. Such elements may or may not be necessary for the function of the DNA, but may provide improved expression of the DNA by affecting transcription, stability of the mRNA, or the like. Such elements may be included in the DNA as desired to obtain the optimal performance of the transforming DNA in the cell.

The general methods for constructing recombinant DNA which can transform target cells are well known to those skilled in the art, and the same compositions and methods of construction may be utilized to produce the DNA useful herein. For example, J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2d ed., 1989), provides suitable methods of construction.

Transformation of Host Cells and Recovery of Recombinant hK2 Polvpeptides The expression cassette or vector comprising the recombinant DNA encoding an hK2 polypeptide can be readily introduced into the target cells by transfection for example, by the modified calcium phosphate precipitation procedure of C. Chen et al., Mol. Cell. Biol., 7, 2745 (1987). Transfection can also be accomplished by lipofectin, using commercially available kits, e.g., provided by BRL.

WO 98/21365 PCT/US97/20948 19 Suitable host cells for the expression ofhK2 polypeptide are derived from multicellular organisms. Such host cells are capable of complex processing and glycosylation activities. In principle, any higher eukaryotic cell culture can be employed in the practice of the invention, whether from vertebrate or invertebrate culture. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. See, Luckow et al., Bio/Technology, 6, 47 (1988); Miller et al., in Genetic Engineering, J. K. Setlow et al., eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al., Nature, 315, 592 (1985). A variety of viral strains for transfection are publicly available, the L-1 variant ofAutographa californica NPV and the strain of Bombyx mori NPV, and such viruses may be used, preferably for transfection of Spodopterafrugiperda cells.

When hK2 polypeptide is expressed in a recombinant cell other than one of human origin, the hK2 polypeptide is completely free of proteins or polypeptides of human origin. However, it is necessary to purify hK2 polypeptide from recombinant cell proteins or polypeptides to obtain preparations that are substantially homogeneous as to hK2 polypeptide. For example, the culture medium or lysate can be centrifuged to remove particulate cell debris. The membrane and soluble protein fractions are then separated. The hK2 polypeptide may then be purified from the soluble protein fraction and, if necessary, from the membrane fraction of the culture lysate. hK2 polypeptide can then be purified from contaminant soluble proteins and polypeptides by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on an anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; or ligand affinity chromatography.

WO 98/21365 PCT/US97/20948 Once isolated from the resulting transgenic host cells, derivatives and variants of the hK2 polypeptide can be readily prepared. For example, amides of the hK2 polypeptides of the present invention may also be prepared by techniques well known in the art for converting a carboxylic acid group or precursor, to an amide. A preferred method for amide formation at the Cterminal carboxyl group is to cleave the polypeptide from a solid support with an appropriate amine, or to cleave in the presence of an alcohol, yielding an ester, followed by aminolysis with the desired amine.

Salts of carboxyl groups of the hK2 polypeptide may be prepared in the usual manner by contacting the peptide with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, sodium hydroxide; a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like.

N-acyl derivatives of an amino group of the present polypeptides may be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected peptide. O-acyl derivatives may be prepared, for example, by acylation of a free hydroxy peptide or peptide resin. Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both Nand O-acylation may be carried out together, if desired. In addition, the internal hK2 amino acid sequence of Figure 1 can be modified by substituting one or two conservative amino acid substitutions for the positions specified, including substitutions which utilize the D rather than L form.

Acid addition salts of the polypeptides may be prepared by contacting the polypeptide with one or more equivalents of the desired inorganic or organic acid, such as, for example, hydrochloric acid. Esters of carboxyl groups of the polypeptides may also be prepared by any of the usual methods known in the art.

WO 98/21365 PCT/US97/20948 21 Variant hK2 Polypeptides It is envisioned that variant hK2 polypeptides have at least one amino acid substitution relative to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, e.g., SEQ ID NO:8 has an alanine to valine substitution at position 217 relative to SEQ ID NO:1. In particular, amino acids are substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of exemplary substitutions. More preferred substitutions are under the heading of preferred substitutions. After the substitutions are introduced, the products are screened for biological activity, ability to generate hK2-specific antibodies or to specifically react with hK2-specific antibodies, antibodies that bind to hK2 and not to hK3 (PSA).

WO 98/21365 PCT/US97/20948 22 TABLE 1 Original Exemplary Preferred Residue Substitutions Substitutions Ala val; leu; ile val Arg lys; gin; asn lys Asn gin; his; lys; arg gin Asp glu glu Cys ser ser Gin asn asn Glu asp asp Gly pro pro His asn; gin; lys; arg arg Ile leu; val; met; ala; phe leu norleucine Leu norleucine; ile; val; met; ile ala; phe Lys arg; gin; asn arg Met leu; phe; ile leu Phe leu; val; ile; ala leu Pro gly gly Ser thr thr Thr ser ser Trp tyr tyr Tyr trp; phe; thr; ser phe Val ile; leu; met; phe; ala; leu norleucine Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, the charge or hydrophobicity of the molecule at the target site, or the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: hydrophobic: norleucine, met, ala, val, leu, ile; neutral hydrophilic: cys, ser, thr; acidic: asp, glu; WO 98/21365 PCT/US97/20948 23 basic: asn, gin, his, lys, arg; residues.that influence chain orientation: gly, pro; and aromatic; trp, tyr, phe.

The invention also envisions hK2 variants with non-conservative substitutions. Non-conservative substitutions entail exchanging a member of one of the classes described above for another. Amino acid substitutions are introduced into the DNA molecules of the invention by methods well known to the art.

Uses of Recombinant hK2 Polvpeptides Once isolated, hK2 polypeptide and its antigenically active variants, derivatives and fragments thereof can be used in assays for hK2 in samples derived from biological materials suspected of containing hK2 or antihK2 antibodies, as disclosed in detail in U.S. Patent No. 5,516,639. For example, the hK2 polypeptide can be labeled with a detectable label, such as via one or more radiolabeled peptidyl residues, and can be used to compete with endogenous hK2 for binding to anti-hK2 antibodies, as a "capture antigen" to bind to anti-hK2 antibodies in a sample of a physiological fluid, via various competitive immunoassay format for hK2 which uses anti-hK2 antibodies which are capable of immobilization is carried out by: providing an amount of anti-hK2 antibodies which are capable of attachment to a solid surface; mixing a physiological sample, which comprises hK2, with a known amount of hK2 polypeptide which comprises a detectable label, to produce a mixed sample; contacting said antibodies with said mixed sample for a sufficient time to allow immunological reactions to occur between said antibodies and said hK2 to form an antibody-hK2 complex, and between said antibodies and said labeled polypeptide to form an antibody-labeled polypeptide complex; separating the antibodies which are bound to hK2 and antibodies bound to the labeled polypeptide from the mixed sample; WO 98/21365 PCT/US97/20948 24 detecting or determining the presence or amount of labeled polypeptide either bound to the antibodies on the solid surface or remaining in the mixed sample; and determining from the result in step the presence or amount of said hK2 in said sample.

In another format which can detect endogenous hK2 in a sample by a competitive inhibition immunoassay, a known amount of anti-hK2 antibody is added to a sample containing an unknown amount of endogenous hK2. The known amount is selected to be less than the amount required to complex all of the hK2 suspected to be present, that would be present in a sample of the same amount of sample material obtained from a patient known to be afflicted with prostate cancer. Next, a known amount of the hK2 polypeptide of the invention or a subunit thereof, comprising a detectable label is added. If endogenous hK2 is present in the sample, fewer antibodies will be available to bind the labeled hK2 polypeptide, and it will remain free in solution. If no endogenous hK2 is present, the added labeled polypeptide will complex with the added anti-hK2 antibodies to form binary complexes. Next, the binary antibodyantigen complexes are precipitated by an anti-mammal IgG antibody (sheep, goat, mouse, etc.). The amount of radioactivity or other label in the precipitate (a ternary complex) is inversely proportional to the amount of endogenous hK2 that is present in the sample, a pellet containing reduced amounts of radioactivity is indicative of the presence of endogenous hK2.

Alternatively to the conventional techniques for preparing polyclonal antibodies or antisera in laboratory and farm animals, monoclonal antibodies against hK2 polypeptide can be prepared using known hybridoma cell culture techniques. In general, this method involves prepared an antibodyproducing fused cell line, of primary spleen cells fused with a compatible continuous line of myeloma cells, and growing the fused cells either in mass culture or in an animal species from which the myeloma cell line used was derived or is compatible. Such antibodies offer many advantages in comparison to those produced by inoculation of animals, as they are highly specific and WO 98/21365 PCT/US97/20948 sensitive and relatively "pure" immunochemically. Immunologically active fragments of the present antibodies are also within the scope of the present invention, the f(ab) fragment, as are partially humanized monoclonal antibodies.

Chimeric and Modified Antibodies Chimeric antibodies comprise the fusion of the variable domains from one immunoglobulin to the constant domains from another immunoglobulin. Usually, the variable domains are derived from an immunoglobulin gene from a different species, perhaps a human. This technology is well known to the art. See, for example, European Patent Applications, EP-A-0 125,023 (Cabilly/Genetech) and EP-A-0 120,694 and U.S.

Patent No. 4,816,567, which disclose the preparation of variations of immunoglobulin-type molecules using recombinant DNA technology.

Another approach to prepare chimeric or modified antibodies is to attach the variable region of a monoclonal antibody to another nonimmunoglobulin molecule, to produce a derivative chimeric molecule (see WO 86/01533, Neuberger and Rabbits/Celltech). A further approach is to prepare a chimeric immunoglobulin having different specificities in its different variable regions (see EP 68763A). Yet another approach is to introduce a mutation in the DNA encoding the monoclonal antibody, so as to alter certain of its characteristics without changing its essential specificity. This can be accomplished by site-directed mutagenesis or other techniques known in the art.

The Winter patent application EP-A-0 239 400 discloses the preparation of an altered, derivative antibody by replacing the complementarity determining regions (CDRs) of a variable region of an immunoglobulin with the CDRs from an immunoglobulin of different specificity, using recombinant DNA techniques ("CDR-grafting"). Thus, CDR-grafting enables "humanization" of antibodies, in combination with the alteration of the framework regions.

Human antibodies can also be prepared by reconstituting the human immune system in mice lacking their native immune system, then WO 98/21365 PCT/US97/20948 26 immunizing the mice so as to yield human antibodies which are specific for the immunogen.

A "humanized" antibody containing the CDRs of a rodent antibody specific for an antigen of interest may be less likely to be recognized as foreign by the immune system of a human. It follows that a "humanized" antibody with the same binding specificity, as HK1G464, may be of particular use in human therapy and/or diagnostic methods.

The manipulation and/or alteration of any given antibody, or gene(s) encoding for the same, to generate a derivative antibody is well known to the art.

Detection of hK2-Specific Transcripts by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) To detect hK2 encoding RNA transcripts, RNA is isolated from a cellular sample suspected of containing hK2 RNA, total RNA isolated from human prostate tissue. RNA can be isolated by methods known to the art, e.g., using TRIZOL" reagent (GIBCO-BRL/Life Technologies, Gaithersburg, MD).

Oligo-dT can be employed as a primer in a reverse transcriptase reaction to prepare first-strand cDNAs from the isolated RNA. Resultant first-strand cDNAs are then amplified in PCR reactions.

"Polymerase chain reaction" or "PCR" refers to a procedure or technique in which amounts of a preselected fragment of nucleic acid, RNA and/or DNA, are amplified as described in U.S. Patent No. 4,683,195.

Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers. These primers will be identical or similar in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, and the like. See generally Mullis et al., Cold Spring Harbor Svmp. Ouant. Biol., 51, 263 (1987); Erlich, ed., PCR Technology, (Stockton Press, NY, 1989). Thus, amplification of specific nucleic acid sequences by PCR relies upon oligonucleotides or "primers" having WO 98/21365 PCT/US97/20948 27 conserved nucleotide sequences, wherein the conserved sequences are deduced from alignments of related gene or protein sequences, a sequence comparison of mammalian hK2 genes. For example, one primer is prepared which is predicted to anneal to the antisense strand, and another primer prepared which is predicted to anneal to the sense strand, of a DNA molecule which encodes an hK2 polypeptide.

In general, the isolated RNA is combined with a primer in a reverse transcriptase (RT) reaction to generate single strand cDNAs. Oligo-dT or random sequence oligonucleotides, as well as sequence specific oligonucleotides, can be employed as a primer in the RT reaction. See Sambrook et al., supra. The single strand cDNAs are then amplified with sequence specific primers to yield an amplified product.

To detect the amplified product, the reaction mixture is typically subjected to agarose gel electrophoresis or other convenient separation technique, and the presence or absence of the hK2-specific amplified DNA detected. Detection of the amplified hK2 DNA may be accomplished by excising or eluting the fragment from the gel (for example, see Lawn et al., Nucleic Acids Res., 9, 6103 (1981), and Goeddel et al., Nucleic Acids Res., 8, 4057 (1980)), cloning the amplified product into a cloning site of a suitable vector, such as the pCRII vector (Invitrogen), sequencing the cloned insert and comparing the DNA sequence to the known sequence of hK2.

Alternatively, for example, the hK2 amplified DNA may be detected using Southern hybridization with an hK2-specific oligonucleotide probe, or comparing its electrophoretic mobility with DNA standards of known molecular weight.

The invention will be further described by reference to the following detailed examples.

WO 98/21365 PCT/US97/20948 28 Example 1.

