MXPA99004504A - Method for detection of metastatic prostate cancer - Google Patents

Abstract

Se detecta cáncer de próstata determinando la presencia del polipéptido hK2 de calicreína glandular específica en próstata o ARN hK2 en una muestra fisiológica.

Description

METHOD FOR THE DETECTION OF METASTATIC PROSTATE CANCER DECLARATION OF GOVERNMENT RIGHTS This invention was made with the support of the United States through guarantees from the National Institutes of Health (Grant Nos. CA70893 and DK41995). The Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION Glandular kallikreins are a subgroup of serine proteases, which are involved in the post-translational processing of polypeptide precursors specific to their biologically active forms. In humans, three members of this family have been identified, and some of their properties have been characterized (Clements, Endoc. Rev., 10, 343 (1989); Clements, Mol. Cell Endo., 99, 1 (1994) Jones et al., Acta Endoc., 127. 481 (1992)). The hKLK1 gene encodes the tissue kallikrein protein hK1, the hKLK2 gene encodes the glandular specific kallikrein protein in the hK2 prostate, and the hKLK3 gene encodes the hK3 prostate specific antigen protein (PSA). Northern blot analysis of mRNA shows that both hK2 and PSA are expressed mainly in the prostate of a human being, while the expression of hK1 is found in the pancreas, submandibular gland, kidney and other non-prosthetic tissues (Chapdelaine et al., FEBS Lett .. 236 205 (1988); Young et al., Biochem. 31. 818 (1992)). Homology of the nucleotide sequence between the exons of hKLK2 and hKLK3 is 80%, while the nucleotide sequence homology between the exons of hKLK2 and hKLK1 is 65%. The amino acid sequence homology deduced from hK2 to PSA is 79%, while the amino acid sequence homology deduced from hK2 to hK1 is 57%. In addition, the deduced amino acid sequence of hK2 suggests that hK2 may be a trypsin-like protease, while PSA is a kimotrypsin-type protease. PSA levels are widely used as a prognostic indicator of prostate carcinoma. However, since serum PSA levels are high in patients with either prostatic cancer (pCa) or benign prostatic hyperplasia (BPH), the detection of elevated PSA levels does not distinguish between these diseases. The degree of homology of hK2 to PSA raises some question of the specificity of antibodies currently used to detect PSA levels.If the levels of hK2 in circulation are not related to pCa or BPH, then antibodies arise to PSA preparations, which are contaminated with hK2, or regions of PSA with homology to hK2, may result in false-positive results.

Although it is now generally accepted that the serum PSA test, combined with digital rectal examination (DRE), is one of the most effective means of clinically detecting significant and confined prostate cancer in the organ, combinations of PSA, DRE and Ultrasonic prostate exam can only detect some tumors in the prostate. For example, up to 40% of patients surgically treated with prostate cancer are subsequently clinically underrepresented. In addition, the actual incidence of histological cancers based on autopsy data in relation to the incidence of clinically important prostate cancer is high. In addition, approximately 30% of patients with localized prostate cancer can have hidden (distant) metastatic disease (Moreno et al., Cancer Res., 52, 6110 (1992)). Of these patients, 80% biochemically experience relapse, that is, elevated PSA levels, or through the reappearance of local disease or the occurrence of frank systemic disease, after therapy. Operational therapy is not the appropriate treatment modality for patients who have established metastases. Classification modalities to determine early metastasis usually fail to identify a significant subset of patients with locally invasive disease involving penetration of the prostate capsule or seminal vesicle. Although immunohistochemical techniques have been used to identify micrometastatic prostate tumor cells, or in circulation, when no obvious metastatic deposit is evidenced through any conventional means, the immunohistochemical methods are laborious and lack the sensitivity necessary for the early detection of metastatic or locally invasive prostate cancer. Therefore, there is a need for early detection of prostate cancer cells with metastatic potential. In addition, there is a need to accurately assess prostate cancer before subjecting a patient to invasive procedures. In particular, there is a need for a marker for prostate cancer that can function independently of, or in combination with, PSA.

COMPENDIUM 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 with the detection of hK2 RNA in the sample. Since the expression of hK2 is specific in prostate tissue, the hK2 DNA theoretically should not be detectable in cells present in body fluids or in non-prostatic tissue, if there is no locally invasive or metastatic disease, or if all tissue Prostatic (benign and malignant) has been removed or destroyed. The method comprises contacting an amount of DNA obtained through reverse transcription (RT) of RNA from a human physiological sample with a plurality of oligonucleotide primers, preferably at least two oligonucleotide primers, at least one of which oligonucleotide specific in hK2, in an amplification reaction in order to produce an amount of amplified hK2 DNA. A preferred amplification reaction is a polymerase chain reaction (PCR). The presence of amplified hK2 DNA is then detected. As described below, the presence of amplified hK2 DNA in blood cells, after RT-PCR, correlates with cancer in the prostate, that is, 67% of patients with prostate cancer expressed hK2, 17% expressed PSA and 17% expressed both hK2 and PSA. Preferably, the source of the sample to be tested is human tissue, for example, prostate, prostate capsule, seminal vesicle, bone marrow or lymph node. Another preferred source of the sample to be tested is a human physiological fluid comprising cells, for example, blood, serum, or seminal fluid. As used herein, "amplified hK2 DNA" is defined as the DNA of hK2 in a sample, which was subjected to an amplification reaction, which is present in an amount that is greater than, ie, 10, preferably 104, and most preferably 106 times more than the amount of hK2 DNA that was present in the sample before amplification. As used herein, the term "specific oligonucleotide in hK2" or specific initiator in hK2"means a DNA sequence having at least about 80%, preferably at least 90%, and most preferably at least about 95% identity or sequence homology to SEQ ID NO: 4, in the regions of SEQ ID NO: 4 which are divergent of nucleotide sequence encoding hK3 (SEQ ID NO: 23) An oligonucleotide or initiator of the invention has at least about 7-50, preferably at least 10-40 and most preferably at least about 15-35 nucleotides Preferably, the oligonucleotide primers of the invention comprise at least 7 nucleotides at 3 'of the initiator of oligonucleotide, which is at least about 80%, preferably at least 85% and most preferably about 90% identity to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. Oligonucleotides of the invention they can also include sequences that are not related to nucleic acid sequences of hK2, for example, can encode restriction endonuclease recognition sequences. A specific oligonucleotide in preferred hK2 of the invention comprises SEQ ID NO: 14. Another preferred hK2-specific oligonucleotide of the invention comprises SEQ ID NO: 17. Still another specific oligonucleotide in hK2 preferred 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. The combined detection of two or more gene products can provide greater diagnostic accuracy or more informative production to evaluate the information. The combined detection can also be useful to differentiate those cells with potential aggressive growth from those that are more indolent. In a particularly preferred embodiment of the method provided by the invention, the detection by RT-PCR of hK2 RNA is combined with the detection by RT-PCR 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 produce DNA. The DNA is contacted with an amount of at least two effective oligonucleotides to amplify the DNA to produce an amount of amplified hK2 DNA, wherein at least one oligonucleotide is a ific oligonucleotide in hK2. The presence of amplified hK2 DNA is then detected. The presence of amplified hK2 DNA is indicative of metastatic prostate cancer in humans. The presence of hK2 RNA, or an RNA level of hK2 that arises over time, in body fluids or non-prostatic tissue can reasonably be expected to indicate the presence of previously undiagnosed metastatic disease. Early detection of metastatic disease provides a "driving time," during which alternative therapeutic strategies can be evaluated, including those that can not exist at the time of surgery but are subsequently developed. In this manner, the present invention provides a method for verifying the progression of prostate cancer. The method comprises contacting an amount of DNA obtained through reverse transcription of RNA from a physiological sample obtained from a human being suffering from prostate cancer with an amount of at least two oligonucleotides, at least one of which is a ific oligonucleotide in hK2, effective to amplify the DNA to produce an amount of amplified hK2 DNA. The presence or amount of amplified hK2 DNA is detected or determined. At least one point after the time, another sample is taken and the amount of amplified hK2 DNA is detected or determined. Next, the amounts of amplified hK2 DNA, obtained from at least two different time points, are compared. A method for pathologically evaluating prostate cancer in a human being is also provided. The method comprises contacting an amount of DNA obtained through reverse transcription of RNA from a physiological sample obtained from the human being suffering from prostate cancer with an amount of at least two oligonucleotides, at least one of which is a ific oligonucleotide in hK2, effective to amplify the DNA to produce an amount of amplified hK2 DNA. The presence or amount of amplified hK2 DNA is then detected or determined. The presence or amount of amplified hK2 DNA is indicative of the pathological stage of prostate cancer. Another embodiment of the invention provides a method for verifying therapeutic interventions involving hormone therapies. For example, since the expression of hK2 is androgen dependent, RNA levels of hK2 in peripheral blood or other tissue or body fluid can be used as a marker during intermittent androgen therapy, or during provocative androgen testing, wherein a patient is temporarily placed in a hyperandrogenized state to stimulate the level of hK2 production through any of the persistent prostate cancer cells sufficient to make them detectable. See, T. K. Takayama et al., Sem. In Oncol., 21, 542-553 (1994), and references cited therein. The levels of hK2 are preferably checked periodically during the course of hormone therapy. It may also be advantageous to determine the levels of hK2 before starting therapy and periodically after the conclusion of a therapeutic regimen. A diagnostic kit for detecting hK2 RNA in a physiological sample suted of containing hK2 RNA is also provided. The kit comprises a package containing (a) a known amount of a ific first oligonucleotide in hK2, wherein the oligonucleotide consists of at least about 7-50 nucleotides, and wherein the oligonucleotide has at least 80% identity to SEQ ID NO: 4, and (b) a known amount of a second specific oligonucleotide in hK2. , 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 isolated, purified peptide comprising SEQ ID NO: 26, a biologically active subunit thereof or a biologically active variant thereof. Also provided is an antibody or purified or isolated antibody preparation that specifically reacts with a protein or polypeptide comprising 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 represent a subunit of a peptide having SEQ ID NO: 22, which has at least about 10%, preferably at least 50% and most preferably at least 90%, of 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 in the art including, but not limited to, the ability of the peptide to produce a specific immunological response in sequence when the peptide is administered to an organism, for example , goat, rabbit, sheep or mice. As used herein, the term "biologically active variant" of a peptide of the invention is preferably defined to represent a peptide which has at least about 80%, preferably at least 90% and most preferably at least about % identity or homology with SEQ ID NO: 22. The biologically active variants of the peptides of the invention have at least about 10%, preferably at least 50% and most preferably at least 90% of the activity of a peptide having SEQ ID NO: 22. The activity of a variant peptide of the invention can be measured by the methods described herein above. The invention further provides a method for detecting or determining the presence of metastatic prostate cancer in a non-prostate human tissue sample. The method comprises mixing an amount of an agent, which binds to a hK2 polypeptide and which does not bind to hK3, with the cells of the mammalian tissue sample in order 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 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 both human and animal mAbs, and polyclonal antibody preparations, as well as antibody fragments, synthetic antibodies, including recombinant antibodies, chimeric antibodies, including humanized antibodies, anti-idiotopic antibodies and derivatives thereof. To prepare antibodies that 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 antibody populations. These antibodies in turn can be used as the basis for direct or competitive assays to detect and quantify hK2 (or "protein") polypeptides in samples derived from tissues such as bone marrow and lymph nodes., and samples of cells such as physiological fluids comprising cells.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the amino acid sequences of mature wild type hK2 (SEQ ID NO: 1) and hK3 (SEQ ID NO: 7). Figure 2 illustrates 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 expression vectors of pGT, pGThK2 and pGThK2v217. Figure 4 illustrates chromatographic profiles of the purification of phK2v217. (A) A DEAE chromatogram of spent medium of 7 days of AV12 cells transfected with a vector encoding pphK2v217. A sample of the spent medium was applied a bicarbonate pH regulator, pH 8 and eluted with a salt gradient. The elution profile of A28o 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 on microtitre plates and developed with a rabbit ati-pphK2 antibody. (B) The hydrophobic interaction profile of combined DEAE fractions. Fractions 24-30 of the DEAE chromatographic eluted products of (A) were combined, concentrated and applied to an HIC column (hydrophobic interaction column) in 1.2 M sodium sulfate, and eluted with a salt gradient. in reduction. The elution profile (A28o) is represented by solid lines. The dotted line represents the results of an ELISA assay of a portion of individual column fractions, which was dried on microtitre plates and developed with a rabbit anti-hK2 antibody. (C) Fractions containing hK2 from the 22 minute peak of (B) were concentrated and applied to a Pharmacia S12 size exclusion column. The fractions were collected and analyzed by SDS / PAGE. The peak of 19.4 minutes seemed homogeneous through SDS-PAGE. Figure 5 depicts an SDS / PAGE analysis of purified hK2 and PSA. A sample of 1.5 mg of phK2v217 or purified PSA was boiled in a sample pH regulator with (R) or without (N) 1% of beta-mercaptotanol. The samples were subjected to SDS / PAGE on a 4-20% gel. The protein bands were visualized by staining the gel with silver. Figure 6 illustrates the Conavalin A staining of phK2. The predicted position of phK2 is designated by an arrow. ZCE (in anti-CEA mAb) and BSA were included as examples of glycosylated and non-glycosylated proteins, respectively. The presence of a band in the predicted position in the phK2 lane shows that this protein is glycosylated. Figure 7 depicts the conversion of mature pro to hK2v217 through trypsin cleavage. Trypsin (1% w / w) was incubated with phK2v217 for 10 minutes at 37 ° C in 10 mM borate pH buffer, pH 8, and then subjected to HIC-HPLC. The faded line represents the profile of phK2v217 before incubation with trypsin. The solid line represents the profile of phK2 after digestion of trypsin. The profiles were superimposed for comparison. The identity of the two forms was confirmed through N-terminal sequencing of the protein. Figure 8 illustrates a Western blot analysis of seminal fluid using the monoclonal antibody (mAb) hK1G 586.1. The 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 an 8-25% gel using PhastSystem (Pharmacia). The protein was transferred to nitrocellulose and incubated with purified G protein with HK1G 586.1 (1 μg / ml) followed by goat anti-mouse IgG-HRP (1: 1000). The staining was developed using the ECL detection system (Amersham). Figure 9 depicts a time course study of the expression of hK2 in AV12 cells. Clone # 27 of AV12-hK2 was developed at a confluence of approximately 60-70%, then the cells were washed with HBSS and a serum free HH4 medium was added. The spent medium was removed every day, concentrated and subjected to SDS / PAGE on a 12% gel. The proteins were electrotroved and were applied with a probe with monoclonal antibody HK1D 106.4, which detects both phK2 and hK2 (1: 1000) and HK1G 464.3, which detects phK2 (1: 000), followed by goat anti-mouse IgG -HRP (1: 500) The staining was developed with ELC (Amersham) according to the manufacturer's instructions. As controls, phK2, v_17 and hK2, V217 were used. The position of hK2 is indicated by the arrow. Figure 10 depicts a time course study of the expression of the variant form of hK2 in transfected AV12 cells. At a confluence of about 60-70%, the AV12-hK2v217 cells were washed with HBSS and a serum free HH4 medium was added. The spent medium was removed every day, concentrated and subjected to SDS / PAGE on a 12% gel. The proteins were electrotrophized and a probe was applied 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 staining was developed with ECL (Amersham) according to the manufacturer's instructions. As controls, purified phK2 217 and hK2v217 were used. The position of hK2 is indicated by the arrow. Figure 11 is a graph of the expression of hK2 and cell viability against time. Clone # 27 of AV12-hK2 was grown to a confluence of 60-70%, washed with HBSS and serum free HH4 medium added. The spent medium was removed every day and the concentration of hK2 was measured through ELISA using HK1D 106.4 or HK1G 464.3 as a primary antibody, in goat anti-mouse IgG of -HRP as a secondary antibody. The reaction was developed with OPD (Sigma, St. Louis, MO). Viable cells were enumerated daily using a trypan blue dye exclusion. Figure 12 illustrates the expression hK2 in PC3 and DU145 cells. PC3 and DU145 cells transfected with pGThK2 were grown to a confluence of about 60-70%, washed and resuspended in a serum free HH4 medium. The spent medium from DU145 cells transfected with pGThK2 was collected 3 days after the additional suspension and the spent medium from PC3 cells transfected with pGThK2 was collected 5 days after the additional suspension. The spent media were concentrated and subjected to SDS / PAGE on 12% gels. The proteins were electrotrown and a probe was applied with HK1D 106.4 or HK1G 464.3 as described above. As controls, purified phK2v217 were used. The position of hK2 is indicated by an arrow. Figure 13 illustrates the expression of hK2 through clones AV12 containing hK2. The hK2 cells containing the AV12 clone numbers 10, 27, 31 and 32 were grown to a confluence of about 60-70% and washed with HBSS, then a serum free HH4 medium was added. The spent medium was removed 7 days after the addition of the serum free medium, concentrated and subjected to SDS / PAGE on a 12% gel. The proteins were electrotended and a probe was applied with HK1D 106.4 or HK1G 464.3. Goat anti-mouse IgG -HRP (1: 500) was used as a secondary antibody and the stain was developed with ECL (Amersham) with the manufacturer's instructions. As controls, purified phK2 217 and hK2v217 were used. The position of hK2 is indicated by an arrow. Figure 14 illustrates the expression of phK2 V217 in clones of AV12-hK v217 selected. The cells of the clone numbers AV12 2, 3, 4, 45 and 48 were grown to a confluence of about 60-70% and washed with HBSS, and a serum free HH4 medium was added. The spent media was removed 7 days after the addition of the serum free medium, concentrated and subjected to SDS / PAGE on a 12% gel. The proteins were electrotrophized and a probe was applied with HK1D 106.4 or HK1G 464.3. Goat anti-mouse IgG -HRP (1: 500) was used as a secondary antibody and the staining was developed with ECL (Amersham) according to the manufacturer's instructions. As controls, purified phK2 217 and hK2v217 were used. The position of hK2 is indicated by an arrow. Figure 15 illustrates the specific amidolytic character of hK2v217, hK2 and PSA for residues 210-236 of hK2. The synthetic peptide (0.63 mM) was digested overnight at 37 ° C with 1 μg / ml hK2, 40 μg / ml hK2 217 or 100 μg / ml PSA, and the digestion products were separated by RP-HPLC. The peaks were normalized to compare the qualitative aspects of the excision.