Materials and Methods Construction of mammalian hK2 expression vectors A cDNA (approximately 820 bp long) encoding the entire prepro-hK2 (pphK2) (from nucleotide #40 to #858 relative to the start site of the pphK2 transcript), as shown in Figure 2, was synthesized from RNA of human BPH tissue using reverse-transcription polymerase chain reaction (RT-PCR) technology with a pair of hK2 specific oligonucleotide primers SEQ ID NO:9 and 5'ACAGCTGCAGTTTACTAGAGGTAGGGGTGGGAC SEQ ID This cDNA was generated such that 5' and 3' ends (with respect to pphK2 sense sequence) were bracketed with BamH1 and Pstl sequences, respectively. The cDNA was then purified by agarose gel electrophoresis, and digested with BamHl and Pstl restriction enzymes. The restricted cDNA was ligated with BamH1-Pstl digested pVL1393 plasmid vector and transformed into the E. coli HB101 strain. E. coli harboring pphK2 cDNA/pVL1393 plasmid vector were selected. The pphK2 containing insert was sequenced. Plasmid pphK2 cDNA/pVL1393 was mass-produced in E. coli and purified by CsCI gradient ultra-centrifugation.

Plasmid pphK2/pVL1393 in E. coli HB101 has been deposited in the American Type Culture Collection, Rockville, MD, USA on May 2, 1994 under the provisions of the Budapest Treaty and has been assigned accession number ATCC 69614.

A 0.8 kb fragment representing the entire pphK2 coding sequence (Figure 2) was generated by PCR using primers A (5'ATATGGATCCATATGTCAGCATGTGGGACCTGGTTCTCTCCA3') (SEQ ID NO:11) and B (5'ATATGGATCCTCAGGGGTTGGCTGCGATGGT3') (SEQ ID NO:12) and plasmid pVL1393 containing pphK2 (gift from Dr. Young, Mayo Clinic) as the template. PCR products were inserted into the TA-cloning vector (Invitrogen Corp., San Diego, CA) and the DNA of the entire insert was sequenced.

WO 98/21365 PCT/US97/20948 29 To obtain the mammalian hK2 expression vectors, the hK2containing inserts were isolated from the corresponding TA clones and inserted into the Bel 1 site of the plasmid pGT-d (Berg et al., Nucl. Acids Res., 20, 54-85 (1992)) (gift from Dr. Brian Grinnell, Lilly) under the control of the GBMT promoter. The mammalian expression vectors, PLNS-hK2 and PLNC-hK2 were obtained by cloning the 0.8 kb wild type hK2 insert from the corresponding TA vector into the plasmids, pLNSX and pLNCX (Miller et al., Biotech., 9, 980 (1989)), respectively. The orientation of the insert in all the mammalian expression vectors was confirmed by DNA sequencing.

Generation of recombinant clones AV 12-664 (ATCC CRL-9595), a cell line derived from adenovirus-induced tumors in Syrian hamster, and DU145 cells were cultured in Dulbecco's modified Eagle's medium (high glucose) supplemented with fetal bovine serum (D1OF). PC3 cells were cultured in Minimal Eagle Medium containing 10% fetal bovine serum. AV12 cells were transfected with the hK2 expression vectors using the calcium phosphate method (Maniatis et al., supra (1989)). Three days after transfection cells were resuspended in DIOF 200 nM methotrexate (MTX). Drug-resistant clonal cell lines were isolated after 2-3 weeks and their spent medium was analyzed by Western blots. PC3 and DU145 cells were transfected with hK2 mammalian expression vectors using lipofectamine (Gibco-BRL, Gaithersburg, MD) and clones (PC3-hK2 and DU145-hK2) were selected in media containing 400 [ig/ml G418.

Purification and sequencing of the protein AV12-hK2 clones were grown in DIOF 200 nM MTX. At about 60% confluency the cells were washed with Hank's balanced salt solution and resuspended in serum-free HH4 medium. The spent medium was collected 7 days after the addition of serum-free spent medium and stored at -20 0 C. To purify the protein, the serum-free spent medium was concentrated and exchanged into 50 mM sodium bicarbonate pH 8. Samples were filtered with 0.2 i filters and then pumped directly onto a TSK DEAE-5PW HPLC column, 21 mm X 150 mm, at a flow rate of 5 ml/minute. Buffer A contained 50 mM Na WO 98/21365 PCT/US97/20948 bicarbonate pH 7.9 and Buffer B contained 50 mM Na bicarbonate plus 0.5 M NaCl pH 7.6. The elution profile was developed with a gradient from 0-50% Buffer B over 35 minutes; 50-100% B from 35-40 minutes and isocratic elution at 100% B for 5 minutes before re-equilibration in Buffer A. The flow rate was 5 mL/minute throughout. In the above procedure, borate buffer could replace bicarbonate buffer with no noticeable difference.

DEAE fractions were assayed for the presence ofhK2 by the dried-down ELISA method (see below) using rabbit anti-pphK2 (Saedi et al., Mol. Cell. Endoc., 109, 237 (1995)). Fractions with hK2 activity were pooled and concentrated by ultrafiltration with membranes (10 kD cut off) to approximately 5-8 mL. Solid ammonium sulfate was then added to a final concentration of 1.2 M. This sample was then injected onto a PolyLC, polypropyl aspartamide column, 1000A pore size, 4.6 mm X 200 mm, to resolve proteins by hydrophobic interaction chromatography (HIC). Buffer A was mM Na phosphate, 1.2 M Na sulfate pH 6.3 and Buffer B was 50 mM Na phosphate, 5% 2-propanol, pH 7.4. The elution gradient was 0-20% B over minutes; 20-55% B from 5-20 minutes, isocratic at 55% B from 20-23 minutes, 55-100% B from 23-25 minutes; isocratic at 100% B for 2 minutes before reequilibration Buffer A. The flow rate was 1 mL/minute. The HIC peak containing hK2 which eluted at about 50% B was exchanged into 50 mM borate buffer pH 8 by repeated concentration with Centricon-10 (Amicon) 10 K MW cutoff ultrafiltration. Purity was assessed by both SDS-PAGE and Western blot analyses. The extinction coefficient used to estimate hK2v 2 17 concentrations was

A

280 of 1.84 1 mg/ml.

In some cases the HIC peak containing hK2 was purified further by size exclusion chromatography (SEC) on a 10/30 Pharmacia S12 column. In this case the HIC peak containing hK2 was concentrated by ultrafiltration as above to less than 1 mL and then applied to the size exclusion column equilibrated in 100 mM ammonium acetate pH 7 or sodium borate pH 8. The flow rate was 0.7 mL/minute. The hK2 peak was then concentrated by ultrafiltration. The peak collected off SEC in ammonium acetate was lyophilized WO 98/21365 PCT/US97/20948 31 to remove the buffer and then was reconstituted in water. An aliquot of this sample was hydrolyzed in gaseous 6 N HCI under vacuum for 20 hours at 112 0

C

then reconstituted in 0.1 N HCI and analyzed on a Hewlett Packard Aminoquant amino acid analyzer utilizing pre-column derivatization of amino acids with OPA for primary and FMOC for secondary amines.

A HK1G 586.1 (see below) affinity resin was used to purify hK2 by affinity chromatography. HK1G 586.1 monoclonal antibody (mAb) was coupled with Pharmacia GammaBind plus Sepharose (cat. no. 17-0886) according to Schneider Biol. Chem., 257, 10766 (1982)). Briefly, HK1G 586.1 mAb and resin were incubated overnight at 4 0 C with rotation. Resin was centrifuged (500 X g for 5 minutes at 4 0 C) and washed twice with 0.2 M triethanolamine, pH 8.2. Amine groups were cross-linked in fresh cross linker solution (25 mM dimethyl pimelimidate dihydrochloride in 0.2 M triethanolamine, pH 8.2) for 45 minutes at room temperature (22 0 The resin was quenched with 20 mM ethanolamine, pH 8.2, for 5 minutes at room temperature and then washed twice with 1 M NaC1, 0.1 M PO4, pH 7.0. The resin was washed two more times with PBS and stored at 4°C with 0.05% NaN3 until use.

An Applied Biosystems Model 477a pulsed liquid phase sequencer was used to sequence the proteins and the peptides. The Model 477a employs automated Edman degradation chemistry to sequentially release amino acids from the N-terminus followed by PTH derivatization and chromatography by reversed-phase HPLC. The peptide samples were applied to the sequencer on biobrene-treated glass fiber filter supports and whole proteins were applied either to biobrene-treated filters or to pre-activated Porton filters (Beckman, Fullerton, CA). Samples sequenced off blots were first run as mini-gels on the Novex system (Novex, San Diego, CA) then transferred to Problot PVDF membrane, visualized with Commassie blue, the appropriate band cut out and sequenced directly from the PVDF membrane.

WO 98/21365 PCT/US97/20948 32 Monoclonal antibody production A/J mice were injected with 50 pl ofphK2 in complete Freund's adjuvant (CFA) i.p. on day 1, 25 gg of phK2 in incomplete Freund's adjuvant (IFA) i.p. on day 14 and 25 pg phK2 in PBS i.p. on day 28. Three days prior to fusion, mice were boosted with 10 lg ofphK2 in PBS i.v. Mice were sacrificed and a single cell suspension was prepared from the spleens. Immune B cells were fused with P3.653 myeloma cells. Cloned hybridomas were screened by ELISA and selected based on the reactivity of supernatants to hk2v 217 and phK2v 2 17 and minimal reactivity with PSA. Two clones selected by these criteria, clones HK1G464 and HK1G586, were subcloned using FACStar plus cell sorter to deposit single cells onto mouse spleen feeder layers. Subclones HK1G464.3 and HK1G586.1 were used for further studies.

Another fusion, which employed the same protocol described above except that the immunogen was hK2v 2 17, alum was used instead of CFA and IFA, BALB/c mice were used instead of A/J mice, produced clone HK1H247.

Hybridoma clones HK1G464.3, HK1G586.1, HK1H247, HK1A523 and HKD106.4 have been deposited with the American Type Culture Collection, in accord with the Budapest Treaty, and granted Accession Nos: HB11983, HB12026, HB12162, HB11876 and HB11937, respectively.

Polvclonal and monoclonal antibody production to hK2 peptide immunogens Sheep and goats were immunized subcutaneously with 100 pg of KLH-conjugated peptide in complete Freund's adjuvant (CFA) and boosted at three week intervals with 100 jig of peptide in incomplete Freund's adjuvant

(IFA).

For monoclonal antibodies to peptide immunogens, Balb/c mice were immunized subcutaneously with 100 gg of KLH-conjugated peptide in complete Freund's adjuvant (CFA) and boosted at three week intervals with 100 g of peptide in incomplete Freund's adjuvant (IFA). Alternatively, A/J mice were immunized twice intraperitoneally with 50 pg of KLH-conjugated peptide WO 98/21365 PCT/US97/20948 33 and mice with positive titers were boosted intravenously with 25 [tg of conjugate.

After the first three immunizations, blood from the animals was tested for the presence of antibody 6 to 10 days following the immunization.

Peptides were immobilized on 0.25 inch polystyrene beads (Clifton, Clifton Heights, PA) by incubating 1 4g of peptide conjugated to BSA (bovine serum albumin) per bead in pH 9.6 carbonate buffer over night at 4 C. The beads were washed three times with 0.01M phosphate buffered saline (PBS), pH 7.4 with 0.1% Tween 20 and blocked with 1% skim milk plus 1% BSA. These beads were incubated for 18 hours with 250 gl of a 1:100, 1:1000 or 1:10,000 dilution of animal sera. Following three washes, 250 pl of rabbit anti-sheep, anti-mouse or anti-goat antibody conjugated to horseradish peroxidase (Cappel- Organon Teknica Corporation, Durham, NC) was incubated with each bead for 3 hours on a horizontal incubator at 150 rpm. The enzyme signal was quantified spectrophotometrically using ortho-phenylene-diamine as a substrate. Nonimmune sera was used as a negative control and immune sera measurements were expressed as multiples of the control reading.

Lymphocytes from the spleens of mice with positive serum titers were fused with myeloma cells to produce hybridoma cells. Antibodies produced by clones of these cells were screened as described above. Positive clones were subcloned by limiting dilution and rescreened. Monoclonal hybridomas were injected into the peritoneal cavities of pristine primed mice to obtain ascitic fluid.

To purify monoclonal and polyclonal antisera, the antisera initially were subjected to an IgG separation by precipitation with saturated ammonium sulfate and size chromatography using an ultragel ACA-34 column.

The polyclonal antisera was further affinity purified using columns produced by cyanogen-bromide coupling of the peptides to Sepharose 4B. The purified antibody was eluted from the column with acidic PBS (pH 2.45).

WO 98/21365 PCT/US97/20948 34 ELISA assays A dried-down ELISA format was used to measure hK2 in the serum-free spent medium of the clones and in the fractions collected during hK2 purification. Microtiter plates (Becton Dickinson Labware, NJ) were coated with 50 ul of spent media or column fractions overnight at 37 0 C. The wells were washed with PBS 0.1% Tween 20 (PBST) and incubated for one hour with 50 [l of primary antibodies. The wells were washed again with PBS+T and incubated for one hour at 37 0 C with 50 ul of goat anti mouse-IgG or goat anti rabbit-IgG Fc antibodies coupled with horseradish peroxidase (1:500, Jackson Immunosearch Laboratories, Inc., West Grove, PA). The wells were washed with PBST, incubated with o-phenylenediamine dihydrochloride (OPD, Sigma, MO) for 5 minutes, and the colorimetric reaction was measured at A 49 0 with an ELISA reader (Biotek Instruments, Inc., model EL310, VT). All samples were assayed in duplicate. The serum-free spent medium from AV12 cells transfected with vector alone was used as negative control.

Antibodies were tested in a solution-based ELISA format using biotinylated phK2v 2 17 hK2v 21 7 and PSA. PSA was purified by the method of Sensabaugh and Blake Urology, 144, 1523 (1990)). Twenty ng of biotinylated hK2v 21 7 or phK2v 21 7 diluted in 50 pl Buffer A (8.82 mM citric acid, 82.1 mM sodium phosphate (dibasic), 10% BSA, 0.1% mannitol, 0.1% Nonidet pH 7.0) or 0.25 ng biotinylated PSA diluted in 10% horse serum (HS) in PBS was incubated with 50 ul of hybridoma supernatants, negative control supematants irrelevant hybridoma supernatant for phK2v 2 17 and hK2' 2 1 7 or ~g/ml irrelevant purified mAb in HS for PSA), or positive control supernatants 20 tg/ml purified PSM773 (anti-PSA) mAb in HS for PSA, or HK1D 104 (anti "hK2") hybridoma supernatant for phK2v 2 17 and hK2v 2 17 HCO514, a mAb against hCG, was used as a negative control in PSA assays, and ZTG085, a mAb against the tau, was used as a negative control in hK2 assays.

These mixtures of antibodies and antigens were allowed to incubate for 1 hour with shaking in a streptavidin coated microtiter plate (Labsystems, Helsinki, Finland). The plate was washed 3 times with 300 l of WO 98/21365 PCT/US97/20948 PBS, 0.1% Tween-20 (PBST), and incubated with 100 pl of gamma-specific goat anti mouse IgG-horseradish peroxidase conjugate (Jackson ImmunoResearch Laboratories, Inc., Westgrove, PA), diluted 1:10,000 in HS, with shaking for 1 hour. After a second PBST washing, color was developed for 30 minutes, with shaking, following the addition of 100 tl of 1 mg/ml ophenylenediamine in 50 mM phosphate-citrate buffer, 0.03% sodium perborate, pH 5.0 (Sigma Chemical, St. Louis, MO). The reaction was quenched by the addition of 50 pl 4 N H2SO4. The color intensity was determined by measuring the absorbance at 490 nm and 540 nm using a microtiter plate reader.

Absorbances above 2.6 at 490 nm were corrected with 540 nm reading. Sample values are averages standard deviation of triplicates. Control values are averages of duplicates.

Western blot assays Western blot analyses were performed using standard procedures.

Serum-free spent media were concentrated ten fold using Centricon 10 (Amicon, Inc., Beverly, MA) and subjected to SDS/PAGE using a 12% gel (Bio-Rad, Inc., Melville, NY). For analytical purposes, SDS/PAGE was performed on a Pharmacia PhastSystem using 8-25% gradient gels. After electrophoresis, proteins were transferred onto nitrocellulose membrane and blocked overnight at 4 0 C with 2% nonfat dried milk in PBS. Blots were rinsed then incubated with primary antibody (1:1000 dilution of ascites, or 1 gg/ml of purified mAbs or polyclonal Abs) for 1 hour at 22 0 C. Blots were then washed and incubated for minutes with secondary antibody (Goat anti-mouse-HRP or goat anti-rabbit- HRP, 1:500, Jackson Immunosearch Laboratories, Inc., West Grove, PA). The immunoreactive bands were detected by developing the blot using DAB (Sigma, St. Louis, MO) plus H202 or by using the ECL (Amersham, Buckinghamshire, England). system accordingto manufacturer's instructions.

Covalent complex formation To test for covalent complex formation, 0.175 .tM hK2 was incubated with 20 pM of inhibitor at pH 8 in 100 mM borate buffer. Inhibitors tested were 1-antichymotrypsin, 1-antitrypsin, 1-antiplasmin, antithrombin and WO 98/21365 PCT/US97/20948 36 2-macroglobulin. To 5 uil ofhK2 (10 tg/ml) was added the calculated tg of inhibitor prepared in 100 mM borate buffer and, if needed, each sample brought up to a total volume of 10 pl. Samples were incubated for 3 hours at 37 0

C

whereupon 1.5 pl of 7 X PhastSystem SDS sample buffer containing 35% 2mercaptoethanol was added and the sample boiled for 3 minutes in a water bath.

Samples were diluted 1/4 in SDS sample buffer prior to application to SDS/PAGE and Western analyses.

Proteolysis of Peptide Substrates To determine the ability of hK2 to cleave peptide substrates, peptides were dissolved in DMSO at 10 mg/ml then diluted 1:10 into 100 mM borate buffer pH 8 containing PSA, hK2 or trypsin. Typical experiments were performed as follows: 1 pl of peptide was added to 7 pl of 100 mM borate buffer and then 2 1l of hK2 (10 pg/ml), PSA (500 pg/ml) or trypsin (0.5 utg/ml) were added. In general, samples were incubated for 16 hours at 37°C.

Samples were quenched with 100 upl of 0.2% TFA/water and the quenched sample was applied directly to a Vydac C-18 reversed-phase column attached to a BioRad Model 800 HPLC equipped with an AS 100 autosampler, dual 1350 pumps and Biodimension scanning UV-VIS detector. Solvent A was 0.1% TFA/water and Solvent B was acetonitrile containing 0.1% TFA. The sample was applied in 90% solvent A and the gradient developed to 60% solvent B in 10 minutes. Absorbance was monitored simultaneously at 220 nm and 280 nm.

Peaks collected off HPLC were concentrated by vacuum centrifugation or lyophilization and then applied to the amino acid sequencer to identify individual fragments. In some cases 10 pl of the quenched sample mixture was applied directly to the sequencing membrane and, since the sequence was known, the cleavage sites were determined from the distribution of amino acids present in each cycle.

Protease assays using chromogenic substrates Assays to measure the hydrolysis of para-nitroanilide derivatized substrates were performed using an HP 8452A UV-VIS spectrophotometer WO 98/21365 PCT/US97/20948 37 equipped with a programmable, thermostated 7-position cell holder. Assays were performed in 100 mM sodium borate pH 8 incubated at 37 0 C, the absorbance increase monitored at 405 nm. Methoxysuccinyl-Arg-Pro-Tyr-paranitroanilide (MeO-Suc-R-P-Y-pNA) and H-D-pro-phe-arg-para-nitroanilide (P- F-R-pNA) were 1 mM in the assay.

An ABI model 431A peptide synthesizer using standard FastMoc chemistry was employed to synthesize all of the peptides listed in Figure 16 except angiotensinogen and oxidized beta-chain of insulin which were obtained from Sigma. The mass of each synthesized peptide was confirmed by mass spectrometry (University of Michigan, Core Facility) using ES/MS. An ABI Model 477a sequencer described above was employed to confirm peptide sequence.

Conversion of phK2v 2 7 to hK2V217 Samples ofphK2v 21 7 at 100-400 jtg/ml in 50 mM sodium borate were incubated with 1% w/w trypsin or hK2 at 37°C. The conversion of pro to mature was monitored by dilution of 1-2 pg of hK2v 2 1 7 starting material into 100 Vtl HIC Buffer A and resolution of the two forms by HIC-HPLC as described above. The incubation of hK2v 2 7 with phK2v 217 was conducted in the same manner except that comparable amounts of the two forms were incubated together as seen in Figure 17B.

Example 2.

Expression and purification of hK2 2 1 7 in mammalian cells To express hK2 in mammalian cell lines, a 0.8 kb fragment encoding the entire coding sequence of hK2 (pphK2) (Figure 2) was amplified using PCR, subcloned into the vector PCR II (TA) and several clones were isolated. The nucleotide sequence of the entire pphK2 insert in a few of these clones was determined to detect any mutations that may have been caused by PCR amplification.

Two clones, one having a wild type hK2 insert, TA-hK2, and one having a mutant hK2 insert, TA-hK2v 21 7 were selected for further analysis. TAhK2 v 217 contains a substitution of T for C at codon 650 of hK2 resulting in a WO 98/21365 PCT/US97/20948 38 conservative substitution of valine (GTT) for alanine (GCT) at amino acid residue 217 of hK2 (Figure To obtain mammalian expression vectors, pphK2 inserts of TA-hK2 and TA-hK2v 217 were subcloned into plasmid PGT-d under the control of the GBMT promoter resulting in plasmids pGThK2 and pGThK2v 21 7 (Figure The GBMT promoter is composed of several regulatory sequences and is activated by the adenovirus Ela protein(s) (Berg et al., supra (1992)).

To determine whether the product of the pphK2 v2 1 7 gene would be expressed in mammalian cells, the plasmid pGThK2 v2 17 was transfected into AV12-664 cells. This cell line is derived from a tumor induced in Syrian hamster by adenovirus type 12 and expresses the adenovirus Ela protein. The Ela protein activates the GBMT promoter which results in the expression of the gene product under the control of this promoter. After 2-3 weeks, MTX-resistant clonal cells were isolated and their spent medium were analyzed by Western blots. Several clones were identified which secreted into the media a polypeptide immunoreactive to anti-pphK2 antiserum. One clone (AV12-pGThK2v 217 #2) was selected for further characterization and protein purification.

To purify hK2 polypeptides, the serum-free spent medium from AV12-pGThK2v 21 7 clone #2 was collected after 7 days, concentrated and subjected to anion exchange chromatography (Figure 4A). The peak of hK2 activity eluted at approximately 0.2 M NaCl as determined by ELISA assays (dotted line). The ELISA assay correlated well with the appearance of about a 34 kD band of protein seen by SDS/PAGE in the same fractions.

The hK2-positive fractions from the anion exchange column were collected and subjected to hydrophobic interaction chromatography (HIC) (Figure 4B). A major portion of the A 2 0 o was not retained on HIC column. The main peak retained on HIC, which eluted at 22 minutes, also showed the highest peak of activity by ELISA assay (dotted line, Figure 4C). A major protein band at about 34 kD was also observed by SDS-PAGE. When the 22 minute peak from HIC was resolved by SEC, typically about 80-90% of the protein A 28 0 eluted at 19.4 minutes, a retention time consistent with a protein of approximately 34 kD (Figure 4C). The only other protein peak on SEC, eluting WO 98/21365 PCT/US97/20948 39 at 16.7 minutes, corresponded to about a 70 kD protein observed in previous purification steps.

To further identify the purified protein, approximately 2.5 tg of the protein was subjected to automated N-terminal analysis which yielded the following sequence: Val-Pro-Leu-Ileu-Gln-Ser-Arg-Ileu-Val-Gly-Gly-Trp-Glu-.

No competing sequence was evident from the profile of amino acids released sequentially by the Edman degradation procedure. By analogy to PSA this protein is phK2v 21 7 since the known sequence of mature PSA (isolated from seminal fluid) begins with Ileu-Val-Gly- and pPSA and phK2 have been postulated to have an extra 7 amino acids at the N-terminus (Figure Amino acid analysis of this protein yielded an amino acid composition consistent with the predicted sequence of phK2v 217 This phK2 polypeptide was isolated and purified in mg quantities.

Example 3.

Characterization of phK2 2 1 7 and generation of hK2 2 17 To examine the efficiency of the purification scheme employed in Example 2, 1.5 tg of purified phK2v 2 17 was subjected to SDS/PAGE in the presence or absence of beta-mercaptoethanol (BME), and the gel was stained with silver. Results showed that the phK2v 21 7 in the sample was about 95% pure (Figure It also showed that phK2v 21 7 migrated at about 30 kD in the absence of BME, and at about 34 kD in the presence of BME. This pattern is similar to that observed for the PSA purified from seminal fluid (Figure The amino acid sequence of hK2, deduced from the cDNA sequence, shows the presence of one potential N-linked glycosylation site at residue 78 To determine if this site is glycosylated, phK2v 217 was subjected to SDS/PAGE, transferred to nitrocellulose paper, reacted with digoxigenin (DIG)-coupled lectins followed by horseradish peroxidase labeled anti-DIG.

In Figure 6 (lane 2 ug of phK2 was stained with concanavalin A (Con A) suggesting the presence of two nonsubstituted or 2-O-substituted amannosyl residues in the protein. Lane 2 shows Con A staining of the positive WO 98/21365 PCT/US97/20948 control glycoprotein, ZCE025 mAB. Both the heavy chains (50 kD) and light chains (25 kD) of this mAb are known to contain N-linked oligosaccharides with mannose cores. Lane 3 shows that a nonglycosylated protein (BSA) fails to react with the Con A lectin. phK2v 217 also reacted with RCA (Gal bl-4GlcNAc specificity) and AAA, linked fucose specificity). This pattern oflectin reactivity is consistent with the presence of complex N-linked oligosaccharides.

The oligosaccharides on phK2v21 7 also contain sialic acid since both SNA (sialic acid linked a(2-6) to galactose) and MAA (sialic acid linked a(2-6) to galactose were reactive with phK2v 21 7 The sequence of the pro region of hK2 is VPLIQSR. An enzymatic cleavage at the carboxy-terminal end of the arginine in this pro sequence would convert phK2 to hK2. A mild trypsin digestion was developed to hydrolyze the peptide bond of purified phK2v 2 17 at this position. phK2v 2 17 was incubated with 1% trypsin and the conversion was monitored by HIC-HPLC (Figure This procedure resulted in a complete conversion of phK2v 2 7 to hK2 217 The peak designated hK2'v2 7 was N-terminally sequenced and shown to begin with the sequence, IVGGWE, which is the N-terminus for the mature form of hK2. No sequence other than the above was detected, demonstrating that this mild trypsin treatment does not result in any significant level of non-specific cleavage. SDS/PAGE of trypsin-treated samples showed a small but discernible increase in mobility, generally consistent with a minor reduction in mass of 826 daltons, the mass of the pro peptide.

Example 4.