Figure 16 illustrates the specificity of hK2 and PSA for different peptide substrates. Open arrows denote peptide bonds divided by PSA; the solid arrows denote links divided by hK2. Peptide # 1 represents amino acid residues 210-236 of hK2. Peptide # 2 represents amino acid residues 1-14 of angiotensinogen, i.e., the substrate tetradecapeptide of renin. Peptide # 3 represents the 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 PSMA. Figure 17 illustrates the activation of phK2v217 through hK2, but not hK2v217. PHk2v217 contains the prolific peptide sequence VPLIQSR, a sequence not present in hK2v217. Panel A shows phk2v217 incubated with 1% w / w of hK2. Panel B is a control with 40% w / w of hK2v217 incubated with phK2v217 for 6 hours. Figure 18 illustrates a Western blot analysis of hK2 incubated with protease inhibitors. Each sample was separated on an SDS / PAGE gradient of 8-25%, stained and a probe was applied with HK1G586.1. hK2 was incubated for 4 hours at 37 ° C with the following inhibitors: lane 1, anti-chymotrypsin (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. Serum inhibitors were used at 20 μM, macroglobulin at 2.8 μM, and hK2 at 0.175 μM. Lane 5 shows the higher Mr complexes representing the formation of a covalent complex of hK2 with alpha-2 macroglobulin. Figure 19 illustrates the complex formation of hK2 in human serum. Western stains of hK2 and PSA were incubated with human serum. A probe was applied to the hK2 samples with HK1G586.1 and the PSA samples with PSM773 anti-PSA mAb. Lanes 1-6 contain samples of hK2 and lanes 7 and 8 are samples of PSA. Lane 1 represents a control of hK2. Lane 2 contains hK2 incubated with ACT for 4 hours. Lane 3 represents a serum lane without 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 alpha-2 macroglobulin purified 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 illustrates the immunoreactivity of the monoclonal antibody HK1G586 in untreated human prostate (n = 257). Figure 21 illustrates the immunoreactivity of the monoclonal antibody HK1G586 in human prostate treated with androgen-free therapy (n = 7). Figure 22 illustrates (A) the detection by RT-PCR of PSA and mRNAs of hK2 in RNA extracted from LNCaP cells diluted in whole human blood, and (B) detection by RT-PCR of PSA and mRNAs of hK2 in RNA extracted from Whole human blood of the following patients: patient 17, age 31, male control; patient 21 age 58 years, clinical stage B; patient 24, age 83 years, known metastatic disease (D2); patient 26, age 75 years, pathological stage C (+ margins); patient 28, age 57 years, pathological stage (+ margins); patient 49, age 64 years, pathological stage C (+ seminal vesicle); patient 59, age 31 years, control man; patient 60, age 73 years, metastatic disease driven.

DETAILED DESCRIPTION OF THE INVENTION The high degree of amino acid sequence homology of hK2a 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 samples of tissue, or measuring the levels of transcripts of hK2 in physiological fluids comprising cells, for example, blood samples, or tissue samples, eg, lymph node, may be useful in the diagnosis and verification of prostatic cancer ( PCa) Definitions As used herein, the term "hK2 peptide" includes pre-pro, pro and mature recombinant hK2 polypeptides.

A mature hK2 peptide having the amino acid sequence shown in Figure 1 (SEQ ID NO: 1) as well as polypeptides "variant", which share at least 90% homology with SEQ ID NO: 1 in regions that are substantially homologous with hK3, ie, regions that 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, ie, a mature hK2 polypeptide having a substitution of alanine to valine at the amino acid position 217. The hK2 polypeptides possess an antigenic function in common with the mature hK2 molecule. Figure 1, since such polypeptides can also be defined by antibodies that specifically bind to them, but which do not cross-react with hK3 (or hK1). Preferably, said antibodies react with antigenic or epitope sites that are also present in the mature hK2 molecule of Figure 1. Antibodies useful for defining a common antigenic function are described in detail in patent series No. 08/096, 946, now E patent. OR . Year . 5, 516, 639, ie, polyclonal antisera separated in vivo against subunit 41-56 of hK2. "Isolated hK2 Nucleic Acid" is RNA or DNA containing more than 7, preferably 1 5 and most preferably 20 or more sequential nucleotide bases that are complementary to the non-coding or encoding structure of RNA or DNA of wild-type hK2 polypeptide , or hybridizes to said RNA or DNA and remains stably bound under severe conditions. Preferably, the isolated nucleic acid encodes a biologically active hK2 polypeptide, a variant thereof or a subunit thereof. The biological activity of a hK2 polypeptide can be detected by methods well known in the art including, but not limited to, the ability to react with antibodies specific for hK2 polypeptides, the ability to divide specific substrates into hK2 (see Example 7). ), or the ability to bind to whey proteins (see Example 9). A polypeptide of hK2 variant or subunit thereof, or a subunit of a hK2 polypeptide, has at least about 10%, preferably at least 50% and most preferably at least about 90% of the biological activity of a polypeptide of hK2 comprising the amino acid sequence of SEQ ID NO: 1. In this way, the RNA or DNA is isolated since it is free of at least one contaminating nucleic acid with which it is normally associated in the natural source of the nucleic acid and preferably is substantially free of any other RNA or mammalian DNA. The phrase "free of at least one nucleic acid from a contaminating source with which it is normally associated" includes the case where the nucleic acid is reintroduced to the source or natural cell but is in a different chromosomal location or otherwise it is flanked through nucleic acid sequences not normally found in the source cell. An example of isolated hK2 nucleic acid is RNA or DNA encoding a biologically active hK2 polypeptide by sharing at least 90% sequence identity with the homologous hK3 regions of the hK2 polypeptide of Figure 1, as described above. The term "isolated, purified" as used with respect to a hK2 polypeptide is defined in terms of the methodologies discussed below. As used herein, the term "recombinant nucleic acid", i.e., "recombinant DNA" refers to a nucleic acid, i.e., DNA that has been derived or isolated from any appropriate tissue source, which may be Subsequently or chemically altered in vitro, and then introduced into target host cells, such as cells derived from animal, plant, insect, yeast, fungal or bacterial sources. An example of recombinant DNA "derived" from a source could 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 an essentially pure form. An example of such DNA "isolated" from a source could be a useful DNA sequence that is excised or removed from said source through chemical means, for example, through the use of restriction endonucleases, so that it can be manipulated additionally, for example, amplified, for use in the invention, through genetic engineering methodology. Therefore, a "recombinant DNA" includes fully synthetic DNA sequences, semi-synthetic DNA sequences, DNA sequences isolated from biological sources, and DNA sequences derived from introduced RNA, as well as their mixtures. 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 is resident in the genome but is not expressed, or is 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 way that does not occur in the "natural" type or wild of the species. "Control sequences" is defined to represent DNA sequences necessary for the expression of an operably coding sequence packaged 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 use promoters, polyadenylation signals, and enhancers. "Operably linked" is defined to represent that the nucleic acids are placed in a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretion leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned in order to facilitate translation. Generally, "operably linked" means that the DNA sequences that are linked are contiguous and, in the case of a secretion leader, contiguous and in the reading phase. However, breeders do not have to be continuous. The link is achieved through ligation at convenient restriction sites. If such sites do not exist, adapters or linkers of synthetic oligonucleotides are used according to conventional practice. "Southern analysis" or "Southern staining" is a method whereby the presence of the DNA sequences in the restriction endonuclease digestion of DNA or DNA-containing composition is confirmed through hybridization to a DNA fragment or oligonucleotide marked, known. Southern analysis typically involves electrophoretic separation of digested DNA on agarose genes, DNA denaturation after electrophoretic separation, and transfer of DNA to nitrocellulose, nylon or other suitable membrane support for analysis with a radiolabeled, biotinylated, or labeled probe. enzyme as described in sections 9.37-9.52 of Sambrook et al., supra. "Northern analysis" or "Northern staining" 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 fragment of RNA. The probe is labeled with a radioisotope such as 32p, through biotinylation or with an enzyme. The RNA that will be analyzed can usually be 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. "Severe conditions" are those that (1) employ a low ionic strength and high temperature for washing, for example 0.015 M NaCl / 0.0015 sodium citrate (SSC); 0.1% sodium lauryl sulfate (SDS) at 50 ° C, or (2) employ a denaturing agent such as formamide during hybridization, eg, 50% formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate pH regulator at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42 ° C. Another example is using 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sperm DNA of salmon with sound application (50 μg / ml), 0.1% SDS, and 10% of dextran sulphate at 42 ° C, washed at 42 ° C in 0.2 x SSC and 0.1% SDS. See Sambrook et al., Supra, for other examples of severe conditions.