Generation of hK2-specific Abs phK2v 2 7 and hK2 v2 17 were used as immunogens to generate mAbs against hK2. Hybridomas were screened based on high reactivity with hK2v 21 7 or phK2v 217 and minimal reactivity with PSA. Representatives of mAbs obtained from the hybridomas are shown in Table 2. Immunization with phK2v 217 resulted in mAb HK1G586.1 and HKIG 464.3. HK1G586.1 was hK2-specific, since it recognized both phK2v 217 and hK2v 2 1 7 but not PSA. On the other hand, WO 98/21365PC1S7204 PCTfUS97/20948 41 HK I G464 was phK2-specific, since it only recognized phK2Q and not hK2v 2 17 or PSA.

Table 2 Specificity of various mnAbs raised to hK2 2 1 7 and phK2 v 2 17 A. mnAbs raised to phK2 2 17 mAbs PSA hK2 2v 2 17 iJVI Irrelevant Ab 0.245 0.162 0.125 positive control 2.242 0.06 9.196 8.91 0.02 HK1 G586.1 0.150 ±0.004 11.154 ±0.18 10.146 ±0.87 HK1G464.3 0.143 0.03 0.245 0.02 6.644 0.17 (Ascites 1:2000) B. mnAb raised to hK-2v2l 7 mAb tested PSA hK2 v 2 1 7 phK2 v217 Irrelevant Ab 0.157 ±0.18 0.132 ±0.01 0.153 ±0.01 Positive control 2.768 0.08 8.342 1.3 9.673 0.99 Media only (neg. 0.129 0.02 0.240 0.02 0.247 0.01 control) HK1H247 0.157 ±0.01 9.34 ±0.7 0.179 ±0.004 Immunization 'with hK2 v 2 1 7 resulted in mAb HK IH247. This mAb was hK2-specific since it recognized only hK2 v 2 17 but not phK2 v 2 1 7 or PSA.

These results show that phK2 v 217 and jhJ( v 2 1 7 are effective as immunogens in generating mAbs specific for different forms of hK2.

Western blot analysis was used to examine if HK1G586 recognizes hK2 in seminal fluid (Figure hK2-immunoreactive bands at about 22 kD, 33 kD, and 85 kD were recognized by this mAb. A similar hK2immunoreactive pattern in seminal fluid was also recently reported by Deperthes et al., Biochem. Biophv. Acta, 1245, 311 (1995). This result indicates that a WO 98/21365 PCT/US97/20948 42 mAb raised to hK2v 217 recognizes native hK2 in seminal fluid. All the antibodies raised to hK2v 2 17 or phK2v 217 also recognized the corresponding form of hK2 and phK2 indicating that hK2 and phK2 are immunologically similar to hK2v 2 17 and phK2v 21 7 respectively (see below).

To prepare additional anti-hK2 antibodies (Abs), a direct primary structure comparison between the members of human kallikrein gene family and computer-aided antigenicity and hydrophobicity analyses was conducted. From this comparison, several hK2 oligopeptide sequences were selected. The selected hK2 peptides correspond to mature hK2 amino acid residues 8-26 (SEQ ID NO:19), 15-26 (SEQ ID NO:26), 41-56 (SEQ ID NO:20), 43-66 (SEQ ID NO:24), 153-167 (SEQ ID NO:21), 17-71 (SEQ ID NO:22) and 210-235 (SEQ ID NO:25). The peptide corresponding to amino acids 17-71 was synthesized in order to increase the likelihood of producing antibodies that recognize the native form of hK2. The peptides were synthesized and HPLC purified in the Protein Core Facility at Mayo Clinic/Foundation. Peptides were conjugated with keyhole limpet hemocyanin (KLH) and BSA for immunogens and assay reagents, respectively. Sheep, goat and mice were immunized with KLH-hK2 peptides for polyclonal (sheep and goat, SEQ ID Nos:20 and 21, and SEQ ID Nos:19, 20, 21, 24, 25 and 26, respectively)) and monoclonal antibody (mice; SEQ ID Nos:19, 20, 21, 24, 25 and 26) production. The 17-71 peptide (SEQ ID NO:22) was oxidized to generate an intramolecular disulfide bond between cys 26 and 42 and used to immunize goats and mice for polyclonal and monoclonal antibody production, respectively.

The hK2 41-56 Ab from sheep was first purified by hK2 41-56 peptide-affinity column and then used for Western blot analysis. The antibody recognized recombinant hK2. The detection of hK2 by the hK2 41-56 Ab was abolished by addition of excess hK2 41-56 peptide but not by PSA 41-56 peptide. Moreover, monoclonal anti-hK2 41-56 peptide antibodies were highly specific for hK2 protein in Western analysis.

WO 98/21365 PCT/US97/20948 43 Anti-hK2 153-167 peptide antiserum (sheep) recognized recombinant hK2. These results suggested that antibodies to peptides 41-56 and 153-167 react with two distinct epitopes in hK2 polypeptides.

Antisera against hK2 amino acid residues 210-235 showed the highest immunoreactivity.

A goat antiserum raised against hK2 peptide 17-71, which has 69% homology with corresponding region of PSA, recognized recombinant hK2 protein but not PSA.

Rabbit antiserum to bacterially expressed recombinant hK2 protein, which recognizes both PSA and hK2, detected a doublet protein band in concentrated LNCaP cell medium from LNCaP cells which were treated with androgen. In contrast, no immunoreactive protein was detected in LNCaP cell medium from LNCaP cells which were not treated with androgen. Thus, the immunoreactive proteins were induced by androgen. Moreover, the upper band in LNCaP media is a PSA-related protein because a PSA-specific antiserum (rabbit anti-PSA antisera, which was raised against bacterially expressed recombinant PSA) detected mainly the upper band. The lower band in LNCaP media is an hK2-related protein because a mouse monoclonal antibody (HK1A523) against hK2 41-56 peptide that has monospecificity for hK2 recognizes the lower protein band. These results were confirmed by sequence analysis of N-terminal amino acids of each protein in the doublet bands.

Immunohistochemistry studies of paraffin-embedded human prostate tissue sections (see Example 10) which employed a monoclonal antibody for hK2 peptide 41-56 (HK1A523) showed that hK2, like PSA, is produced in the epithelia, but not in stroma. Moreover, the immunostaining is specific for hK2 protein in the prostate, as other tissues tested were negative for hK2.

Example Expression of hK2 in mammalian cells To express wild type hK2 (hK2) in mammalian cells pGThk2 (Figure 3) was transfected into AV 12 cells. Several clones expressing an hK2 WO 98/21365 PCT/US97/20948 44 polypeptide were identified by Western analysis using HK1D 106.4 (a hK2specific mAb raised to a polypeptide corresponding to amino acid residues 17-71 ofhK2). Clone AV12-hK2#27 (AV12-hK2) was selected for further analysis based on its higher hK2 expression level. Cells transfected with vector alone (pGTD) showed no reactivity with HKlD 106.4.

ELISA using HK1D 106.4 mAb indicated the presence of about 0.5-1 pg/ml of an hK2 polypeptide in the serum-free spent medium of AV12hK2 at day 7. The same method used in purification ofphK2v 2 1 7 from AV12hK2 v 2 17 was used to purify hK2 polypeptides from the day 7 spent medium of AV12-hK2. This resulted in low yields of purified hK2 polypeptides which were unstable to the purification procedures.

hK2 polypeptides were partially purified using the above method, subjected to SDS/PAGE, electroblotted and subjected to N-terminal amino acid sequencing. This analysis indicated that the hK2 polypeptide in the spent medium of AV12-hK2 at day 7 has the sequence, IVGGWECEK at N-terminus.

No competing sequence was evident from the profile of amino acids released sequentially by the Edman degradation procedure. By comparison to PSA, this sequence corresponds to mature hK2 (hK2). Amino acid analysis of this protein was also consistent with that of hK2.

This finding demonstrates that phK2v 21 7 was predominantly present in the serum-free spent medium of AV12-hK2v 217 at day 7, whereas predominantly hK2 was present in the serum-free spent medium of AV 12-hK2 at day 7. To examine the form of hK2 present in the serum-free medium of AV12hK2 at day 1 this material was partially purified by affinity purification using HK1G 586.1 mAbs. The 34 kD protein was transferred onto PVDF and was subjected to N-terminal analysis, yielding a sequence, VPLIQSRIVGG. No competing sequence was evident from the profile of amino acids released sequentially by the Edman degradation procedure. Compared with PSA, this sequence corresponds to phK2. This suggests that the hK2 polypeptide is secreted as the pro form by both AV12-hK2 and AV12-hk2 v 21 7 cells. However, WO 98/21365 PCT/US97/20948 while phK2v 217 is stable and is not converted to hK2v 21 7 phK2 is unstable and is easily converted to hK2 extracellularly.

Example 6.

Biosynthesis of hK2 To further study the biosynthesis of hK2 in mammalian cells, a time course study was conducted where serum-free spent media from AV12-hK2 clone #27 was collected each day for 8 consecutive days, concentrated and subjected to SDS/PAGE. The proteins were transferred to nitrocellulose membrane and probed with either HK1D 106.4 or HKlG 464.3 mAbs (Figure As also shown in Figure 9, HKlD 106.4 recognizes both phK2 and hK2 whereas HK1G 464.3 recognizes only phK2 as its epitope lies in -7 to +7 region of hK2. Expression of hK2 polypeptides (about 34 kD) peaked by day 3 and plateaued thereafter as detected by HKID 106.4 mAbs. Two other immunoreactive bands migrating at about 70 kD and about 90 kD were also detected from day 4 onwards.

On the other hand, when the same samples were blotted and probed with HKIG 464.3, a gradual reduction in the level of hK2 was detected by day 4. By day 8, very low levels of hK2 were found in the spent medium.

This result shows that phK2 is being secreted into the media by AV12-hK2 cells and is gradually converted to hK2 extracellularly. Curiously, the 70 kD and kD bands were not observed with HK1G 464.3 mAbs indicating that these bands are either homo-oligomers of hK2 or are hK2 covalently complexed with a yet unknown protein(s). Even though the identity of these bands is not known at this time, they can serve as markers for the presence of hK2 in the spent media. In Figure 9, purified phK2 217 and hK2v 217 proteins were used as controls.

To study the biosynthesis of hK2 2 17 in AV12 cells a similar time course study was conducted on AV12-hK2 v217 clone As shown in Figure expression of hK2' 2 17 polypeptides peaked by day 3 and did not vary much from day 4 onwards, as detected by HK1D 106.4 mAbs. Similar results were obtained when the blot was probed with HK1G 464.3 mAbs (Figure 10). This indicated that AV12-pGThK2v 21 7 clone #2 cells are expressing phK2' 217 from day 1 WO 98/21365 PCT/US97/20948 46 onwards and that for at least 8 days thereafter, this protein is not converted to the mature form. These results are in contrast with those ofphK2, which is converted to hK2 if left in the media for 8 days, indicating that phK2v 21 7 is stable in media at 37 0 C for 8 days.

To study whether extracellular conversion of phK2 to hK2 correlates with the viability of AV12-hK2 clone #27 cells in culture, clone #27 cells were counted using trypan blue exclusion. Expression of hK2 in the spent medium was measured by ELISA using both HK1D 106.4 and HK1G 464.3 mAbs. As shown in Figure 11, the number of viable cells peaked at 38 million in culture by day 3 and gradually decreased thereafter. By day 8, the number of viable cells were reduced to less than 10 million. The expression of phK2 (measured by HK1G 464.3) also peaked by day 3 and gradually declined thereafter.

On the other hand, expression of hK2 (measured by HK1D 106.4) peaked by day 3 but plateaued thereafter. This result indicates that phK2 is secreted by AV12-hK2 cells and a fraction of it is gradually converted extracellularly to hK2 by day 4. Moreover, it shows that conversion of phK2 to hK2 clearly correlates with a decrease in cell viability, indicating that the extracellular proteases released by the dying cells may be one of the factor(s) responsible for this conversion. Expression of hK2 was highest at the point in which cells were most viable. A decrease in hK2 paralleled a decrease in cell viability, suggesting the hK2 is secreted by these cells, as opposed to being released following cell death and lysis. Also, a rise in hK2 corresponded to a drop in phK2, indicating that the pro form of hK2 was automatically converted to the mature form over time.

To examine the biosynthesis of hK2 in prostate carcinoma cells hK2 was expressed in DU145 and PC3 cell lines. DNA encoding pphK2 was cloned into plasmids pLNCX and pLNSX (Miller and Rosman, BioTechniques, 7, 980 (1989)), under the control of the CMV and SV40 promoters, respectively.

The resulting plasmids, pLNC-hK2 and pLNS-hK2, respectively, were transfected into PC3 and DU145 cells, respectively, and clones were selected in WO 98/21365 PCT/US97/20948 47 media containing G418. Clones expressing high levels of hK2 were selected (PC3-hK2 and DU145-hK2) by ELISA and Western blots.

To assess the level of hK2 and phK2 in the media, serum-free medium of PC3-hK2 and DU145-hK2 cells were subjected to Western blot analysis using HKlD 106.4 (hK2-specific) and HKIG 464.3 (phK2-specific) mAbs (Figure 12). Results showed that phK2 is present in the spent medium of both DU145-hK2 and PC3-hK2. This indicates that in prostate carcinoma cells hK2 is secreted as phK2 and is converted to the mature form extracellularly.

This finding confirms the results previously obtained with AV12 cells.

Predominantly phK2 was detected in the spent medium of PC3-hK2 cells even after 7 days, however, predominately hK2 was present in the serum-free medium of DU145-hK2 starting from day 1. This is probably due to abundance of extracellular proteases in DU145 spent medium.

To examine whether the above results were limited to just one clone, 3 other independently isolated clones of AV12-hK2 and 4 other independently isolated clones of AV12-hK2v 21 7 were tested for the expression of hK2 polypeptides. Serum-free spent medium of the clones were collected at day 7 and tested for the expression ofhK2 by Western blots using HK1D 106.4 (hK2-specific) and HK1G 464 (phK2-specific) mAbs (Figures 13 and 14). In all of the AV12-hK2 clones, HK1D 106.4 mAb detected not only the major 34 KD band but also the 70 kD and the 90 kD bands that are indicative of the presence of hK2 (Figure 13). HKIG 464.3 detected very low levels of phK2 in all of the AV12-hK2 clones (Figure 14). This result indicates that predominantly hK2 is present in the spent medium of all the AV12-hK2 clones verifying the biosynthetic mechanism established for AV12-hK2 #27 clone. The same analyses were used on AV12-hK2v 21 7 clones (Figure 14). Results indicated that only phK2v 2 17 was present in the spent medium of these clones at day 7 verifying our findings with the AV12-hk2 v2 1 7 clone.

The above results collectively suggest that hK2 is expressed as the pro form in mammalian cells and is converted to mature form extracellularly by as yet unknown proteases. These results also suggest that phK2 may be WO 98/21365 PCT/US97/20948 48 present in the biological fluids and therefore can be a useful diagnostic marker for pCa and BPH.

Example 7.

Enzymatic activity and specificity of hK2 and hK2 2 1 7 A small amount of hK2 was purified to sufficient purity to determine its enzymatic activity and substrate specificity. The general activity of hK2 was measured by determining its amidolytic activity chromogenically on p-nitroanilide derivatives of peptides (Table The p-nitroanilide released by proteolytic digestion of these substrates is measured at absorbance A 4 05 The substrate methoxysuccinyl-Arg-Pro-Tyr-para-nitroanilide (MeO-Suc-R-P-YpNA) is used to measure chymotrypsin-like proteases which cleave at the phenylalanine. This substrate has been used previously to measure the activity of PSA (Christensson et al., Eur. J. Biochem., 194, 755 (1990)). The substrate H-D-Pro-Phe-Arg-para-nitroanalide (P-F-R-pNA) is specific for trypsin-like proteases which cleave at arginine hK2 was found to have overall activity more than 10 times higher than hK2v 217 on P-F-R-pNA and neither protein showed an ability to hydrolyze MeO-Suc-R-P-Y-pNA, the chymotrypsin substrate. Other comparable substrates containing trypsin-like sites for cleavage (lysine, arginine) were also tested and hK2 was found to hydrolyze the substrate P-F-R-pNA with the highest rate.

These findings indicate that hK2 has trypsin-like activity.

WO 98/21365 PCT/US97120948 49 Table 3 Amidolytic Activity of hK2, hK2v 2 1 7 PSA and trypsin on Chromogenic Substrates Protease MeO-Suc-R-P-Y-pNA P-F-R-pNA xmol/min/gg/ml xmol/min/ig/ml hK2v 217 0 8.7 pmol hK2 0 4.1 nmol PSA 13.3 pmol 2.2 pmol Trypsin 3.8 pmol 25.5 nmol The specificity ofhK2 and hK2 v217 was examined in more detail by the use of peptide substrates together with N-terminal amino acid sequence analysis to determine which peptide bonds had been hydrolyzed. Figure shows amidolytic activity on the polypeptide CALPEKPAVYTKVVHYRKWIKDTIAAN, which has both potential trypsin and chymotrypsin cleavage sites. hK2v 21 7 cleaved at both a trypsin and chymotrypsin site with the trypsin-like cleavage at a 2:1 ratio over the chymotrypsin-like cleavage. As a control in these experiments phK2v 2 17 was also incubated with this peptide and showed no amidolytic activity. hK2 showed specificity different than hK2v 2 1 7 towards this peptide substrate. No chymotrypsin-like specificity was seen for hK2 on this substrate and its activity was exclusive for the trypsin-like site None of the other lysine (K) residues in this polypeptide were hydrolyzed indicating that the specificity of hK2 was exclusive for the arginine residue.

As a control trypsin was also studied on this substrate and cleaved all lysine and arginine sites except the K-P bond which is known not to be a site suitable for trypsin cleavage. Trypsin cleaved the R-K site of the 210- 236 substrate (peptide Figure 16) at rates approximately 4X faster than hK2 and about 4000X faster than hK2v 2 17 No chymotrypsin-like bonds were cleaved by trypsin. PSA cleaved the Y-R bond primarily. A minor trypsin-like activity WO 98/21365 PCT/US97/20948 on the R-K bond was also seen for PSA (Figure 15). This was consistent with the minor trypsin-like activity previously seen for PSA on the chromogenic substrate (Table 2).

Several other peptide substrates were also incubated with hK2 and PSA (Figure 16). In all of the peptides tested, hK2 had specificity only for selected arginines, and PSA primarily for selected tyrosine phenylalanine and leucine residues. Only peptide #1 in Figure 16 was cleaved by hK2v 2 17 as detailed by the chromatograms in Figure Example 8.

Activation of phK 21 7 by hK2 The sequence of peptide #3 in Figure 16 corresponds to amino acid residue -7 to +7 ofphK2. This region contains the pro peptide, VPLIQSR, which is found as an N-terminal leader peptide in phK2v 21 7 As mentioned above, hK2 was able to cleave this peptide releasing the propeptide region, but hK2 v 2 17 was not. To delineate if hK2 can cleave this pro sequence on a native substrate, its ability to convert phK2v 21 7 to hK2v 2 17 was monitored. phK2' 217 was incubated with 1% hK2 and the conversion was monitored by the HIC-HPLC method (Figure 17A). Results showed that hK2 was able to convert phK2v 2 17 to hK2v 21 7 albeit at a rate about 30X slower than trypsin. When phK2 21 7 was incubated with 40% hK2 v2 17, no difference in the ratios of the two hK2 forms was detected even after 6 hours (Figure 17B). This corroborated previous observations with the peptide substrate and showed that, even on a native substrate, only hK2 and not hK2v 21 7 cleaved the pro region of hK2.

These results collectively demonstrate the stability of phK2v 217 and hK2v 2 1 7 upon extended incubation. When compared with hK2 2 7 hK2 was shown to have a higher proteolytic activity, higher degree of specificity and, in particular, to have a specificity for the pro form of hK2 as demonstrated by activity on the pro peptide in Figure 15 and its activity toward phK2v2 1 7 in Figure 17.

These results demonstrate a significant difference in enzymatic activity between hK2 and hK2v 2 7 and may help explain the low yields associated WO 98/21365 PCT/US97/20948 51 with attempts to purify hK2 from the medium compared to phK2v 2 17 Highly purified preparation ofhK2 may not be stable due to autolysis as seen for other active proteases. These results further suggest that, in addition to immunological tests, enzymatic activity on hK2-specific substrates could be used to monitor the level of this protein in bodily fluids.

Example 9.

Formation of inhibitor complexes with hK2 PSA has been shown to form complexes with a2 macroglobulin (MG) and the serine protease inhibitor, antichymotrypsin (ACT). To explore its complex formation, hK2 was incubated with a series of common proteases present in human plasma (ACT, a2-antiplasmin, antithrombin III, and alantitrypsin (Travis and Salvesen, Ann. Rev. Biochem., 52, 655 (1983)) and the mixtures were analyzed by Western blot (Figure 18). Any covalent complex of hK2 with these serpins should result in about a 80-100 kD band on SDS/PAGE under reducing conditions.

ACT and a2-antiplasmin formed significant complexes with hK2 (Figure 18, lane 1 and Antithrombin III (lane 3) and al-antitrypsin (al protease inhibitor, lane 4) formed no detectable complex with hK2. MG, a major component of blood plasma, also rapidly complexed with hK2 (lane This complex corresponds to Mr of about 200 kD and 120 kD, which were also formed when PSA was incubated with purified MG (Figure 18, lane 8, see below). It was particularly interesting that hK2 did not form complexes with a lantitrypsin, even though this protein inhibits a wide range of trypsin-like proteases (Loebermann et al., J. Mol. Biol., 177, 531 (1984); Carrell and Travis, TIBS, 10, 20 (1985)).

It was not surprising that hK2 formed a complex with a2antiplasmin since this protein has arginine residues in its inhibitor active site (Hunt and Dayhoff, Biochem. Biophy. Res. Comm., 95, 864 (1980); Chandra et al., Biochemistry, 22, 5055 (1983); Potempa, et al., Science, 241, 699 (1985); Shieh et al., J. Biol. Chem., 264, 13420 (1989); Mast et al., Biochemistry, 1723 (1991)). However, it was also not expected that hK2 would form a WO 98/21365 PCT/US97/20948 52 complex with ACT, since ACT has a leucine in its inhibitor active site. Clearly the structural similarities between PSA and hK2 influence their complex formation with a common inhibitor even though their proteolytic specificity is entirely different as demonstrated in Figure 16 and Table 2.

When spiked into human female serum hK2 formed a rapid complex with MG as detected by Western blot (Figure 18). Lane 1 and lane 3 are hK2 and serum only controls, respectively. Lane 2 is hK2 incubated with ACT showing the 90 kD hK2-ACT complex and residual hK2. Lanes 4 and are hK2 spiked into serum for 15 minutes and 1 hour, respectively. Lane 6 is hK2 incubated with purified MG for 4 hours. Lane 7 is PSA spiked into serum for 15 minutes and Lane 8 is PSA incubated with purified MG for 4 hours.

These results show that MG is the major hK2 or PSA complex formed when hK2 or PSA are spiked into human serum in in vitro experiments.

Since PSA complex with ACT is known to occur in the blood serum of patients with prostate disease, it is believed that hK2 present in serum would also form some level of ACT complex.

Discussion The in vivo protein processing and secretion mechanisms for PSA or hK2 are not known. The results presented herein show that phK2 is secreted by AV12-hK2, DU145-hK2, and PC3-hK2 cells, indicating that hK2 is normally secreted as phK2 and the propeptide is cleaved extracellularly. This suggests that phK2 exists in biological fluids and thus could be a useful diagnostic marker for pCa or BPH.

Both the mutant form of hK2 (hK2 2 17 and the wild type form of hK2 were purified from AV12 cells. hK2 was very unstable to the purification procedures employed which, as found with other proteases, may be due to its autocatalytic property, and makes it very difficult to purify hK2 or phK2 in quantities sufficient for use as immunogens and calibrators. In contrast, phK2v 2 1 7 is highly stable and is converted to hK2v 21 7 which was also stable, by trypsin digestion. Purified phK2v21 7 and hK2v 217 provided immunogens to generate mAbs specific for hK2 and phK2.

WO 98/21365 PCT/US97/20948 53 Example Immunoreactivity of monoclonal antibody 586 with prostate tissue Immunohistochemistry of normal prostate tissue with HK1523 showed staining in epithelia but not stroma. Moreover, hK2 expression is prostate-specific as other tissues, kidney and pancreas, showed no staining.

To determine if hK2 is expressed in prostate tissue and, if so, is correlated with prostate cancer, 264 radical prostatectomy specimens, of which 257 were from untreated patients (Figure 20) and 7 were from androgen deprivation therapy treated patients (Figure 21), were analyzed in a comparative study. Each specimen was analyzed for the cytoplasmic expression of hK2 in areas with benign epithelium, high grade prostatic intraepithelial neoplasia (PIN) and adenocarcinoma.

Prostate tissue was weighed, measured in three dimensions and inked. The apex and base were amputated at a thickness of 4-5 mm and serially sectioned at 3 mm. The remaining prostate was serially sectioned at 4-5 mm intervals by knife perpendicular to the long axis of the gland from the apex of the prostate to the tip of the seminal vesicles. Transverse sections were prepared and stained with hematoxylin and eosin. A single slice of the radical prostatectomy of each patient which encompassed cancer and benign tissue was fixed in neutral buffered formalin and embedded in paraffin by methods well known to the art.

Tissue sections on slides were deparaffinized by immersion in xylene and then in 95% ethanol. Endogenous peroxidase activity was blocked by incubating sections for 10 minutes in methanol/H 2 0 2 and then rinsing sections in tap water. Sections were then placed in 10 mM citrate buffer, pH 6.0, and steamed for 30 minutes. Sections were cooled for 5 minutes prior to rinsing in cold running tap water. Nonspecific protein binding was blocked by incubating sections for 10 minutes with 5% goat serum. Slides were then gently drained.

Primary antibody, hK1G586 or PSM773 at 0.5 [lg/ml, was added to the sections for 30 minutes at room temperature and then the sections were rinsed with tap water. Tissue sections were then incubated with biotinylated WO 98/21365 PCT/US97/20948 54 rabbit anti-mouse antibody for one hour and rinsed with water. Sections were incubated with peroxidase conjugated streptavidin (1:500) for 30 minutes, then rinsed in tap water. Subsequently, sections were incubated with 3-amino-9ethylcarbazole (ABC) chromagen solution for 15 minutes prior to rinsing in tap water. Sections were then counterstained with mercury-free hematoxylin for one minute and rinsed for 5 minutes in running water. Slides were mounted with aqueous mounting media (glycerol gelatin). The percentage of cells staining was recorded at 10% increments from 0-100% for benign epithelium, high grade PIN and adenocarcinoma.

Benign atrophic glands showed the least amount of staining, particularly in areas of inflammation, in which there was virtually no immunoreactivity. In hyperplastic acini and benign acini with no evidence of atrophy, there was moderate to intense immunoreactivity, usually appearing in a granular pattern in the secretory luminal cell layer just above the nuclei, often extending to the luminal surface. There was no staining of the uroepithelium of the urethra or the viva montanum, although the underlying glands often showed immunoreactivity. Basal cells were usually negative.