Expression Cassettes or Expression Vectors A cassette or expression vector comprising a recombinant DNA sequence encoding hK2, which is operably linked to a functional promoter in a host cell, can be circular, or linear, double-stranded structure or of individual chain structure. Generally, the cassette or expression vector is in the form of chimeric DNA, such as plasmid DNA, which may also contain coding regions flanked by control sequences that promote the expression of the recombinant DNA present in the resulting cell line. For example, the expression cassette itself may comprise a promoter that is active in mammalian cells, or may use a promoter already present in the genome that is the target of transformation. Such promoters include the CMV promoter, as well as the SV40 back promoter and retroviral LTRs (long terminal repeat elements). In addition to the recombinant DNA sequences that serve as transcription units for hK2 or portions thereof, a portion of the recombinant DNA may be non-transcribed, serving as a regulatory or structural function. The expression cassettes or expression vectors that will be introduced into the cells will generally also contain either a selectable marker gene or a reporter gene or both to facilitate identification and cells transformed from the population of cells that are searched for transformation. . Alternatively, the selectable marker can be carried on a separate piece of DNA and used in a co-transformation procedure. Both selectable markers and reporter genes can be flanked with appropriate regulatory sequences to allow expression in host cells. Useful selectable markers are well known in the art and include, for example, antibiotic resistance and herbicide genes, such as neo, hpt, dhfr, bar, aroA and the like. See also Table 1 of Lundquist et al. (US Patent No. 5,554,798). Reporting genes are used to identify potentially transformed cells to evaluate the functionality of regulatory sequences. Reporting genes encoding easily assayable proteins are well known in the art. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a protein whose expression is manifested by some easily detectable property, for example, an enzymatic activity. Preferred genes include the chloramphenicol acetyl transferase (cat) gene from E. coli Tn9, the beta-glucuronidase gene (gus) from the uidA site of E. Coli, and the firefly luciferase gene Photinus pyralis. The expression of the reporter gene is analyzed at an appropriate time after the DNA has been introduced into the recipient cells. Other functional elements in host cells, such as introns, enhancers, polyadenylation sequences and the like, can also be part of the recombinant DNA. Such elements may or may not be necessary for the function of DNA, but may provide improved expression of DNA by transcription, stability of mRNA, or the like. Such elements may be included in the DNA as desired to obtain the optimum performance of the DNA transformation in the cell.

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 can be used 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 construction methods. Host Cell Transformation and Recovery of Recombinant hK2 Polypeptides The expression cassette or expression vector comprising the recombinant DNA encoding a hK2 polypeptide can be easily introduced into the target cells through transfection, for example, through the modified calcium phosphate precipitation process of C. Chen et al., Mol. Cell. Biol., 7., 2745 (1987). Transfection can also be achieved through lipofectin, using commercially available equipment, for example through BRL. Suitable host cells for the expression of hK2 polypeptide are derived from multicellular organisms. Said host cells are capable of complex glycosylation and processing activities. In principle, any higher eukaryotic cell culture can be employed in the practice of the invention, whether of vertebrate or invertebrate culture. Examples of invertebrate cells include plant cells and insects. Numerous baculoviral strains and variants and host cells of corresponding permissible insects of hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fly), and Bombyx mori have been identified. See, for example, Luckow and others, Bio / Technology. 6, 47 (1988); Miller et al., In Genetic Engineering, J.K. Setlow et al., Eds., Vol. 8 (Plenum Publishing, 1986), p. 277-279; and Maeda et al., Nature, 315, 592 (1985). A variety of viral strains for transfection are publicly available, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used, preferably for the transfection of Spodoptera frugiperda cells. . When the 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 the hK2 polypeptide from recombinant cell proteins or polypeptides to obtain preparations that are substantially homogeneous as the hK2 polypeptide. For example, the culture medium or lysate can be centrifuged to remove waste from cell to particle. The membrane and the soluble protein fractions are then separated. The hK2 polypeptide can then be purified from the soluble protein fraction and, if necessary, from the membrane fraction of the culture lysate. The hK2 polypeptide can then be purified from soluble contaminating proteins and polypeptides through fractionation and immunoaffinity columns or ion exchange; precipitation with ethanol; Reverse phase HPLC; chromatography on silica or anion exchange resin such as DEAE; chromate focus; SDS / PAGE; precipitation of ammonium sulfate; gel filtration using, for example, Sephadex G-75; or ligand affinity chromatography. Once isolated from the resulting transgenic host cells, the derivatives and variants of the hK2 polypeptide can be easily prepared. For example, amides of the hK2 polypeptides of the present invention can also be prepared through techniques well known in the art to convert a carboxylic acid group or precursor to an amide. A preferred method for the formation of amide in the C-terminal carboxyl group is to separate the polypeptide from a solid support with an appropriate amine, or separate in the presence of an alcohol, yielding an ester, followed by aminolysis with the desired amine. Salts of carboxyl groups of hK2 peptide can be prepared in a usual manner by contacting the peptide with one or more equivalents of a desired base such as, for example, a metal hydroxide base, for example sodium hydroxide; a base of bicarbonate or bicarbonate of metal 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 polypeptides herein can be prepared using an N-acyl-protected amino acid for final condensation, or through the acylation of a protected or unprotected peptide. O-acyl derivatives can be prepared, for example, through the acylation of a free hydroxy peptide or peptide resin. Any acylation can be performed using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N and O-acylation can be performed 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 specified positions, including substitutions using form D instead of L. Acid addition salts can be prepared from polypeptides by contacting the polypeptide with one or more equivalents of inorganic or organic acid, such as, for example, hydrochloric acid. Esters of carboxyl groups of the peptides can also be prepared by any of the usual methods known in the art.

Polypeptides hK2 Variants It is noted that the 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, for example SEQ ID NO: 8 has a substitution of alanine to valine at position 217 in relation to SEQ ID NO: 1. In particular, amino acids are substituted in a relatively conservative manner. Said conservative substitutions are shown in Table 1 under the heading of illustrative substitutions. Very preferred substitutions are under the heading of preferred substitutions. After the substitutions are introduced, the products are classified for biological utility, for example, ability to generate specific antibodies in hK2 or to specifically react with specific antibodies in hK2, ie, antibodies that bind to hK2 and not to hK2 ( PSA). TABLE 1 Residual Substitutions Substitutions Original Illustrative Preferred Wing (A) val; leu; ile val Arg (R) lys; gln; asn lys ASN (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C) be Gln (Q) asn asn Glu (E) asp asp Gly (G) pro pro His (H) asn; gln; lys; arg arg lie (I) leu; val; met; to; phe leu norleucine Leu (L) norleucine; ile; val; met; ile ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; wing leu Pro (P) giy gly Ser (S) thr thr Thr (T) be Trp (W) tyr tyr Tyr (Y) trp; phe; thr; to be phe Val (V) ile; leu; met; phe; to; leu norleucine Amino acid substitutions that fall within the scope of the invention, in general, are achieved by selecting substitutions that do not differ significantly in their effect in maintaining (a) the base structure of the polypeptide in the substitution area, eg, as a conformation. of sheet or helical, (b) the load or hydrophobic character of the molecule at the target site, or (c) the volume of the side chain. The residues of natural existence are divided into groups based on common side chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acid: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence the chain orientation: gly, pro; and (6) aromatic; trp, tyr, phe. The invention also encompasses variants of hK2 with non-conservative substitutions. Non-conservative substitutions refer to the exchange of a member of one of the classes described above by another. Amino acid substitutions are introduced into the DNA molecules of the invention through methods well known in the art.

Uses of Recombinant hK2 Polypeptides Once isolated, the hK2 polypeptide and its variants, derivatives and antigenically active fragments can be used in assays for hK2 and samples derived from biological materials suspected of containing hK2 or anti-hK2 antibodies, as described in detail in U.S. Patent No. 5,516,639. For example, the hK2 polypeptide can be labeled with a detectable label, such as through one or more radiolabeled peptidyl residues, and can be used to compete with endogenous hK2 to bind anti-hK2 antibodies, ie, as a " Capture antigen "to bind anti-hK2 antibodies in a sample of the physiological fluid, through various formats of competitive immunoassays for hK2 using anti-hK2 antibodies whare capable of immobilization is performed: (a) provide an amount of anti-hK2 antibodies that are capable of binding to a solid surface; (b) mixing a physiological sample, whcomprises hK2, with a known amount of the hK2 polypeptide comprising a detectable label to produce a mixed sample; (c) contacting said antibodies with the mixed sample for a sufficient time to allow immunological reactions to occur between the antibodies and hK2 to form an antibody-hK2 complex, and between the antibodies and the labeled polypeptide to form a complex of the polypeptide labeled with antibody; (d) separating the antibodies that are bound to hK2 and the antibodies bound to the labeled polypeptide from the mixed sample; (e) detecting or determining the presence or amount of the labeled polypeptide either bound to the antibodies on the solid surface or remaining in the mixed sample; and (f) determining from the result in step (e) the presence or amount of said hK2 in the sample. In another format, which can detect endogenous hK2 in a sample through a competitive inhibition immunoassay, a known amount of the anti-hK2 antibody is added to a mixture containing an unknown amount of endogenous hK2. The known amount is selected to be less than the amount required to complex the entire hK2 with suspicion of being present, for example, which could be present in a sample of the same amount of sample amount obtained from a patient known to be who has prostate cancer Then, a known amount of the hK2 polypeptide of the invention or a subunit thereof is added, comprising a detectable label. If endogenous hK2 is present in the sample, fewer antibodies will be available to bind to the labeled hK2 polypeptide, and will remain free in solution. If no endogenous hK2 is present, the aggregated labeled polypeptide will complex with the added anti-hK2 antibodies to form binary complexes. Then, the binary antibody-antigen complexes are precipitated through an anti-mammalian IgG antibody (sheep, goat, mouse, etc.). The amount of radioactivity or other mark in the precipitate (a ternary complex) is inversely proportional to the amount of endogenous hK2 that is present in the sample, for example, a pellet containing reduced amounts of radioactivity is indicative of the presence of endogenous hK2. . Alteratively to 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 the preparation of a fused cell line of production and antibody, for example, of primary spleen cells fused with a compatible continuous line of myeloma cell, and developing the fused cells either in a mass culture or in an animal species from which the myeloma cell line used was derived or is compatible. Such antibodies offer many advantages compared to those produced through inoculation of animals, since they are highly specific and sensitive and relatively "pure" immunochemically. Immunologically active fragments of the antibodies of the present invention are also within the scope of the present invention, for example, the f (ab) fragment, since they are partially humanized monoclonal antibodies.