Specimens with high grade PIN showed intense immunoreactivity throughout the cytoplasm and in the cytoplasmic apical blebs in a majority of cases. There was no apparent difference in the immunoreactivity among the different patterns of PIN except for the cribiform pattern which was usually decreased in intensity centrally when compared to the periphery.

Carcinoma specimens showed intense cytoplasmic reactivity in virtually all cases. Cells with abundant cytoplasmic vacuoles showed less staining, including signet ring cells and areas of mucin; otherwise the cytoplasm was intensely stained. The greatest intensity was observed in the highest grade adenocarcinoma (Gleason pattern 4) which showed immunoreactivity in virtually every case. Foci with cribiform carcinoma were similar to cribiform PIN in that there was a greater intensity in the periphery than centrally. The peripheral edge and the advancing edge of the carcinomas were always intensely immunoreactive.

WO 98/21365 PCT/US97/20948 In seven specimens from patients who had undergone androgen deprivation therapy, there was little or no immunoreactivity in the majority of benign atrophic acini, although PIN and adenocarcinoma occasionally showed intense cytoplasmic staining.

A summary of the number of cells staining with monoclonal antibody HK1G586 in the benign epithelium, high grade PIN and adenocarcinoma is shown in Table 4. Pair wise analysis, benign versus PIN, benign versus carcinoma, and PIN versus carcinoma, revealed significant differences for each category 0.001, Spearman Rank Correlation).

Table 4 Immunoreactivitv with hK1G586 mean standard deviation Benign epithelium 44.3% 10-90 High grade PIN 69.1% 20-100 Adenocarcinoma 80.0% 20-100 (untreated) Thus, an increase in cytoplasmic hK2 expression in prostate tissue is correlated with prostatic neoplasia and prostate cancer. Although the data from prostate obtained from androgen deprivation therapy treated patients is not statistically significant due to the small sample size, there is a decrease in hK2 expression in benign epithelium, high grade PIN and adenocarcinoma in these patients relative to untreated patients. Thus, an increase in hK2 expression in prostate is a novel marker for high grade PIN and prostate cancer.

Example 11.

RT-PCR detection of hK2 RNA in prostate cancer cells Because a large percentage of prostate cancers are understaged, it is of interest to detect hK2 expressing cells present in tissue biopsies, e.g., prostate capsule, bone marrow or lymph node, or in physiological fluid, e.g., blood, serum or seminal fluid. Such a detection method preferably can detect a single hK2 expressing cell in a large number of non-hK2 expressing cells.

Preferably, the method can detect a single hK2 expressing cell in a sample WO 98/21365 PCT/US97/20948 56 comprising at least about 104, more preferably at least about 106, or even more preferably 107, cells. To provide such a sensitive detection method, a reverse transcriptase-polymerase chain reaction (RT-PCR) specific for hK2 transcripts is employed.

A. LNCaP cell line To determine the sensitivity of detection of an hK2-specific transcript by RT-PCR, cells of the human PSA- and hK2-expressing LNCaP cell line were serially diluted in samples of buffy coat cells. The buffy coat cells were isolated from whole blood from normal females and males. Venous blood (5-7 ml) was collected in citrate-dextrose tubes. Samples were centrifuged at 1000 x g for 15 minutes at 4°C. Buffy coat cells were recovered from the top of the cell pellet.

The mixture of buffy coat cells and LNCaP cells was centrifuged at 1,500 rpm for five minutes, and RNA extracted from the pelleted cells. RNA was isolated by an acidic phenol-chloroform-guanidium thiocyanate method (Chomczynski et al., Anal. Biochem., 162, 156 (1987)). RNA samples were further extracted with chloroform-butanol v/v) to remove residual heme, which can inhibit both reverse transcription and polymerase chain reactions. The isolated RNA was then treated with RNase-free DNase.

To prepare first strand cDNAs, an aliquot containing 1 Pg of total RNA was added to a reverse transcription reaction containing 100 pmol of a PSA-specific oligonucleotide primer TCATCTCTGTATCC SEQ ID NO:13) or 100 pmol of an hK2-specific oligonucleotide primer GAGTAAGCTCTA SEQ ID NO:14), and Moloney murine leukemia virus reverse transcriptase (GIBCO BRL), and brought to a final volume of 25 uL mM Tris-HC1, pH 8.3, 75 mM KC1, 3 mM MgCl 2 10 mM dithiothreitol, 0.5 mM each dNTP, and 800 U Moloney murine leukemia virus reverse tranascriptase).

The reaction was incubated at 42 0 C for fifteen minutes and the enzyme was heat inactivated at 95 0 C for fifteen seconds.

To amplify PSA first strand cDNAs, 10 Rl of the PSA-specific oligonucleotide primed first strand cDNAs was amplified in a PCR (0.2 mM WO 98/21365 PCT/US97/20948 57 each dNTP, 0.5 U AmpliTaq polymerase, 50 mM KC1, 10 mM Tris-HCl, pH 8.3, mM MgCl 2 0.1% gelatin) with a PSA-specific primer pair. The PSA PCR employed 50 pmol of PSA-1 GATGACTCCAGCCACGACCT SEQ ID NO:15) and 50 pmol of PSA-2 CACAGACACCCCATCCTATC SEQ ID NO:16). To amplify hK2 first strand cDNAs, 10 ul of the hK2 PSA-specific oligonucleotide primed first strand cDNAs was amplified in a PCR (0.2 mM each dNTP, 0.5 U AmpliTaq polymerase, 50 mM KC1, 10 mM Tris-HC1, pH 8.3, mM MgCI 2 0.1% gelatin) with an hK2-specific primer pair. The hK2 PCR employed 50 pmol of hK2-1 GAGGGTTGTGTACAGTCATGGAT 3'; SEQ ID NO:17) and 50 pmol of hK2-2 ACACACTGAAGACTCCTGGGGCG SEQ ID NO:18)).

The cycling parameters employed were: thirty-five to forty cycles of 94 0 C for 1 minute; 58 0 C (PSA) or 60 0 C (hK2) for 90 seconds, and 72 0 C for seconds. The final cycle was at 72 0 C for ten minutes. Aliquots of the reaction were electrophoresed on 1.0% agarose gels The gels were stained with ethidium bromide and were viewed under ultraviolet light. Some of the amplified products were excised from the gel and subcloned into a pCRII vector (Invitrogen, San Diego, CA) for sequencing.

The PSA-specific PCR yielded a 710 bp product while the hK2specific PCR yielded a 405 bp product. The results of the dilution analysis showed that PSA and hK2 RNA was detectable at approximately 1 LNCaP cell in 106 and 107 white blood cells, respectively (Figure 22A). This result was unexpected because the RT-PCR detected LNCaP-derived hK2 transcripts at a ten fold high dilution than LNCaP-derived PSA transcripts.

B. Prostate cancer patients The blood from six patients with prostate cancer and from two normal males was analyzed by RT-PCR. Buffy coat cells and isolated RNA were obtained from all eight males, and RT-PCR was performed, as described above. The six prostate cancer patients included one with clinical stage B prostate cancer, two with known metastatic disease (clinical stage D2) and three with pathological stage C. Prostate cancer pathological stages A-C are localized WO 98/21365 PCT/US97/20948 58 forms of prostate cancer. Pathological stage D 1 is prostate cancer which has spread to the nodes (nodal metastases). Pathological stage D2 is systemic (systemic metastases) prostate cancer. For a further description of the pathological stages of prostate cancer, see Moreno et al. (Cancer Res., 52, 6110 (1992)) Deguchi et al. (Cancer Res., 53, 5350 (1993)) and Katz et al.

(Urology 43, 765 (1994)).

The results showed that 67% of the prostate cancer patients expressed hK2, 17% expressed PSA, and 17% expressed both hK2 and PSA. No detectable levels of PSA or hK2 RNA were found in normal controls.

Thus, the detection of hK2 RNA may serve as a useful marker for early detection of micrometastasis of prostate cancer.

The invention is not limited to the exact details shown and described, for it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention defined by the claims.

WO 98/21365 PCT/US97/20948 59 SEQUENCE LISTING GENERAL INFORMATION APPLICANT: Mayo Foundation for Medical Education and Research, and Hybritech Incorporated (ii) TITLE OF THE INVENTION: METHOD FOR DETECTION OF METASTATIC PROSTATE CANCER (iii) NUMBER OF SEQUENCES: 26 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Schwegman, Lundberg, Woessner Kluth, P.A.

STREET: P.O. Box 2938 CITY: Minneapolis STATE: MN COUNTRY: U.S.A ZIP: 55402 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette COMPUTER: IBM Compatible OPERATING SYSTEM: DOS SOFTWARE: FastSEQ Version (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: Unknown FILING DATE: November 14, 1997

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER:08/759,354 FILING DATE:14-NOV-1996 APPLICATION NUMBER: PCT/US96/06167 FILING DATE: 02-MAY-1996 APPLICATION NUMBER: 08/622,046 FILING DATE: 26-MAR-1996 APPLICATION NUMBER: 08/427,707 FILING DATE: 02-MAY-1995 (viii) ATTORNEY/AGENT INFORMATION: NAME: Embretson, Janet E REGISTRATION NUMBER: 39,665 REFERENCE/DOCKET NUMBER: 545.005W01 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 612-359-3260 TELEFAX: 612-359-3263

TELEX:

WO 98/21365 PCT/US97/20948 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 237 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Ile Val Gly Gly Trp Glu Cys Glu Lys His Ser Gin Pro Trp Gin Val 1 5 10 Ala Val Tyr Ser His Gly Trp Ala His Cys Gly Gly Val Leu Val His 25 Pro Gin Trp Val Leu Thr Ala Ala His Cys Leu Lys Lys Asn Ser Gin 40 Val Trp Leu Gly Arg His Asn Leu Phe Glu Pro Glu Asp Thr Gly Gin 55 Arg Val Pro Val Ser His Ser Phe Pro His Pro Leu Tyr Asn Met Ser 70 75 Leu Leu Lys His Gin Ser Leu Arg Pro Asp Glu Asp Ser Ser His Asp 90 Leu Met Leu Leu Arg Leu Ser Glu Pro Ala Lys Ile Thr Asp Val Val 100 105 110 Lys Val Leu Gly Leu Pro Thr Gin Glu Pro Ala Leu Gly Thr Thr Cys 115 120 125 Tyr Ala Ser Gly Trp Gly Ser Ile Glu Pro Glu Glu Phe Leu Arg Pro 130 135 140 Arg Ser Leu Gin Cys Val Ser Leu His Leu Leu Ser Asn Asp Met Cys 145 150 155 160 Ala Arg Ala Tyr Ser Glu Lys Val Thr Glu Phe Met Leu Cys Ala Gly 165 170 175 Leu Trp Thr Gly Gly Lys Asp Thr Cys Gly Gly Asp Ser Gly Gly Pro 180 185 190 Leu Val Cys Asn Gly Val Leu Gin Gly Ile Thr Ser Trp Gly Pro Glu 195 200 205 Pro Cys Ala Leu Pro Glu Lys Pro Ala Val Tyr Thr Lys Val Val His 210 215 220 Tyr Arg Lys Trp Ile Lys Asp Thr Ile Ala Ala Asn Pro WO 98/21365 PCTIUS97/20948 230 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 711 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

ATT

Ile 1 GTG GGA GGC Val Gly Gly

TGG

Trp 5 GAG TGT GAG AAG Glu Cys Glu Lys

CAT

His 10 TCC CAA CCC TGG Ser Gin Pro Trp CAG GTG Gin Val GCT GTG TAC Ala Val Tyr CCC CAG TGG Pro Gin Trp CAT GGA TGG GCA His Gly Trp Ala TGT GGG GGT GTC Cys Gly Gly Val CTG GTG CAC Leu Val His AAT AGC CAG Asn Ser Gin 96 144 GTG CTC ACA GCT Val Leu Thr Ala

GCC

Ala CAT TGC CTA AAG His Cys Leu Lys GTC TGG Val Trp CTG GGT CGG CAC Leu Gly Arg His CTG TTT GAG CCT Leu Phe Glu Pro

GAA

Glu GAC ACA GGC CAG Asp Thr Gly Gin

AGG

Arg GTC CCT GTC AGC Val Pro Val Ser AGC TTC CCA CAC Ser Phe Pro His CTC TAC AAT ATG Leu Tyr Asn Met 192 240 288 336 CTT CTG AAG CAT Leu Leu Lys His

CAA

Gin AGC CTT AGA CCA Ser Leu Arg Pro

GAT

Asp 90 GAA GAC TCC AGC Glu Asp Ser Ser CAT GAC His Asp CTC ATG CTG Leu Met Leu AAG GTC CTG Lys Val Leu 115 CGC CTG TCA GAG Arg Leu Ser Glu

CCT

Pro 105 GCC AAG ATC ACA GAT GTT GTG Ala Lys Ile Thr Asp Val Val GGC CTG CCC ACC Gly Leu Pro Thr

CAG

Gin 120 GAG CCA GCA CTG Glu Pro Ala Leu GGG ACC ACC TGC Gly Thr Thr Cys 125 TTC TTG CGC CCC Phe Leu Arg Pro 384 TAC GCC Tyr Ala 130 TCA GGC TGG GGC Ser Gly Trp Gly

AGC

Ser 135 ATC GAA CCA GAG Ile Glu Pro Glu

GAG

Glu 140

AGG

Arg 145 AGT CTT CAG TGT Ser Leu Gin Cys AGC CTC CAT Ser Leu His CTC CTG Leu Leu 155 TCC AAT GAC ATG Ser Asn Asp Met WO 98/21365 PCT/US97/20948