Chimeric and Modified Antibodies Chimeric antibodies comprise the fusion of the variable domains of one immunoglobulin to the constant domains of another immunoglobulin. Usually, the variable domains are derived from an immunoglobulin gene from a different species, perhaps a human being. This technology is well known in the field. See, for example, European Patent Applications EP-A-0 125,023 (Cabilly / Genetech) and EP-A-0 120,694, and US Patent No. 4,816,567, which describe the preparation of variations of immunoglobulin-like molecules using recombinant DNA technology. Another aspect for preparing chimeric or modified antibodies is to bind the variable region of a monoclonal antibody to another non-immunoglobulin molecule, to produce a chimeric derivative molecule (see WO 86/01533, Neuberger and Rabbits / Celltech). An additional aspect is to prepare a chimeric immunoglobulin having different specific characters in its different variable regions (see EP 68763A). Another aspect is introducing a mutation in the DNA that encodes the monoclonal antibody, so that it alters certain of its characteristics without changing its specific essential character. This can be achieved through site-directed mutagenesis or other techniques known in the art. The patent application of Winter EP-A-0 239 400 describes the preparation of a derivatized antibody, altered replacing the complementarity determining regions (CDRs) of a variable region of an immunoglobulin with the CDRs of an immunoglobulin of different specific character, using recombinant DNA techniques ("CDR-graft"). In this way, CDR-grafting allows the "immunization" of antibodies, in combination with the alteration of framework regions. Human antibodies can also be prepared by reconstituting the human immune system in mice lacking their natural immune system, then immunizing the mice in order to produce human antibodies that are specific for the immunogen. A "humanized" antibody that contains the CDRs of a rodent antibody specific for an antigen of interest may probably be less recognized as foreign by the immune system of a human being. Then a "humanized" antibody with the same specific binding character, as for example HK1G464, can be of particular use in therapy for human beings and / or diagnostic methods. The manipulation and / or alteration of any given antibody, or gene encoding thereto, to generate a derivatized antibody is well known in the art.

Detection of Specific Transcripts in hK2 through Reverse Transcriptase Chain-Polymerase Reaction (RT-PCR) To detect transcripts of RNA encoding hK2, the RNA is isolated in a cellular sample suspected of containing hK2 RNA, for example, the Total RNA isolated from human prostate tissue. The RNA can be isolated through methods known in the art, for example, using the TRIZOL ™ reagent (GIBCO-BRL / Life Technologies, Gaithersburg, MD). Oligo-dT can be used as an initiator in a reverse transcriptase reaction to prepare first strand structure cDNAs from the isolated RNA. The first chain structure cDNAs are then amplified in PCR reactions. The "polymerase chain reaction" or "PCR" refers to a method or technique in which amounts of a pre-selected fragment of nucleic acid, RNA and / or DNA, are amplified as described in the patent of US Pat. No. 4,683,195. Generally, sequence information from the ends of the region of interest or beyond is used to design oligonucleotide primers. These primers will be identical or similar in sequence to opposite string structures of the template that will be amplified. PCR can be used to amplify specific RNA sequences, DNA sequences specific to total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, and the like. See in general Mullis et al., Cold Spring Harbor Svmp. Quant. Biol .. 51, 263 (1987); Erlich, ed., PCR Technology, (Stockton Press, NY, 1989). In this way, the amplification of specific nucleic acid sequences via PCR is based on oligonucleotides or "primers" having conserved nucleotide sequences, wherein the conserved sequences are deduced from alignments of the related genoprotein sequences, for example, a sequence comparison of mammalian hK2 genes. For example, an initiator is prepared, which is predicted to recose the antisense strand structure, and another primer is prepared, which is predicted to recose the sense strand structure of a DNA molecule encoding a hK2 polypeptide. In general, the isolated RNA is combined with an initiator in a reverse transcriptase (RT) reaction to generate cDNAs of individual chain structure. Oligo-dT or oligonucleotides of random sequence, as well as sequence-specific oligonucleotides, can be used as an initiator in the RT reaction. See Sambrook et al., Supra. The individual chain structure cDNAs are then amplified with sequence specific primers to produce 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 specific amplified DNA in hK2 is detected. The detection of the amplified hK2 DNA can be achieved by separating 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 to the cloning site of a suitable vector, such as the pCRI1 vector (Invitrogen), sequencing the cloned insert and comparing the DNA sequence with the known sequence of hK2. Alternatively, for example, DNA amplified with hK2 can be detected using Southern hybridization with a specific oligonucleotide probe in hK2, or by comparing its electrophoretic mobility with DNA standards of known molecular weight. The invention will be further described with reference to the following detailed examples.

EXAMPLE 1 Materials and Methods Construction of Mammalian hK2 Expression Vectors A cDNA (length of approximately 820 bp) encoding all prepro-hK2 (pphK2) (from nucleotide # 40 to # 858 relative to the start site of transcription pphK2) as shown in Figure 2, was synthesized from human BPH tissue RNA using polymerase-reverse transcription chain reaction (RT-PCR) with a pair of specific oligonucleotide primers in hK2 (5'ACGCGGATCCAGCATGTGGGACCTGGTTCTCT 3 '; SEQ ID NO: 9 and 5' ACAGCTGCAGTTTACTAGAGGTAGGGG TGGGAC 3 '; SEQ ID NO: 10 This cDNA was generated so that the 5' and 3 'ends (with respect to the sense sequence pphK2 are with the sequence) BamH1 and Pst1, respectively The cDNA was then purified through agarose gel electrophoresis, and was digested with restriction enzymes BamH1 and Pst1 The restricted cDNA was ligated with the plasmid vector pVL1393 digested with BamH1-Pst1 and transformed E. coli strain HB101. The plasmid vector pphK2 cDNA / pVL1393 carrying E. coli was selected. The insert containing pphK2 was sequenced. Plasmid pphK2 cDNA / pVL1393 was mass produced in E. coli encoded by ultra CsCi gradient centrifugation. Plasmid pphK2 / pVL1393 in E. Coli HB101 was deposited in the American Type Culture Collection, Rockville, MD, USA, on May 2, 1994, under the provisions of the Budapest Treaty and with access number assigned ATCC 69614. It was generated a 0.8 kb fragment representing the entire coding sequence of pphK2 (Figure 2) via PCR using the primers A (5? TATGGATCCATATGTCAGCATGTGGGACCTGGTTCTCTCCA3 ') (SEQ ID NO: 11) YB (5? TATGGATCCTCAGGGGTTGGCTGCGATGGT3') (SEQ ID NO: 12), and plasmid PvL1393 containing pphK2 (a gift from DR Young, Mayo Clinic) as the template. The PCR products were inserted into the TA cloning vector (Invitrogen Corp., San Diego, CA) and the DNA of the entire insert was sequenced. To obtain the mammalian hK2 vectors, the inserts containing hK2 were isolated from the corresponding TA clones and inserted into the Bc11 site of plasmid pGT-d (Berg et al., Nucí Acids Res., 20, 54-85 (1992) ) (gift of Dr. Brian Grinnell, Lilly) under the control of the GBMT promoter. The mammalian expression vectors, PLANS-hK2 and PLNC-hK2, were obtained by cloning the 0.8 kb wild-type hK2 insert of the TA vector corresponding to the plasmids, pLNSX and pLNCX (Miller et al., Biotech .. 9. 980 (1989)), respectively. The orientation of the insert in all mammalian expression vectors was confirmed through DNA sequencing.

Generation of Recombinant Clones AV12-664 (ATCC CRL-9595), a cell line derived from adenovirus-induced tumors in Surian hamster, and DU145 cells were cultured in a Dulbecco-modified Eagle (high glucose) supplemented with 10 % fetal bovine serum (D10F). PC3 cells were cultured in minimal Eagle's 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, the cells were resuspended in D10F + 200 nM methotrexate (MTX). Clone cell lines resistant to the drug were isolated after 2-3 weeks and their spent medium was analyzed through Western stains. PC3 and DU145 cells were transfected with mammalian expression vectors hK2 using lipofectamine (Gibco-BRL, Gaithersburg, MD) and the clones (PC3-hK2 and DU145-hK2) were selected in medium containing 400 μg / ml G418.

Purification and Sequencing of the Protein AV12-hK2 clones were developed in D10F + 200 nM of MTX. At a confluence of about 60%, the cells were washed with a balanced salt solution of Hanks and resuspended in serum free HH4 medium. The spent medium was collected 7 days after the addition of spent medium free of serum and stored at -20 ° C. To purify the protein, the spent serum-free medium was concentrated and exchanged in 50 nM sodium bicarbonate, pH8. The samples were filtered with 0.2 μ 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. The pH regulator A contained 50 mM sodium bicarbonate, pH 7.9, and the pH B regulator contained 50 mM sodium bicarbonate plus 0.5 M NaCl, pH 7.6. The elution profile was developed with a gradient of 0-50% of pH B regulator for 35 minutes. % 0-100% B of 35-40 minutes and 100% Socratic elution of B for 5 minutes before rebalancing the pH regulator A. The flow rate was totally 5 ml / minute. In the above procedure, the borate pH regulator can replace the bicarbonate pH regulator without any noticeable difference.

DEAE fractions were analyzed for the presence of hK2 through the dry ELISA method (see below) using rabbit anti-pphK2 (Saedi et al., Mol.Cell.Endoc., 109, 237 (1995)). The fractions with the activity of hK2 were combined and concentrated through ultrafiltration with membranes (10 kD cut) to about 5-8 ml. Then, solid ammonium sulfate was added to a final concentration of 1.2 M. This sample was then injected onto a polypropylpartamide column, PolyLC, pore size 1000A, 4.6 mm X 200 mm, to solve the proteins through integration chromatography hydrophobic (HIC). The pH A regulator was 20 mM sodium phosphate, 1.2 M sodium sulfate, pH 6.3, and the pH regulator B was 50 mM sodium phosphate, 5% 2-propanol, pH 7.4. The elution gradient was 0-20% B for 5 minutes; 20-55% of B of 5-20 minutes, Socratic to 55% of B of 20-23 minutes, 55-100% of B of 23-25 minutes; 100% Socratic at B for 2 minutes before re-equilibrating with pH regulator A. The flow rate was 1 ml / minute. The hK2 containing the HIC peak, which was eluted at approximately 50% B, was exchanged in the pH buffer of 50 mM borate, pH 8, through repeated concentration with an ultra-cut filtration of 10 K MW Centricon-10 (Amicon) purity was analyzed by both SDS-PAGE and Western stain analysis. The extinction coefficient used to estimate hK2 concentrations was A28o of 1.84 = 1 mg / ml. In some cases, hK2 containing the peak HIC was further purified through size exclusion chromatography (SEC) on a 10/30 Pharmacia S12 column. In this case, the hK2 containing the peak HIC was concentrated through ultra filtration as previously done 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 through ultra filtration. The peak SEC collected in ammonium acetate was removed to remove the pH regulator and then reconstituted in water. An aliquot of this sample was hydrolyzed in 6 N gaseous HCl under vacuum for 20 hours at 120 ° C, then reconstituted in 0.1N HCl and analyzed on a Hewlett Packard Aminoquant amino acid analyzer using pre-column derivatization of amino acids with OPA for primary amines and FMOC for secondary amines. An affinity resin HK1G 586.1 (see below) was used to purify hK2 by affinity chromatography. The monoclonal antibody HK1G 586.1 (mAb) was coupled with Pharmacia GammaBind plus Sepharose (cat. No. 17-0886) according to Schneider (J. Biol. Chem. 257, 10766 (1982)). Briefly, HK1G 586.1 mAb and the resin were incubated overnight at 4 ° C with rotation. The resin was centrifuged (500 X g for 5 minutes at 4 ° C) and washed twice with 0.2 M triethanolamine, pH 8.2. The amine groups were entangled in a fresh linker solution (25 mM dimethyl pimelimidate dihydrochloride in 0.2 M triethanolamine, pH 8.2) for 45 minutes at room temperature (22 ° C). The resin was quenched with 20 mM ethanolamine, pH 8.2, for 5 minutes at room temperature and then washed twice with 1 M NaCl, 0.1 M PO 4, pH 7.0. The resin was washed twice more with PBS and stored at 4 ° C with 0.5% NaN3 until used. An Applied Biosystems Model 477a pulsed liquid phase sequencer was used to sequence the proteins and peptides. Model 477a used automatic Edman degradation chemistry to sequentially release amino acids from the N term followed by PTH derivatization and reverse phase HPLC chromatography. Peptide samples were applied to the sequencer on fiberglass filter supports treated with biobrew and the complete proteins were applied either to the filters treated with biobrew or to the pre-activated gate filters (Beckman, Fullerton, CA). Samples of staining sequences were first operated as mini gels in the Novex system (Novex, San Diego, CA), then transferred to the Problot PVDF membrane, visualized with Commassie blue, the appropriate band was cut and sequenced directly from the PVDF membrane.

Production of Monoclonal Antibody A / J mice were injected with 50 μl of phK2 in complete Freund's assistant (CFA) i.p. on day 1, 1.25 μg of phK2 in incomplete Freund's assistant (IFA) i.p. on day 14 and 25 μg of phK2 in PBS i.p. on day 28. Three days before the fusion, the mice were boosted with 10 μg of phK2 in PBS i.v. The mice were sacrificed and a single cell suspension of the spleens was prepared. Immune B cells were fused with P3.653 myeloma cells. The cloned hybridomas were classified by ELISA and selected based on the reactivity of the supernatants at hK2v217 and phK2v217 and the minimal reactivity with PSA. The clones selected through these criteria, clones HK1G464 and HK1G586, were subcloned using the sealed FACStar plus cell sorter to deposit the individual cells on the mouse spleen feeder layers. Subclones HK1G464.3 and HK1G586 were used for further studies. Another fusion, which used the same protocol described above, except that the immunogen was hK2 217, alum was used in place of CFA and IFA, BALB / c mice were used instead of A / J mice, producing the clone HK1H247. Hybridoma clones HK1G464.3, Hk1G586.1, HK1H247, HK1A523 and HKD106.4 were deposited with the American Type Culture Collection, in accordance with the Budapest Treaty and with the access numbers granted: HB11983, HB12026, HB12162, HB11876 and HB11937, respectively.

Production of Polyclonal and Monoclonal Antibody for Peptide Immunogens hK2 Sheep and goats were immunized subcutaneously with 100 μg of peptide conjugated with KLH in complete Freund's Assay (CFA) and boosted at 3-week intervals with 100 μg of Peptide in Freund's Assay incomplete (IFA). For monoclonal antibodies to peptide immunogen, Balb / c mice were immunized subcutaneously with 100 μg of KLH-conjugated peptide in complete Freund's Assay (CFA) and boosted at 3-week intervals with 100 μg of incomplete Freund peptide (IFA). . Alternatively, A / J mice were immunized twice intraperitoneally with 50 μg of the peptide conjugated with KLH and mice with positive titers were reinforced intravenously with 25 μg of the conjugate. After the first three immunizations, the blood of the animals was tested for the presence of the antibody, 6 to 10 days after the immunization. Peptides were immobilized on 0.635 cm polystyrene beads (Clifton, Clifton Heights, PA) by incubating 1 μg of the peptide conjugated to BSA (bovine serum albumin) per bead in carbonate pH regulator, pH 9.6, overnight at 4 ° C. ° C, the beads were washed three times with 0.01 M saline regulated at pH with phosphate (PBS), pH 7.4, with 0.1% Tween and blocked with 1% skim milk plus 1% BSA. These beads were incubated for 18 hours with 250 μl of a dilution of 1: 100, 1: 1000 or 1: 10,000 of animal sera. After three washes, 250 μl of rabbit anti-mouse, anti-mouse or anti-goat antibody conjugated to horseradish peroxidase (Cappel-Organon Teknica Corporation, Durham, NC) were incubated with each bead for three hours in a horizontal incubator at 150 rpm. The enzyme signal was quantified spectrophotometrically using ortho-phenylene diamine as a substrate. No non-immune sera were used as a negative control and measurements of immune sera were expressed as multiple of the control reading. Spleen lymphocytes from mice with positive serum titers were fused with myeloma cells to produce hybridoma cells. The antibodies produced through the clones of these cells were classified as described above. Positive clones were subcloned by limiting dilution and reclassified. The monoclonal hybridomas were injected into the perifoneal cavities of mice initiated with pristine to obtain ascitic fluid. To purify monoclonal and polyclonal antisera, the antisera were initially subjected to a separation of IgG through precipitation with saturated ammonium sulfate and size chromatography using an ACA-34 ultragel column. The polyclonal antisera were further purified by affinity 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).

ELISA assays A dry ELISA format was used to measure hK2 in a serum-free spent medium of the clones and in the fractions collected during the purification of hK2. Microtiter plates (Becton Dickinson Labware, NJ) were coated with 50 μl of spent medium or column fractions overnight at 37 ° C. The cavities were washed with PBS + 0.1% Tween 20 (PBST) and incubated for one hour with 50 μl of primary antibodies. The wells were washed again with PBS + T and washed with one hour at 37 ° C with 50 μl goat anti-mouse-IgG or goat anti-rabbit-IgG antibodies coupled with horseradish peroxidase (1 : 500, Jackson Immunosearch Laboratories, Inc., West Grove, PA). The cavities were washed with PBST, incubated with o-phenylenediamine dihydrochloride (OPD, Sigma, MO) for 5 minutes, and the colorimetric reaction was measured at A490 with an ELISA reader (Biotek Instruments, Inc., model EL310, VT ). All samples were analyzed in duplicate. Serum-free spent medium from AV12 cells transfected with only the vector was used as a negative vector. Antibodies were tested in an ELISA-based solution using phK2v? , hK2v¿, and biotinylated PSA. PSA was purified by the method of Sensabaugh and Blake (J. Urology, 144, 1523 (1990)). Twenty ng of phK2v217 or biotinylated hK2v217 diluted in 50 μl of buffer pH A (8.82 mM citric acid, 82.1 mM sodium phosphate (dibasic), 10% BSA, 0.1% mannitol, 0.1% Nonidet P-40, pH 7.0 ) or 0.25 ng of biotinylated PSA diluted in 10% horse serum (HS) in PBS, incubated with 50 μl of hybridoma supernatants, negative control supernatants (i.e. irrelevant hybridoma supernatant for phK2v217 and hK2v217, or μg / ml of irrelevant purified mAb in HS for PSA), or positive control supernatants (ie, 20 μg / ml purified PSM773 (anti-PSA) mAb in HS for PSA, or hybridoma supernatant HK1D 104 (anti "hK2") for phK2v217 and hK2v217). HCO514, a mAb against hCG, was used as a negative control in PSA assays, and ZCG085, a mAb against tau was used as a negative control in the hK2 assays. These mixtures of antibodies and antigens were allowed to incubate for 1 hour with shaking in a microtiter plate coated with streptavidin (Labsystems, Helsinki, Finland). The plate was washed three times with 300 μl of PBS, 0.1% Tween-20 (PBST), and incubated with 100 μl of gamma-specific goat anti-mouse IgG-horseradish peroxidase conjugate (Jackson I mmunoResearch Laboratories, Inc., Westgrove, PA), was diluted 1: 10,000 in HS, with stirring for 1 hour. After a second wash with PBST, the color was developed for 30 minutes, with shaking, after the addition of 100 μl of 1 mg / ml of o-phenylenediamine in 50 mM pH regulator of phosphate citrate, 0.03% of Sodium perborate, pH 5.0 (Sigma Chemical, St. Louis, MO). The reaction was quenched by the addition of 50 μl 4 N H2SO4. The intensity of the color was determined by measuring the absorbance at 490 nm and 540 nm using a microtiter plate reader. Absorbances above 2.6 to 490 nm were corrected with a reading of 540 nm. The sample values are average standard deviations +. of triplicate. The control values are averages of duplicates.

Western Dye Assays Western blot analyzes were performed using standard procedures. Serum-free spent media were concentrated 10 times using Centricon 10 (Amicon, Inc., Beverly, MA) and subjected to SDS / PAGE using 12% gel (Bio-Rad, Inc., Melville, NY). For analytical purposes, SDS / PAGE was performed in a Pharmacia PhastSystem using 8-25% gradient gels. After electrophoresis, the proteins were transfected on nitrocellulose membrane and blocked overnight at 4 ° C with 2% dry milk without fat in PBS. The stains were rinsed, then incubated with primary antibody (dilution 1: 1000 of ascites, or 1μl / ml of purified mAbs or polyclonal Abs) for 1 hour at 22 ° C. The stains were then washed and incubated for 45 minutes with secondary antibody (goat anti-mouse-HRP or goat anti-rabbit-HRP, 1: 500, Jackson Immunosearch Laboratories, Inc., West Grobe, PA). The immunoreactive bands were detected by revealing the staining using DAB (Sigma, St. Louis, MO) plus H2O2 or using the ECL system (Amersham, Buckinghamshire, England) according to the manufacturer's instructions.

Covalent Complex Formation To test for covalent complex formation, 0.175 μM of hK2 was incubated with 20 μM of inhibitor at a pH of 8 in 100 mM of borate pH buffer. The inhibitors tested were 1-antichromotripsome, 1-antitrypsin, 1-antiplasmin, antithrombin and 2-macroglobulin. To 5 μl of hK2 (10 μg / ml) was added the calculated μg of inhibitor prepared in 100 mM of borate pH buffer and, if necessary, each sample was brought to a total volume of 10 μl. The samples were incubated for 3 hours at 37 ° C after which 1.5 μl of a 7 pH PhastSystem SDS sample buffer containing 35% 2-mercaptoethanol was added and the sample boiled for 3 minutes in a water bath. The samples were diluted in% SDS sample buffer before application to SDS / PAGE and Western analysis.

Proteolysis of Peptide Substrates To determine the ability of hK2 to separate peptide substrates, the peptides were dissolved in DMSO at 10 mg / ml, then diluted 1:10 in 100 mM borate pH buffer, pH 8, containing PSA , hK2 or trypsin. Typical experiments were performed as follows: 1 μl of peptide was added to 7 μl of 100 mM borate pH buffer and then 2 μl of hK2 (10 μg / ml), PSA (500 μg / ml) or trypsin ( 0.5 μg / ml). In general, the samples were incubated for 16 hours at 37 ° C. Samples were quenched with 100 μl of 0.2% TFA / water and the extinguished sample was applied directly to a Vydac C-18 reverse phase column attached to the BioRad Model 800 HPLC equipped with an AS 100 autosampler, dual pumps 1350 and detector UV-VIS of Biodimension exploration. Solvent A was 0.1% TFA / water and solvent B was acetonitrile containing 0.1% TFA. The sample was applied in 90% of solvent A and the gradient was developed to 60% of solvent B in 10 minutes. The absorbance was verified simultaneously at 220 nm and 280 nm. The collected HPLC peaks were concentrated through vacuum centrifugation or leophilized and then applied to the amino acid sequencer to identify individual fragments. In some cases, 10 μl of the extinguished sample mixture was applied directly to the sequencing membrane and, since the sequence was known, the cleavage sites were determined from the distribution of the amino acids present in each cycle.

Protease Assays Using Chromogenic Substrates Tests were performed to measure the hydrolysis of derivatized substrates with para-nitroanilide using an HP 8452A UV-VIS spectrophotometer equipped with a programmable, heat settable 7-position cell holder. The assays were performed in 10 mM sodium borate, pH 8, incubated at 37 ° C, the increase in absorbance was verified at 404 mM. Methoxysuccinyl-Arg-Pro-Tyt-para-nitroanilide (MeO-Suc-R-P-Y-pNA) and H-D-pro-phe-arg-para-nitroamilide (P-F-R-pNA) were at 1 mM in the assay. An ABI model 431A peptide synthesizer using standard FastMoc chemistry was used to synthesize all the peptides listed in Figure 16, except # 2, angiotensin and # 5, oxidized beta-chain insulin, which were obtained from Sigma. The mass of each peptide synthesized was confirmed by mass spectrometry (University of Michigan, Core Facility), using ES / MS. An ABI sequencer model 477a described above was used to confirm the peptide sequence.

Conversion of phK2 Vv2¿117 'to hK2, v217 Samples of phK2 .V217 were incubated at 100-400 μg / ml in 50 mM sodium borate with 1% w / w trypsin or hK2 at 37 ° C. The conversion from pro to mature was verified through the division of 1-2 μg of the starting material hK2v217 to 100 μl of buffer A HIC and the resolution of the two forms through HIC-HPLC was as described above . Incubation of hK2 217 with phK2v217 was conducted in the same manner except that comparable amounts of the two forms were incubated together as shown in Figure 17B.