GCT

Ala

CTC

Leu

CTT

Leu

CCA

Pro

TAC

Tyr 225 (2) AGA GCT TAC TCT GAG AAG GTG ACA GAG TTC Arg Ala Tyr Ser Glu Lys Val Thr Glu Phe 165 170 TGG ACA GGT GGT AAA GAC ACT TGT GGG GGT Trp Thr Gly Gly Lys Asp Thr Cys Gly Gly 180 185 GTC TGT AAT GGT GTG CTT CAA GGT ATC ACA Val Cys Asn Gly Val Leu Gin Gly Ile Thr 195 200 TGT GCC CTG CCT GAA AAG CCT GCT GTG TAC Cys Ala Leu Pro Glu Lys Pro Ala Val Tyr 210 215 CGG AAG TGG ATC AAG GAC ACC ATC GCA GCC Arg Lys Trp Ile Lys Asp Thr Ile Ala Ala 230 235 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 261 amino acids TYPE: amino acid TOPOLOGY: linear

ATG

Met

GAT

Asp

TCA

Ser

ACC

Thr 220

AAC

Asn

TTG

Leu

TCT

Ser

TGG

Trp 205

AAG

Lys

CCC

Pro

GGG

Gly

CCA

Pro

GAG

Glu

CAT

His 528 576 624 672 (ii) MOLECULE TYPE: protein Met 1 Ala Lys His His Phe Pro Pro (xi) SEQUENCE Trp Asp Leu Val 5 Val Pro Leu Ile His Ser Gin Pro Cys Gly Gly Val Cys Leu Lys Lys Glu Pro Glu Asp His Pro Leu Tyr 100 Asp Glu Asp Ser 115 DESCRIPTION: SEQ ID Leu Ser Ile Ala Leu Gin Ser Arg Ile Val Trp Gin Val Ala Val 40 Leu Val His Pro Gin 55 Asn Ser Gin Val Trp 70 Thr Gly Gin Arg Val Asn Met Ser Leu Leu 105 Ser His Asp Leu Met 120 NO:3: Ser Val Gly Gly Tyr Ser Trp Val Leu Gly 75 Pro Val Lys His Leu Leu Gly Trp His Leu Arg Ser Gin Arg 125 Gly Glu Ala Ala Leu Phe Arg Glu WO 98/21365 PCTIUS97/20948 63 Pro Ala Lys Ile Thr Asp Val Val Lys Val Leu Gly Leu Pro Thr Gin 130 135 140 Glu Pro Ala Leu Gly Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile 145 150 155 160 Glu Pro Glu Glu Phe Leu Arg Pro Arg Ser Leu Gin Cys Val Ser Leu 165 170 175 His Leu Leu Ser Asn Asp Met Cys Ala Arg Ala Tyr Ser Glu Lys Val 180 185 190 Thr Glu Phe Met Leu Cys Ala Gly Leu Trp Thr Gly Gly Lys Asp Thr 195 200 205 Cys Gly Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gin 210 215 220 Gly Ile Thr Ser Trp Gly Pro Glu Pro Cys Ala Leu Pro Glu Lys Pro 225 230 235 240 Ala Val Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile Lys Asp Thr 245 250 255 Ile Ala Ala Asn Pro 260 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 832 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GGATCCAGC ATG TGG GAC CTG GTT CTC TCC ATC GCC TTG TCT GTG GGG 48 Met Trp Asp Leu Val Leu Ser Ile Ala Leu Ser Val Gly 1 5 TGC ACT GGT GCC GTG CCC CTC ATC CAG TCT CGG ATT GTG GGA GGC TGG 96 Cys Thr Gly Ala Val Pro Leu Ile Gin Ser Arg Ile Val Gly Gly Trp 20 GAG TGT GAG AAG CAT TCC CAA CCC TGG CAG GTG GCT GTG TAC AGT CAT 144 Glu Cys Glu Lys His Ser Gin Pro Trp Gin Val Ala Val Tyr Ser His 35 40 GGA TGG GCA CAC TGT GGG GGT GTC CTG GTG CAC CCC CAG TGG GTG CTC 192 Gly Trp Ala His Cys Gly Gly Val Leu Val His Pro Gin Trp Val Leu 55 WO 98/21365 PTU9104 PCTfUS97/2.0948 ACA GCT GCC CAT TGC CTA AAG AAG AAT AGC Ser CAG GTC TGG CTG GGT CGG Thr Ala Ala CAC AAC CTG His Asn Leu His Cys Leu Lys Lys Asn 70 Gin Val Trp Leu Gly Arg CCT GTC AGC Pro Val Ser 240 288 TTT GAG CCT GAA Phe Glu Pro Giu ACA GGC CAG AGG Thr Gly Gin Arg

GTC

Val CAC AGC His Ser TTC CCA CAC CCG Phe Pro His Pro

CTC

Leu 100 TAC AAT ATG AGC Tyr Asn Met Ser

CTT

Leu 105 CTG AAG CAT CAA Leu Lys His Gin

AGC

Ser 110 CTT AGA CCA GAT Leu Arg Pro Asp

GAA

Glu 115 GAC TCC AGC CAT Asp Ser Ser His CTC ATG CTG CTC Leu Met Leu Leu

CGC

Arg 125 336 384 432 CTG TCA GAG CCT Leu Ser Glu Pro

GCC

Ala 130 AAG ATC ACA GAT Lys Ile Thr Asp

GTT

Val 135 GTG AAG GTC CTG Val Lys Val Leu GGC CTG Gly Leu 140 CCC ACC CAG Pro Thr Gin GGC AGC ATC Gly Ser Ile 160

GAG

Glu 145 CCA GCA CTG GGG Pro Ala Leu Gly

ACC

Thr 150 ACC TGC TAC GC Thr Cys Tyr Ala TCA GGC TGG Ser Gly Trp 155 CTT CAG TGT Leu Gin Cys 480 528 GAA CCA GAG GAG Glu Pro Glu Glu TTG CGC CCC AGG Leu Arg Pro Arg

AGT

Ser 170 GTG AGC Val Ser 175 CTC CAT CTC CTG Leu His Leu Leu

TCC

Ser 180 AAT GAC ATG TGT Asn Asp Met Cys

GCT

Ala 185 AGA GCT TAC TCT Arg Ala Tyr Ser

GAG

Glu 190 AAG GTG ACA GAG Lys Val Thr Glu ATG TTG TGT GCT Met Leu Cys Ala CTC TGG ACA GGT Leu Trp Thr Gly

GGT

Gly 205 AAA GAC ACT TGT Lys Asp Thr Cys

GGG

Gly 210 GGT GAT TCT GGG Gly Asp Ser Gly

GGT

Gly 215 CCA CTT GTC TGT Pro Leu Val Cys AAT GGT Asn Gly 220 GTG CTT CAA Val Leu Gin GAA AAG CCT Glu Lys Pro 240

GGT

Gly 225 ATC ACA TCA TGG Ile Thr Ser Trp

GGC

Gly 230 CCT GAG CCA TGT Pro Glu Pro Cys GCC CTG CCT Ala Leu Pro 235 AAG TGG ATC Lys Trp Ile 720 768 GCT GTG TAC ACC Ala Val Tyr Thr

AAG

Lys 245 GTG GTG CAT TAC Val Val His Tyr

CGG

Arg 250 AAG GAC Lys Asp 255 ACC ATC GCA GCC Thr Ile Ala Ala AAC CCC Asn Pro 260 TGAGTGCCCC TGTCCCACCC CTACCTCTAG 822 832

TAAACTGCAG

INFORMATION FOR SEQ ID WO 98/21365 WO 98/ 1365PCTIUS97/20948 SEQUENCE CHARACTERISTICS: LENGTH: 244 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID, Val. Pro Leu Ile Gin Ser Arg Ile Val Gly Gly Trp Glu Cys Giu Lys His Cys Cys Giu His Asp Ala Pro Pro 145 Leu Giu Giy Ile Val Ser Gly Leu Pro Pro Giu Lys Ala 130 Glu Leu Phe Gly Thr 210 Tyr Gin Gly Lys Giu Leu Asp Ile 115 Leu Giu Ser Met Asp 195 Ser Thr Pro Val Lys Asp Tyr Ser 100 Thr Gly Phe Asn Leu 180 Ser Trp Lys Trp Leu Asn Thr Asn Ser Asp Thr Leu Asp 165 Cys Gly Gly Val Gin Val Ser Gly 70 Met His Val Thr Arg 150 Met Ala Gly Pro Val His Gin 55 Gin Ser Asp Val.

Cys 135 Pro Cys Giy Pro Glu 215 Al a Pro 40 Val Arg Leu Leu Lys 120 Tyr Arg Ala Leu Leu 200 Pro Vai 25 Gin Trp Val Leu Met 105 Val Ala Ser Arg Trp 185 Val.

Cys Tyr Trp, Leu Pro Lys 90 Leu Leu Ser Leu Al a 170 Thr Cys Al a Ser Val Gly Val 75 His Leu Gly Gly Gin 155 Tyr Gly Asn Leu His Leu Arg Ser Gin Arg Leu Trp 140 Cys Ser Gly Gly Pro 220 Gly Thr His His Ser Leu Pro 125 Gly Va.

Giu Lys Val.

205 Glu Trp Aia Asn Ser Leu Ser 110 Thr Ser Ser Lys Asp 190 Leu Lys Ala Ala Leu Phe Arg Giu Gin Ile Leu Val 175 Thr Gin Pro His His Phe Pro Pro Pro Giu Glu His 160 Thr Cys Gly Ala Val His Tyr Arg Lys Trp Ile Lys Asp Thr Ile 225 Ala Ala Asn Pro WO 98/21365 PCTIUS97/20948 66 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 766 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..732 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

GTG

Val 1 CCC CTC ATC CAG TCT CGG ATT GTG Pro Leu Ile Gin Ser Arg Ile Val 5

GGA

Gly 10 GGC TGG GAG TGT Gly Trp Glu Cys GAG AAG Glu Lys CAT TCC CAA His Ser Gin TGT GGG GGT Cys Gly Gly TGG CAG GTG GCT Trp Gin Val Ala TAC AGT CAT GGA Tyr Ser His Gly TGG GCA CAC Trp Ala His GTC CTG GTG CAC Val Leu Val His

CCC

Pro 40 CAG TGG GTG CTC ACA GCT GCC CAT Gin Trp Val Leu Thr Ala Ala His 144 TGC CTA Cys Leu AAG AAG AAT AGC Lys Lys Asn Ser GTC TGG CTG GGT Val Trp Leu Gly CAC AAC CTG TTT His Asn Leu Phe

GAG

Glu CCT GAA GAC ACA Pro Glu Asp Thr CAG AGG GTC CCT Gin Arg Val Pro AGC CAC AGC TTC Ser His Ser Phe

CCA

Pro 192 240 288 CAC CCG CTC TAC His Pro Leu Tyr

AAT

Asn ATG AGC CTT CTG Met Ser Leu Leu

AAG

Lys 90 CAT CAA AGC CTT His Gin Ser Leu AGA CCA Arg Pro GAT GAA GAC Asp Glu Asp GCC AAG ATC Ala Lys Ile 115 AGC CAT GAC CTC Ser His Asp Leu

ATG

Met 105 CTG CTC CGC CTG Leu Leu Arg Leu TCA GAG CCT Ser Glu Pro 110 ACC CAG GAG Thr Gin Glu 336 ACA GAT GTT GTG AAG GTC CTG GGC CTG Thr Asp Val Val Lys Val Leu Gly Leu 120 CCA GCA Pro Ala 130 CTG GGG ACC ACC TGC TAC GCC TCA GGC TGG GGC AGC ATC GAA Leu Gly Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile Glu CCA GAG GAG TTC TTG CGC CCC AGG AGT CTT CAG TGT GTG AGC CTC CAT WO 98/21365 PCT/US97/20948 Pro 145

CTC

Leu

GAG

Glu

GGG

Gly

ATC

Ile

GTG

Val 225

GCA

Ala Glu

CTG

Leu

TTC

Phe

GGT

Gly

ACA

Thr 210

TAC

Tyr

GCC

Ala Glu

TCC

Ser

ATG

Met

GAT

Asp 195

TCA

Ser

ACC

Thr

AAC

Asn Phe

AAT

Asn

TTG

Leu 180

TCT

Ser

TGG

Trp

AAG

Lys

CCC

Pro Leu Arg Pro Arg Ser Leu G1n Cys Val Ser 150 155 GAC ATG TGT GCT AGA GCT TAC TCT GAG AAG Asp Met Cys Ala Arg Ala Tyr Ser Glu Lys 165 170 TGT GCT GGG CTC TGG ACA GGT GGT AAA GAC Cys Ala Gly Leu Trp Thr Gly Gly Lys Asp 185 190 GGG GGT CCA CTT GTC TGT AAT GGT GTG CTT Gly Gly Pro Leu Val Cys Asn Gly Val Leu 200 205 GGC CCT GAG CCA TGT GCC CTG CCT GAA AAG Gly Pro Glu Pro Cys Ala Leu Pro Glu Lys 215 220 GTG GTG CAT TAC CGG AAG TGG ATC AAG GAC Val Val His Tyr Arg Lys Trp Ile Lys Asp 230 235 TGAGTGCCCC TGTCCCACCC CTACCTCTAG TAAA Leu