Example 2 Expression and Purification of hK2v217 in Mammalian Cells To express hK2 in mammalian cell lines, a 0.8 kb fragment encoding all hK2 (pphK2) coding sequences (Figure 2) was amplified using PCR, subcloned to PCR II vector (TA) and several clones were isolated. The nucleotide sequence of the whole pphK2 insert in some of these clones was determined to detect any mutation that might have been caused by the PCR amplification. Two clones, one having an insert wild type hK2, TA-hK2 and one having the insert hK2 mutant, TA-hK2 v217 were selected for further analysis. TA-hK2 contains a substitution of T for C at codon 650 of hK2 resulting in a conservative substitution of valine (GTT) for alanine (GCT) at amino acid residue 217 of hK2 (Figure 2). To obtain mammalian expression vectors, the pphK2 inserts of TA-hK2 and TA-hK2v217 were cloned into plasmid PGT-d under the control of the GMBT promoter using the plasmids pGThK2 and pGThK2v217 (Figure 3). The GBMT promoter is composed of several regulatory sequences and is activated through the adenovirus E1a protein (Berg et al., Supra (1992)). To determine whether the product of the pphK2v217 gene can be expressed in mammalian cells, the plasmid GThK2 217 was transfected into AV12-664 cells. This cell line is derived from a tumor induced in Syrian hamster through type 12 of adenovirus and expresses the E1a protein of adenovirus. The E1a protein activates the GMBT 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 media analyzed by Western staining. Several clones were identified that secreted into the medium a polypeptide immunoreactive to the anti-pphK2 antiserum. Once cloned (AV12-pGThK2v217 # 2) it was selected for further characterization and protein purification. To purify hK2 polypeptides, the spent serum-free medium from clone # 2 AV12-pGThK2v217 was collected 7 days later, concentrated and subjected to anion exchange chromatography (Figure 4A). The peak activity of hK2 was eluted at approximately 0.2 M NaCl as determined by ELISA assays (dotted line). The ELISA assay co-related well with the appearance of a band of approximately 34 kD of protein seen through SDS / PAGE in the same fractions. The positive hK2 fractions from the anion exchange column were collected and subjected to hydrophobic interaction chromatography (HIC) (Figure 4B). A major portion of A28o was not retained in the HIC column. The main peak retained in HIC, which was eluted at 22 minutes, also showed the highest peak of activity through ELISA assay (dotted line, Figure 4C). A major protein band of approximately 34 kD was also observed through SDS-PAGE. When the 22 minute peak of HIC resolved through SEC, typically about 80-905 of the A280 protein eluted in 19.4 minutes, a retention time consistent with a protein of approximately 34 kD (Figure 4C). The only other protein spot in SEC was eluted at 16.7 minutes, and corresponds to a protein of approximately 70 kD observed in the previous purification steps. To identify the purified protein, approximately 2.5 μg of the protein was subjected to automatic N-terminal analysis, which produced the following sequence: Val-Pro-Leu-lleu-GIn-Ser-Arg-lleu-Val-Gly-Gly- Trp-Glu-. No sequence of competition was evident from the profile of the amino acids released sequentially by the Edman degradation procedure. By analogy to PSA, this protein is phK2 217, since the known sequence of mature PSA (isolated from seminal fluid) starts with lleu-Val-Gly- and pPSA and phK2 has been postulated to have 7 extra amino acids in the N term ( Figure 2). The amino acid analysis of this protein produced an amino acid composition consistent with the predicted sequence of phK2v217. This phK2 polypeptide was isolated and purified in amounts of mg.

Example 3 Characterization of phK2v217 and generation of hK2, v'217 To examine the efficiency of the purification scheme employed in Example 2, purified 1.5 μg phK2v217 was subjected to SDS / PAGE in the presence or absence of beta-mercaptoethanol (BME), and the gel was stained with silver. The results showed that phK2v217 in the sample was approximately 95% pure (Figure 5). It was also shown that phK2v217 migrated to approximately 30 kD in the absence of BME, and approximately 34 kD in the presence of BME. This pattern is similar to that observed for PSA purified from seminal fluid (Figure 5). The amino acid sequence of hK2, deduced from the cDNA sequence shows the presence of a potential N-linked glycosylation site at residue 78 (N-M-S). To determine if this site is glycosylated, phK2v217 was subjected to SDS / PAGE, transferred to nitrocellulose paper, reacted with lecithins coupled with digoxigenin (DIG) followed by anti-DIG labeled with horseradish peroxidase. In Figure 6 (lane 1), 2 μg of phK2 was stained with concanavalin A (Con A) suggesting the presence of two unsubstituted or 2-O-substituted alpha-mannosyl residues in the protein. Lane 2 shows Con A staining of the positive control glycoprotein, ZCE025 mAB. Both the heavy chains (50 kD) and the light chains (25 kD) of this mAb are known to contain N-linked oligosaccharides with mannose nuclei. Lane 3 shows that a non-glycosylated protein (BSA) fails to react with lecithin With A. PhK2v2i7 was also reacted with RCA (specific character of Gab b1-4GlcNAc) and AAA, (specific character of fucose linked with alpha (1-) 6)). This pattern of lecithin reactivity is consistent with the presence of the N-linked oligosaccharide complex. The oligosaccharides in phK2v217 also contain sialic acid since both SNA (sialic acid bound alpha (2-6) to galactose) and MAA (sialic acid bound alpha (2-6) to galactose) were reactive with phK2 217.

The sequence of the pro region of hK2 is VPLIQSR. A cleavage at the carboxy-terminal end of arginine in this pro sequence can convert phK2 to hK2. A moderate trypsin digestion was developed to hydrolyse the peptide binding of phK2v217 purified in this position. phK2v217 was purified with 1% trypsin and the conversion was verified through HIC-HPLC (Figure 7). This procedure resulted in a complete conversion of phK2v217 to hK2v217. The hK2v217 designated peak was sequenced N-terminally and showed to start with the sequence, IVGGWE, which is the N-term for the mature form of hK2. No other sequence than the previous one was detected, demonstrating that this moderate trypsin treatment does not result in any significant level of non-specific cleavage. The SDS / PAGE of samples treated with trypsin showed a small but discernible increase in morbidity, generally consistent with a smaller mass reduction of 826 daltons, the mass of the pro peptide.

Example 4 Generation of specific hK2 Abs. PhK2 217 and hK2v217 were used as immunogens to generate mAbs against hK2. Hybridomas were classified based on high reactivity with hK2v217 or phK2v217 and minimal reactivity with PSA. The representatives of mAbs obtained from the hybridomas are shown in Table 2. Immunization with phK2 217 resulted in mAb HK1G586.1 and HK1G464.3. HK1G586.1 was specific in hK2, since it recognized both phK2v217 but not PSA. On the other hand, HK1G464 was specific in phK2, since it only recognized phK2v217 and not hK2v217 or PSA.

TABLE 2 Specificity of several mAbs raised to hK2 and phK2 A. mAbs raised to phK2 v217 B. mAbs raised to phK2 V217 Immunization with hK2v217 yielded mAb HK1H247. This mAb was specific in hK2 since it recognized only hK2v217 but not phK2v217 or PSA. These results show that phK2v217 and hK2v217 are effective as immunogens to generate specific mAbs for different forms of hK2.

It was used in Western staining analysis to examine whether HK1G586 recognizes hK2 in seminal fluid (Figure 8). Immunoreactive bands of hK2 at approximately 22 kD, 33 kD, and 85 kD were recognized through this mAb. Recently, an immunoreactive pattern to hK2 in seminal fluid was also reported by Deperthes et al., Biochem. Biophy. Acta. 1245, 311 (1995). This result indicates that mAb raised hK2v217 and recognizes natural hK2 in the seminal fluid. All antibodies raised to hK2v217 or phK2v217 also recognized the corresponding form of hK2 and phK2 indicating that hK2 and phK2 are immunologically similar to hK2v217 and phK2v217, respectively (see below). To prepare additional anti-hK2 antibodies (Abs), a direct primary structure comparison between the members of the human kallikrein gene family and the computer-assisted antigenic character and hydrophobicity analysis was conducted. From this comparison, several sequences of the oligonucleotide hK2 were selected. The selected hK2 peptides correspond to the amino acid residues of mature hK2 8-26 (SEQ ID NO: 19), 15-26 (SEQ ID NO: 26), 41-56 8SEQ ID NO: 20), 43-66 (SEC 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 probability of producing antibodies that recognize the natural form of hK2. Peptides were synthesized and HPLC purified at Protein Core Facility at Mayo Clinic / Foundation. The peptides were conjugated with key limpet hemocyanin (KLH) and BSA for immunogens and assay reagents, respectively. Sheep, goats and mice were immunized with KLH-hK2 peptides for polyclonal antibody (sheep and goats SEQ ID Nos: 20 and 21, and SEQ ID Nos19, 20, 21, 24, 25 and 26, respectively)) and monoclonal antibody (mice; SEQ ID Nos: 19, 20, 21, 24, 25 and 26). Peptide 17-71 (SEQ ID NO: 22) was oxidized to generate an intramolecular disulfide bond between cys 26 and 42 and was used to immunize goats and mice for the production of monoclonal and polyclonal antibody, respectively. The goat hK2 41-56 Ab was first purified by column activity of peptide hK2 41-56 and then used for Western staining analysis. The antibody recognized the recombinant checker. The detection of hK2 through hK2 41-56 Ab was abolished through the addition of an excess of peptide hK2 41-56 but not of peptide PSA 41-56. In addition, monoclonal anti-hK2 41-56 peptide antibodies were highly specific for hK2 protein in Western analysis. Antiserum of the anti-hK2 peptide 153-167 (sheep) recognized recombinant hK2. These results suggested that the antibodies for peptides 41-56 and 153-167 react with two distinct epitopes on hK2 polypeptides. Antisera against the amino acid residues of hK2 210-235 showed the highest immunoreactivity. N goat antiserum raised against peptide hK2 17-71, which has 69% homology with the corresponding region of PSA, recognized the recombinant hK2 protein but not PSA. The rabbit antiserum to the bacterially expressed recombinant hK2 protein, which recognizes both PSA and hK2, detected a double band of protein in the LNCaP cell medium concentrated from LNCaP cells that were treated with androgen. In contrast, no immunoreactive protein was detected in the LNCaP cell medium from LNCaP cells, which were not treated with androgen. In this way, the immunoreactive proteins were induced by the androgen. In addition, the upper band in the LNCaP medium is a PSA-related protein since a specific antiserum in PSA (rabbit anti-PSA antiserum which was raised against bacterially expressed recombinant PSA) mainly detected the upper band. The lower band in the LNCaP medium is a hK2-related protein since a mouse monoclonal antibody (HK1A523) against the peptide hK2 41-56 having mono-specific character for hK2 recognizes the lower protein band. These results were confirmed through N-terminal amino acid sequence analysis of each protein in the double bands. Immunohistochemistry studies of sections of human prostate tissue embedded in paraffin (see example 10), which used a monoclonal antibody for peptide hK2 41-56 (HK1A523) showed that hK2, as well as PSA, is produced in the epithelium, but not in the stroma. In addition, immunostaining is specific for the hK2 protein in the prostate, since other tissues tested were negative for hK2.

Example 5 Expression of hK2 in Mammalian Cells To express wild-type hK2 (hK2) in mammalian cells pGThk2 (Figure 3) was transfected into AV12 cells. Several clones expressing an hK2 polypeptide were identified through Western analysis using HK1D 106.4 (a specific mAb in hK2 raised to a polypeptide corresponding to amino acid residues 16-71 of hK2). Clone AV12-hK2 # 27 (AV12-hK2) was selected for further analysis based on its higher hK2 expression level. Cells transfected only with the vector (pGTD) showed no reactivity with HK1D 106.4. ELISA using HK1D 106.4 mAb indicated the presence of approximately 0.5-1 μg / ml of an hK2 polypeptide in the spent serum-free medium of AV12-hK2 on day 7. The same method used in the purification of phK2v1 'from of AV12-hK2, Vv2¿1p7 'was used to purify hK2 polypeptides from day 7 of the spent medium of AV12-hK2. This resulted in low yields of purified hK2 polypeptides, which were unstable to the purification procedures. The hK2 polypeptides were partially purified using the above method, subjected to SDS / PAGE, electroti? Ed and subjected to N-terminal amino acid sequencing. This analysis indicated that the hK2 polypeptide in the spent medium of AV12-hK2 on day 7 has the sequence, IVGGWECEK, in the N-terminus. No competition sequence was evident from the amino acid profile released sequentially by the degradation procedure Edman In comparison with PSA, this sequence corresponds to mature hK2 (hK2). The amino acid analysis of this protein was also consistent with that of hK2. This finding demonstrates that phK2v217 was predominantly present in serum-free spent medium of AV12-hK2v217 on day 7, while predominant hK2 was present in serum-free spent medium of AV12-hK2 on day 7. To examine the form of hK2 present in the serum free medium of AV12-hK2 on day 1, this material was partially purified through affinity purification using HK1G586.1 mAbs. The 34 kD protein was transferred into PVDF and subjected to N-terminal analysis, yielding a VPLIQSRIVGG sequence. No sequence of competition 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-hK2v217 cells. However, since phK2v217 is stable and is not converted to hK2v217, 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 wherein the serum free spent medium of AV12-hK2 clone # 27 was collected every day for 8 consecutive days, concentrated and subjected to SDS / PAGE. The proteins were transfected to nitrocellulose membrane and a probe was applied with either HK1D 106.4 or HK1G 464.3 mAbs (Figure 9). As also shown in Figure 9, HK1D 106.4 recognizes both phK2, and hK2, while HK1G 464.3 recognizes only phK2 as its epitope lying in region -7 to +7 of hK2. Expression of hK2 polypeptides (approximately 34 kD) formed peak on day 7 and plaque after as detected by HK1D 106.4 mAbs. Two other immunoreactive bands migrating at approximately 70 kD and approximately 90 kD were also detected from day 4. On the other hand, when the same samples were stained and a probe was applied with HK1G 464.3, a gradual reduction in the level was detected. of hK2 on 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 medium through AV12-hK2 cells and is gradually converted to hK2 extracellularly. curiously, the 70 kD and 90 kD bands were not observed with HK1G 464.3 mAbs, indicating that these bands are either homo-oligomers of hK2 or are hK2 covalently formed in complex with a yet unknown protein. Although the identity of these bands is not known at this time, they can serve as markers for the presence of hK2 in the spent medium. In Figure 9, the purified phK2v217 and hK2v217 proteins were used as controls. To study the biosynthesis of hK2v217 in AV12 cells, a similar study of time course in clone # 2 AV12-hK2v217 was conducted. As shown in Figure 10, the expression of the hK2v217 polypeptides formed peak on day 3 and did not vary much from day 4 onward, as detected by HK1D 106.4 mAbs. Similar results were obtained when the staining was placed on a probe with HK1G 464.3 mAbs (Figure 10). This indicates that the cells of clone # 2 AV12-pGThK2v217 are expressing phK2v217 from day 1 forward, and that at least during the 8 following days, this protein was not converted to the mature form. These results are in contrast to those of phK2, which was converted to hK2 if left in the medium for 8 days, indicating that phK2v217 is stable in the medium at 37 ° C for 8 days. To study whether the extracellular conversion of pHk2 to hK2 correlates with the viability of clone # 27 AV12-hK2 cells in culture, clone # 27 cells were counted using trypan blue exclusion. The expression of hK2 in the spent medium was measured through ELISA using both HK1D 106.4 and HK1G 464.3 mAbs. As shown in Figure 11, the number of viable cells formed a peak at 38 million in culture on day 3 and gradually decreased thereafter. By day 8, the number of viable cells was reduced to less than 10 million. The expression of phK2 (measured through HK1G 464.3) also formed a peak on day 10 and gradually declined later.

On the other hand, the expression of hK2 (measured by HK1D 106.4) formed peak on day 3, but formed plaque afterwards. This result indicates that phK2 is secreted by AV12-hK2 cells and a fraction of this was gradually and extracellularly converted to hK2 on day 4. In addition, it shows that the conversion of phK2 to hK2 clearly correlates with a reduction in the viability of cell, indicating that the extracellular proteases released by the stained cells may be one of the factors responsible for this conversion. The expression of hK2 was higher at the point where the cells were more viable. A reduction in hK2 was made parallel in a reduction in cell viability, suggesting that hK2 is secreted by these cells, as opposed to being released from the cell. death and cell lysis. Also, an increase in hK2 corresponded to a fall in pHk2, indicating that the pro form of hK2 was automatically converted to mature form over time. To examine the biosynthesis of hK2 in prostate carcinoma cells, hK2 was expressed in cell lines DU145 and PC3. The DNA encoding pphK2 was cloned into the plasmids pLNCX and pLNSX (Miller and Rosman, BioTechnigues, 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 the clones were selected in the medium containing 418 gene. Clones expressing high levels of hK2 were selected (PC3-hK2 and DU145-hK2) through ELISA and Western stains. To determine the level of hK2 and phK2 in the medium, the serum free medium of PC3-hK2 and DU145-hK2 cells was subjected to Western blot analysis using HK1D 106.4 (specific in hK2) and HK1G 464.3 (specific in phK2) mAbs (Figure 12). The 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 previously obtained results with AV12 cells. Predominantly, phK2 was detected in the spent medium of PC3-hK2 cells even after 7 days, however, hK2 was predominantly present in serum-free medium of DU145-hK2 starting on day 1. This is probably due to the abundance of extracellular proteases in spent medium DU145. To examine whether the above results were limited to a single clone, three other clones independently isolated from AV12-hK2 and another 4 clones independently isolated from AV12-hK2v217 were tested for the expression of hK2 polypeptides. The spent serum-free medium of the clones was collected on day 7 and tested for the expression of hK2 through Western stains using HK1D 106.4 (specific in hK2) and HK1G 464 (specific in phK2) mAbs (synthesis Figures 13 and 14 ). In all clones of AV12-hK2, HK1D 106.4 mAb detected not only the main band of 34 kD ("hK2") but also the 70 kD and 90 kD bands that are indicative of the presence of hK2 (Figure 13) . HK1G 464.3 detected very low levels of phK2 in all AV12-hK2 clones (Figure 14). This result indicates that hK2 is predominantly present in the spent medium of all clones AV12-hK2, verifying the biosynthetic mechanism established for clone # 27 AV12-hK2. The same analyzes were used in clones AV12-hK2v217 (Figure 14). The results indicated that only phK2v217 was present in the spent medium of these clones on day 7, verifying the findings with clone AV12-hK2v217. The above results collectively suggest that hK2 is expressed as the pro form in mammalian cells and is converted to mature form extracellularly through yet unknown proteases. These results also suggest that phK2 may be present in biological fluids and therefore may be a useful diagnostic marker for pCa and BPH.

Example 7 Enzymatic Activity and Specific Character of hK2 and hK2v217 A small amount of hK2 was purified for sufficient purity to determine its enzymatic activity and specific substrate character. The general activity of hK2 was measured by determining its amidolytic activity chromogenically in p-nitroanilide derivatives of peptides (Table 3). The p-nitroanilide released by proteolytic digestion of these substrates was measured in absorbance A405. The substrate methoxysuccinyl-Arg-Pro-Tyr-para-nitroanilide (MeO-Suc-R-P-Y-pNA) was used to measure proteases of the chymotrypsin type that divide into phenylalanine. This substrate has previously been used to measure the activity of PSA (Christenson et al., Eur. J. Biochem., 1994, 755 (1990)). The substrate H-D-Pro-Phe-Arg-para-nitronilide (P-F-R-pNA) is specific for trypsin-like proteases which are divided into arginine (R). It was found that hK2 has all the activity more than 10 times greater than hK2 in P-F-R-pNA and no protein showed an ability to hydrolyze MeO-Suc-R-P-Y-pNA, the substrate of chymotrypsin. Other comparable substrates containing trypsin-like cleavage sites (lysine, arginine) were also tested and it was found that hK2 hydrolyzes the P-F-R-pNA substrate at the highest rate. These findings indicate that hK2 has trypsin-like activity.

TABLE 3 Amidolytic Activity of hK2, hK2, and Trypsin in Chromogenic Substrates The specific character of hK2 and hK2v217 was examined in more detail through the use of peptide substrates together with N-terminal amino acid sequence analysis to determine which peptide bonds have been hydrolyzed. Figure 15 shows amidolytic activity in the peptide CALPEKPAVYTKVHYRKWIKD TLAAN, which has potentials for both trypsin and chymotrypsin cleavage sites. hK2v217 was divided into both a trypsin (R-K) and chymotrypsin (Y-R) site with trypsin-like cleavage at a 2: 1 ratio on chymotrypsin-like cleavage. As a control in these experiments, phK2v217 was also incubated with this peptide and showed no amidolytic activity. HK2 showed specific character different to hK2 217 towards this peptide substrate. No specific character of chymotrypsin type for hK2 was seen in this substrate and its activity was exclusive to the trypsin-like site (R-K). None of the other lysine (K) residues in this polypeptide was hydrolyzed indicating the specific character of hK2 that was exclusive for the arginine (R) residue. As a control trypsin was also studied in this substrate and all lysine (K) and arginine (R) sites were divided except for the K-P bond which is not known to be a suitable site for trypsin cleavage. Separate cleavage of the R-K site from substrate 210-236 (peptide # 1, Figure 16) at speeds of approximately 4 times greater than hK2 and approximately 4000 times greater than hK2v217. No chymotrypsin-type bond was separated by trypsin. PSA separated the Y-R link mainly. A minor trypsin-like activity at the R-K bond was also observed for PSA (Figure 15). This was consistent with the minor trypsin-like activity previously seen for PSA in the chromogenic substrate (Table 2). Several other peptide substrates were also incubated in hK2 and PSA (Figure 16). In all the peptides tested, hK2 had a specific character only for selected arginines, and PSA mainly for selected residues of tyrosine (Y), phenylalanine (F) and leusin (L). Only peptide # 1 in Figure 16 was separated by hK2 as detailed by the chromatographs in Figure 15.

Example 8 Activation of phK2, Vv21"7 by hK2 The sequence of peptide # 3 in Figure 16 corresponds to amino acid residue -7 to +7 of phK2 This region contains the pro peptide, VPLIQSR, which is found as a peptide N-terminal leader in phK2v217.As mentioned above, hK2 was able to separate this peptide by releasing the propeptide region, but hK2v217 was not. To delineate whether hK2 can separate this pro sequence into a natural substrate, its ability to convert phK2v217 to hK2v217 was verified. phK2v217, was incubated with 1% hK2 and the conversion was verified through the HIC-HPLC method (Figure 17A). The results showed that hK2 was able to convert phK2v217 to hK2 217, despite a speed about 30 times lower than trypsin. When phK2v217 was incubated with 40% hK2v217, no difference in the ratios of the two forms of hK2 was detected even after 6 hours (Figure 17B). This corroborated previous observations with the peptide substrate and showed that, even in a natural substrate, only hK2 and not hK2v217 STparo | a pro region of hK2. These results collectively demonstrate the stability of phK2 217 and hK2v217 after extended incubation. When compared to hK2v217, hK2 showed to have a higher proteolytic activity, a higher degree of specific character, and, in particular, to have a specific character for the pro form of hK2 as demonstrated by the activity in the pro peptide in Figure 15 and its activity towards phK2v217 in Figure 17. These results demonstrate a significant difference in enzyme activity between hK2 and hK2v217 and may help explain the low yields associated with attempts to purify hK2 from the medium compared to phK2v217. The highly purified hK2 preparation may not be stable due to autolysis as seen for other active proteases. These results also suggest that, in addition to immunological tests, enzymatic activity can be used on specific substrates in hK2 to verify the level of this protein in body fluids.

Example 9 Formation of Inhibitory Complexes with hK2 It has been shown that PSA forms complexes with alpha-2-macroglobulin (MG) and the serine protease inhibitor, anti-chymotrypsin (ACT). To explore this complex formation, hK2 was incubated with a series of common proteases present in human plasma (ACT, alpha-2-antiplasmin, antithrombin III, and alpha-1-antitrypsin (Travis and Salvesen, Am. Rev. Biochem., 52, 655 (1983)), and the mixtures were analyzed through Western staining (Figure 18) Any covalent complex of hK2 with these serpins should result in a band of approximately 80-100 kD in SDS / PAGE under conditions of reduction ACT and alpha-2-antiplasmin formed significant complexes with hK2 (Figure 18, lane 1 and 2) Antithrombin III (lane 3) and alpha-1 -antritrypsin (protease inhibitor alpha 1, lane 4) did not form any detectable complex with hK2. MG, a major component of blood plasma also easily complexed with hK2 (lane 5). This complex corresponds to Mr of approximately 200 kD and 120 kD, which were also formed when PSA was incubated with purified MG (Figure 18, lane 8, see below). Particularly interesting was that hK2 did not complex with alpha-1-antitrypsin, although this protein inhibits a wide variety 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 alpha-2-antiplasmin, since this protein has arginine residues in its active inhibitor site (Hunt and Dayhoff, Biochem, Biophy, Res. Comm., 95, 864 (1980); Chandra; and others, Biochemistry, 22. 5055 (1983), Potempa, et al., Science, 241, 699 (1985), Shieh et al., J. Biol. Chem. 264. 13420 (1989), Mast et al., Biochemistry, 30, 1723 (1991)). However, it was also not expected that hK2 could form a complex with ACT, since ACT has a leucine in its active inhibitor site. Clearly, the structural similarities between PSA and hK2 have an influence on their complex formation with a common inhibitor although their specific proteolytic character is completely different as demonstrated in Figure 16 and Table 2. When spliced into serum hK2 of human female formed a rapid complex with MG as detected by Western staining (Figure 18). Lane 1 and lane 3 are hK2 and serum controls only, respectively. Lane 2 is hK2 incubated with ACT showing the 90 kD hK2-ACT complex and residual hK2. Lanes 4 and 5 are hK2 spliced in serum for 15 minutes and 1 hour, respectively. Lane 6 is hK2 incubated with purified MG for 4 hours. Lane 7 is PSA spliced in serum for 15 minutes and lane 8 is PSA incubated with purified MG for 4 hours. These results show that MG is the largest complex of hK2 or PSA formed when hK2 or PSA are spliced in human serum in in vitro experiments. Since the PSA complex with ACT is known to occur in the blood serum of patients with prostate disease, it is believed that hK2 present in the serum could also form some level of ACT complex.