GTG

Val 175

ACT

Thr

CAA

Gin

CCT

Pro

ACC

Thr His 160

ACA

Thr

TGT

Cys

GGT

Gly

GCT

Ala

ATC

Ile 240 528 576 624 672 720 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 237 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) Ile Val 1 Leu Val Pro Gin Ile Leu Val Phe SEQUENCE DESCRIPTION: SEQ ID NO:7: Gly Gly Trp Glu Cys Glu Lys His Ser 5 Ala Ser Arg Gly Arg Ala Val Cys Gly Trp Val Leu Thr Ala Ala His Cys Ile Leu Gly Arg His Ser Leu Phe His Pro 55 Gin Val Ser His Ser Phe Pro His Pro 70 75 Gin Gly Arg Glu Leu Val His Val Gln Ser WO 98/21365 WO 9821365PCTIUS97/20948 Leu Leu Lys Tyr Lys 145 Ala Arg Leu Pro Tyr Leu Met Val1 Ala 130 Lys Gin Trp Val Cys 210 Arg Lys Leu Met 115 Ser Leu Val1 Thr Cys 195 Ala Lys Asn Arg Phe Leu Arg Pro Giy Asp Asp Ser Ser His Leu 100 Asp Giy Gin His Gly 180 Asn Leu Trp Arg Leu Trp Cys Pro 165 Gly Gly Pro Ile Leu Pro Gly Vai 150 Gin Lys Val Glu Lys Ser Thr Ser 135 Asp Lys Ser Leu Arg 215 Asp Giu Gin 120 Ile Leu Val Thr Gin 200 Pro Thr Pro 105 Giu Glu His Thr Cys 185 Gly Ser Ile Giu Ala Giu Ile 155 Phe Gly Thr Tyr Ala 235 Thr Gly 125 Phe Asn Leu Ser Trp 205 Lys Pro Asp Val Cys Pro Cys 160 Giy Pro Giu His INFORMATION FOR SEQ ID NO:8: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 237 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Ile Vai Giy Gly Trp Giu Cys Giu Lys His Ser Gin Pro Trp Gin Val 1 5 10 Ala Vai Tyr Ser His Giy Trp Ala His Cys Gly Gly Val Leu Val His 25 Pro Gin Trp Vai Leu Thr Ala Ala His Cys, Leu Lys Lys Asn Ser Gin 40 Vai Trp Leu Gly Arg His Asn Leu Phe Giu Pro Glu Asp Thr Gly Gin 55 WO 98/21365 PCT/US97/2.0948 69 Arg Val Pro Val Ser His Ser Phe Pro His Pro Leu Tyr Asn Met Ser 70 75 Leu Leu Lys His Gin Ser Leu Arg Pro Asp Glu Asp Ser Ser His Asp 90 Leu Met Leu Leu Arg Leu Ser Glu Pro Ala Lys Ile Thr Asp Val Val 100 105 110 Lys Val Leu Gly Leu Pro Thr Gin Glu Pro Ala Leu Gly Thr Thr Cys 115 120 125 Tyr Ala Ser Gly Trp Gly Ser Ile Glu Pro Glu Glu Phe Leu Arg Pro 130 135 140 Arg Ser Leu Gin Cys Val Ser Leu His Leu Leu Ser Asn Asp Met Cys 145 150 155 160 Ala Arg Ala Tyr Ser Glu Lys Val Thr Glu Phe Met Leu Cys Ala Gly 165 170 175 Leu Trp Thr Gly Gly Lys Asp Thr Cys Gly Gly Asp Ser Gly Gly Pro 180 185 190 Leu Val Cys Asn Gly Val Leu Gin Gly Ile Thr Ser Trp Gly Pro Glu 195 200 205 Pro Cys Ala Leu Pro Glu Lys Pro Val Val Tyr Thr Lys Val Val His 210 215 220 Tyr Arg Lys Trp Ile Lys Asp Thr Ile Ala Ala Asn Pro 225 230 235 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: ACGCGGATCC AGCATGTGGG ACCTGGTTCT CT 32 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 98/21365 PCTIUS97/20948 (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID ACAGCTGCAG TTTACTAGAG GTAGGGGTGG GAC 33 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: ATATGGATCC ATATGTCAGC ATGTGGGACC TGGTTCTCTC CA 42 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: ATATGGATCC TCAGGGGTTG GCTGCGATGG T 31 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TCATCTCTGT ATCC 14 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 12 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 98/21365 PCT/US97/20948 71 (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GAGTAAGCTC TA 12 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID GATGACTCCA GCCACGACCT INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CACAGACACC CCATCCTATC INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GAGGGTTGTG TACAGTCATG GAT 23 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 98/21365 PCTIUS97/20948 72 (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ACACACTGAA GACTCCTGGG GCG 23 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Glu Lys His Ser Gln Pro Trp Gln Val Ala Val Tyr Ser His Gly Trp 1 5 10 Ala His Cys INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID His Cys Leu Lys Lys Asn Ser Gln Val Trp Leu Gly Arg His Asn Leu 1 5 10 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: His Leu Leu Ser Asn Asp Met Cys Ala Arg Ala Tyr Ser Glu Lys 1 5 10 WO 98/21365 PCT/US97/20948 73 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 55 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Ala 1 Val Tyr Ser His Gly Trp Ala His Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala His 25 Val Trp Leu Gly Arg His Asn Leu Phe Arg Val Pro Val Ser His Ser Leu Lys Lys Asn Ser Gln Thr Gly Gln Glu Pro Glu INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 711 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

ATTGTGGGAG

CGTGGCAGGG

CACTGCATCA

GACACAGGCC

CTCCTGAAGA

CGCCTGTCAG

GAGCCAGCAC

TTCTTGACCC

GCGCAAGTTC

GGCAAAAGCA

GGTATCACGT

AAGGTGGTGC

GCTGGGAGTG

CAGTCTGCGG

GGAACAAAAG

AGGTATTTCA

ATCGATTCCT

AGCCTGCCGA

TGGGGACCAC

CAAAGAAACT

ACCCTCAGAA

CCTGCTCGGG

CATGGGGCAG

ATTACCGGAA

CGAGAAGCAT

CGGTGTTCTG

CGTGATCTTG

GGTCAGCCAC

CAGGCCAGGT

GCTCACGGAT

CTGCTACGCC

TCAGTGTGTG

GGTGACCAAG

TGATTCTGGG

TGAACCATGT

GTGGATCAAG

TCCCAACCCT GGCAGGTGCT GTGCACCCCC AGTGGGTCCT CTGGGTCGGC ACAGCCTGTT

AGCTTCCCAC

GATGACTCCA

GCTGTGAAGG

TCAGGCTGGG

GACCTCCATG

TTCATGCTGT

GGCCCACTTG

GCCCTGCCCG

GACACCATCG

ACCCGCTCTA

GCCACGACCT

TCATGGACCT

GCAGCATTGA

TTATTTCCAA

GTGCTGGACG

TCTGTAATGG

AAAGGCCTTC

TGGCCAACCC

TGTGGCCTCT

CACAGCTGCC

TCATCCTGAA

CGATATGAGC

CATGCTGCTC

GCCCACCCAG

ACCAGAGGAG

TGACGTGTGT

CTGGACAGGG

TGTGCTTCAA

CCTGTACACC

C

120 180 240 300 360 420 480 540 600 660 711 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 24 amino acids WO 98/21365 PCT/US97/20948 74 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Leu Lys Lys Asn Ser Gln Val Trp Leu Gly Arg His Asn Leu Phe Glu 1 5 10 Pro Glu Asp Thr Gly Gln Arg Val INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 26 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Cys Ala Leu Pro Glu Lys Pro Ala Val Tyr Thr Lys Val Val His Tyr 1 5 10 Arg Lys Trp Ile Lys Asp Thr Ile Ala Ala INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Gin Val Ala Val Tyr Ser His Gly Trp Ala His Cys 1 5

Claims (24)

1. A method for detecting hK2 DNA, comprising: contacting an amount of DNA obtained by reverse transcription of RNA from a human physiological sample that is not obtained from the prostate and which comprises cells suspected of containing hK2 RNA, with an amount of at least two oligonucleotides under conditions effective to amplify the DNA by a polymerase chain reaction so as to yield an amount of amplified hK2 DNA, wherein at least one oligonucleotide is an hK2-specific oligonucleotide; and detecting the presence of the amplified hK2 DNA.

2. A method for detecting metastatic prostate cancer in a human, comprising: contacting an amount of DNA obtained by 0 reverse transcription of RNA from a human physiological 20 sample that is not obtained from the prostate and which comprises cells suspected of containing hK2 RNA, with an amount of at least two oligonucleotides under conditions effective to amplify the DNA by a polymerase chain reaction so as to yield an amount of amplified hK2 DNA, wherein at least one oligonucleotide is an hK2-specific oligonucleotide; and detecting the presence of the amplified hK2 DNA, wherein the presence of hK2 DNA is indicative of the presence of metastatic prostate cancer in said human.

3. The method of claim 1 or 2, wherein the physiological sample is a tissue sample.

4. The method of claim 3, wherein the tissue is selected from the group consisting of prostate, prostate capsule, seminal vesicle, bone marrow and lymph node. \\.elb-files\homeS\cintae\Keep\speci\52062.9adoc 11/01/01 76 The method of claim 3, wherein the tissue is non- prostate tissue.

6. The method of claim 1 or 2, wherein the physiological sample is a fluid.

7. The method of claim 6, wherein the fluid is selected from the group consisting of whole blood, blood serum and seminal fluid.

8. The method of claim 7, wherein the fluid is whole blood.

9. The method of claim 1 or 2, wherein the amplified hK2 DNA is subjected to agarose gel electrophoresis prior to detection. The method of claim 1 or 2, further comprising quantifying the amount of amplified hK2 DNA.

11. The method of claim 1 or 2, further comprising: contacting a second amount of the DNA obtained by reverse transcription of RNA from the human physiological sample with an amount of at least two oligonucleotides under conditions effective to amplify prostate specific antigen (PSA) DNA, but not hK2 DNA, by polymerase chain reaction so as to yield amplified PSA DNA; and detecting the presence of the amplified PSA 30 DNA.

12. A diagnostic method for detecting hK2 RNA comprising: extracting RNA from a physiological sample obtained from a human, wherein the sample is not obtained from the prostate; reverse transcribing the extracted RNA to \\melbfiles\home$\cintae\Keep\speci\52062.98doc 11/01/01 77 yield DNA; contacting the DNA with an amount of at least two oligonucleotides under conditions effective to amplify the DNA by polymerase chain reaction so as to yield an amount amplified hK2 DNA, wherein at least one oligonucleotide is an hK2-specific oligonucleotide; and detecting the presence of the amplified hK2 DNA, wherein the presence of the amplified hK2 DNA is indicative of metastatic prostate cancer in the human.

13. The method of claim 12, wherein the sample is a tissue sample.

14. The method of claim 12, wherein the sample is a physiological fluid sample. The method of claim 12, wherein the human has had a radical prostatectomy, and wherein the presence of hK2 :DNA is indicative of the presence of persistent prostate 20 cancer in the human.

16. A method for monitoring the progression of prostate cancer, comprising: contacting an amount of DNA obtained by reverse transcription of RNA from a physiological sample obtained from a human afflicted with prostate cancer with an amount of at least two oligonucleotides under conditions effective to amplify the DNA by a polymerase o ,chain reaction so as to yield an amount of amplified hK2 30 DNA, wherein at least one oligonucleotide is an hK2- specific oligonucleotide, and wherein the sample is not obtained from the prostate; detecting or determining the amount of the amplified hK2 DNA; repeating steps and at a point later in time; and comparing the result of step with the \\melb.files\home$\cintae\Keep\speci\52062.98.doc 11/01/01 78 result of step wherein an increase in the amount of hK2 DNA is indicative of the progression of prostate cancer in said human.

17. A method for pathologically staging prostate cancer, comprising: contacting an amount of DNA obtained by reverse transcription of RNA from a physiological sample obtained from a human afflicted with prostate cancer with an amount of at least two oligonucleotides under conditions effective to amplify the DNA by a polymerase chain reaction so as to yield an amount of amplified hK2 DNA, wherein at least one oligonucleotide is an hK2- specific oligonucleotide, and wherein the sample is not obtained from the prostate; and detecting or determining the presence or amount of the amplified hK2 DNA, wherein the presence or amount of amplified hK2 DNA is indicative of the pathological stage of the prostate cancer.

18. The method of claim 16 or 17, wherein the human is a candidate for radical prostatectomy.

19. The method of claim 16, wherein the sample of step is obtained prior to the human undergoing hormone therapy. S* 20. The method of claim 19, wherein the hormone therapy is androgen therapy.

21. The method of claim 20, wherein the androgen therapy is androgen provocative therapy.

22. The method of claim 16 or 17, wherein the sample is a non-prostate tissue sample.

23. The method of claim 16 or 17, wherein the sample \\melb-fies\hoeS\cintae\Keep\peci\52062.98.doc 11/01/01 79 is a physiological fluid sample.

24. The method of claim 23, wherein the fluid is whole blood. A method for detecting or determining the presence of metastatic prostate cancer in a human non- prostate tissue sample, comprising: mixing an amount of an agent, which binds to an hK2 polypeptide and which does not bind to hK3, with the cells of the human tissue sample, so as to form a binary complex comprising the agent and the cells; and determining or detecting the presence or amount of complex formation in the sample, wherein the presence or amount of said complex provides an indication of the presence of micrometastatic prostate cancer.

26. The method of claim 25, wherein the agent is an go antibody.

27. The method of claim 26, wherein the antibody is a member of a population of polyclonal antibodies.

28. The method of claim 26, wherein the antibody is a S 25 monoclonal antibody.

29. A method according to any one of claims 1, 2, 12, 16, 17 and 25, substantially as herein described with reference to the Examples. Dated this 10th day of July 2001 MAYO FOUNDATION FOR MEDICAL EDUCATION AND RESEARCH and HYBRITECH, INC. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia )1 H:\cintae\Keep\speci\52602.98.doc 10/07/01 '^SJ/ 7