Discussion In vivo protein processing and secretion mechanisms for PSA or hK2 are not known. The results presented here 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 extracellularly separated. This suggests that phK2 exists in biological fluids and thus can be a useful diagnostic marker for pCa or BPH. Both the mutant form of hK2 (hK2 217) 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 make it very difficult to purify hK2 or pHk2 in sufficient quantities to be used as immunogens and calibrators. In contrast, phK2v217 is highly stable and converted to hK2 217, which is also stable, through trypsin digestion. Purified phK2v217 and hK2v217 provided immunogens to generate specific mAbs for hK2 and phK2.

Example 10 Immunoreactivity of Monoclonal Antibody 586 with Prostate Tissue Immunohistochemistry of normal prostate tissue with HK1523 showed staining in epithelium but not in stroma. In addition, the expression of hK2 is specific in prostate as well as other tissues, for example, kidney and pancreas, did not show any staining. To determine if hK2 is expressed in prostate tissue and, if so, correlates with prostate cancer, 264 radical prostatectomy specimens, of which 257 were from untreated patients (Figure 20) and 7 were from patients treated with therapy of androgen elimination (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. The prostate tissue was weighed, measured in three dimensions and stained. The tip and base were amputated to a thickness of 4-5 mm and serially sectioned at 3 mm. The remaining prostate was sectioned serially at 4-5 mm intervals through a knife perpendicular to the long axis of the gland from the apex of the prostate to the tip of the seminal vesicles. Cross sections were prepared and stained with hematoxylin and eosin. An individual slice of each patient's radical prostatectomy, encompassing cancer and benign tissue, was fixed at 10% formalin regulated at its neutral pH and embedded in paraffin through methods well known in the art. The sections of tissue on porta objects were deparaffinized through immersion in xylene and then in 95% ethanol. The endogenous peroxidase activity was blocked by incubating sections for 10 minutes in methanol / H2O2 and then the sections were rinsed in tap water. Then, the sections were placed in 10 mm citrate pH regulator, pH 6.0, and steam was applied for 30 minutes. The sections were cooled for 5 minutes before rinsing in cold running water. The non-specific protein binding was blocked by incubating the sections for 10 minutes with 5% goat serum. Afterwards, the object holders drained moderately. The primary antibody, hK1G586 or PSM773 at 0.5 μg / ml, was added to the sections for 30 minutes at room temperature and then the sections were rinsed with tap water. Then, the tissue sections were incubated with biotinylated rabbit anti-mouse antibody for 1 hour and rinsed with water. The sections were incubated with streptavidin conjugated with peroxidase (1: 500) for 30 minutes, then rinsed with tap water. Subsequently, the sections were incubated with a solution of 3-amino-9-ethylcarbazole (ABC) chromagen for 15 minutes before rinsing in tap water. The sections were then counterstained with mercury-free hematoxylin for 1 minute and rinsed for 5 minutes in tap water. The porta porta objects were mounted with aqueous mounting media (glycerol, gelatin). The percentage of cell staining was recorded in increments of 10% from 0-100% for benign epithelium, high-grade PIN and adenocarcinoma. The benign atrophic glands showed the last amount of staining, particularly in areas of inflammation, and where there was virtually no immunoreactivity. In hyperplastic acini and benign acini without any evidence of atrophy, there is a moderate intense immunoreactivity, usually appearing in a granular pattern in the luminal cell layer of secretion just above the nuclei, usually extending to the luminal surface. There was no staining of the uroepithelium of the urethra or viva montanum, although the underlying glands usually showed immunoreactivity. The basal cells were usually negative. Specimens with a high degree of PIN showed intense immuno-reactivity through the cytoplasm and cytoplasmic apical bladders in most cases. This was a difference not evident in the immuno-reactivity between different PIN patterns, except for the cribriform pattern that was usually reduced in intensity centrally when compared to the periphery. The 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 highest intensity was observed in the highest grade adenocarcinoma (4 Gleason pattern), which showed immuno-reactivity in virtually every case. The foci with cribiform carcinoma were similar to cribiform PIN since there is a higher intensity in the periphery than in the central part. The peripheral border and advancing edge of the carcinomas were always intensely immunoreactive. In seven specimens of patients who underwent androgen deficiency therapy, there is little or no immunoreactivity in most of the benign atrophic acini, although PIN and adenocarcinoma occasionally showed intense cytoplasmic staining. A summary of the number of cells stained with the monoclonal antibody HK1G586 in the benign epithelium, high-grade PIN and adenocarcinoma is shown in Table 4. Paired analysis, ie, benign against PIN, benign against carcinoma and PIN against carcinoma, revealed significant differences for each category (P <0.001), Spearman Rank Correlation).

TABLE 4 Immuno-reactivity with hK1G586 Medium% 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 the expression of cytoplasmic hK2 in prostate tissue correlates with prostatic neoplasia and prostate cancer. Although the prostate data obtained from patients treated with androgen-free therapy is not statistically significant due to the small sample size, there is a reduction in the expression of hK2 in benign epithelium, high-grade PIN and adenocarcinoma in these patients in relation to patients not treated. Thus, an increase in the expression of hK2 in the prostate is a novel marker for high-grade PIN and prostate cancer.

EXAMPLE 11 Detection by RT-PCR of hK2 RNA in Prostate Cancer Cells Since a large percentage of prostate cancers are subtracted, it is of interest to detect cells expressing hK2 present in tissue biopsies, eg, prostate capsule, bone marrow or lymph node, or in physiological fluid, for example, blood, serum or seminal fluid. Said detection method can preferably detect an individual hK2 expression cell in a large number of cells expressing no hK2. Preferably, the method can detect an individual hK2 expression cell in a sample comprising at least about 104, preferably at least 106, and most preferably 107 cells. To provide such a sensitive detection method, a reverse transcriptase polymerase chain reaction (RT-PCR) specific for transcripts of hK2 was employed.

A. LNCap cell line To determine the sensitivity of the detection of a specific transcription in hK2 through RT-PCR, the cells of the LNCaP cell line expressing PSA and human hK2 were serially diluted in samples of yellow cell cover . The yellow coat cells were isolated from whole blood of normal men and women. Venous blood (5-7 ml) was collected in citrate-dextrose tubes. The samples were centrifuged at 1000g for 15 minutes at 4 ° C. The yellow coat cells were recovered from the top of the cell pellet. The mixture of yellow coat cells and LNCaP cells was centrifuged at 1500 rpm for 5 minutes, and RNA was extracted from the cells in pellet form. The RNA was isolated through a guanidinium phenol-chloroform-thiocyanate acid method (Chomczynski et al., Anal. Biochem 162, 156 (1987)). The RNA samples were further extracted with chloroform-butanol (4: 1 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 cDNAs of first strand structure, an aliquot containing 1 g of the total RNA was added to a reverse transcription reaction containing 10O pmoles of a specific oligonucleotide primer to PSA (5'TCATCTCTGTATCC 3 '; SEQ ID NO: 13) or 100 pmoles of a specific oligonucleotide primer in hK2 (5 'GAGTAAGCTCTA 3 ?; SEQ ID NO: 14), and Moloney murine leukemia virus reverse transcriptase (GIBCO BRL), and brought to a final volume of 25 μL ( 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl 2, 10 mM dithioerythritol, 0.5 mM, from each dNTP, and 800 U reverse transcriptase from Moloney murine leukemia virus). The reaction was incubated at 42 ° C for 15 minutes and the enzyme was inactivated with heat at 95 ° C for 15 seconds. To apply PSA, the first strand structure cDNAs, 10 μl of the specific oligonucleotide in PSA initiated with first strand structure cDNAs was amplified in PCR (0.2 mM of each dNTP, 0.5 U AmpliTaq polymerase, 50 mM KCl, 10 mM Tris -HCl, PH 8.3, 1.5 mM MgCl2, 0.1% (w / v) gelatin) with a specific primer pair to PSA. The PSA PCR used 50 pmoles of PSA-1 (5'GATGACTCCAGCCACGACCT 3 '; SEQ ID NO: 15) and 50 pmoles of PSA-2 (5 'CACAGACACCCCATCCTATC 3'); SEQ ID NO: 16). To amplify A.DNcs from the first strand structure of hK2, 10 μL of the specific oligonucleotide in PSA hK2 initiate cDNAs from first strand structure and was amplified in PCR (0.2 mM of each dNTP, 0.5 U AmpliTaq polymerase, 50 mM KCl, 10 mM Tris-HCl, PH 8.3, 1.5 mM MgCl 2, 0.1% (w / v) gelatin) with a specific initiator pair in hK2. The hK2 PCR employed 50 pmoles of hK2-1 (5'GAGGGTTGTGYACAGTCATGGAT 3 '; SEQ ID NO: 17) and 50 pmoles of hK2-2 (5'ACACACTGAAGACTCCTGGGGCG 3'; SEQ ID NO: 18)). Parameters of cyclization used were: 35 to 40 cycles of 94 ° C for 1 minute; 58 ° C (PSA) or 60 ° C (hK2) for 90 seconds, 72 ° C for 90 seconds. The final cycle was at 72 ° C for 10 minutes. The aliquots of the reaction were electrophoresed in 1.0% agarose gels. The gels were stained with ethidium bromide and then viewed under ultraviolet light. Some of the amplified products were separated from the gel and subcloned into a PCR II vector (Invitrogen, San Diego, CA) for sequencing. The specific PCR in PSA produced a product of 710 bp, while the specific PCR in hK2 introduced a product of 405 bp. The results of the dilution analysis showed that PSA and hK2 RNA was detectable at approximately one LNCaP cell in 106 and 107 white blood cells, respectively (Figure 22A). This result was unexpected, since the RT-PCR detected hK2 transcripts derived from LNCaP at a 10-fold higher dilution than the PCN transcripts derived from LNCaP.

B. Patients with Prostate Cancer The blood of six patients with prostate cancer and two normal men was analyzed by RT-PCR. Yellow coated cells and RNA isolated from the eight men were obtained, and RT-PCR was performed as described above. The six patients with prostate cancer included one with clinical stage B prostate cancer, two with known metastatic disease (clinical stage D2) and three with pathological stage C. The pathological AC stages of prostate cancer are located in cancer forms of prostate. The pathological stage D1 is prostate cancer, which has spread to the nodes (nodal metastasis). The pathological stage D2 is systemic prostate cancer (systemic metastasis). For an additional 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 patients with prostate cancer 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. In this way, the detection of hK2 RNA can serve as a useful marker of early detection of prostate cancer micrometastases. The invention is not limited to the exact details shown and described, it should be understood that many variations and modifications may be made as long as they remain within the spirit and scope of the invention defined by the appended claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Mayo Foundation for Medical Education and Research, and Hybritech Incorporated (ii) TITLE OF THE INVENTION: METHOD FOR DETECTING METASTATIC PROSTATE CANCER (iii) NUMBER OF SEQUENCES: 26 (iv) ADDRESS OF CORRESPONDENCE: (A) RECIPIENT: Schwegman, Lundberg, Woessner & Kluth, P.A. (B) STREET: P.O. Box 2938 (C) CITY: Minneapolis (D) STATE: MN (E) COUNTRY: E.U.A. (F) CODE: 55402 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: floppy disk (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: TWO (D) SOFTWARE: FastSEQ Version 2.0 (iv) REQUEST DATA CURRENT: (A) NUMBER OF APPLICATION: unknown (B) DATE OF SUBMISSION: November 14, 1197 (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATA: (A) NUMBER OF APPLICATION: 08 / 759,354 (B) DATE OF PRESENTATION: 14-NOV-1996 (A) NUMBER OF APPLICATIONS: PCT / US96 / 06167 (B) DATE OF SUBMISSION: 02-MAY-1996 (A) NUMBER OF APPLICATION: 08 / 622,046 (B) DATE OF SUBMISSION: MARCH 25, 1996 (A) APPLICATION NUMBER: 08 / 427,707 (B) SUBMISSION DATE: MAY 2, 1995 (viii) EMPLOYEE / AGENT INFORMATION: (A) NAME: Embretson, Janet E (B) REGISTRATION NUMBER: 39,665 (C) ) DO NOT. REFERENCE / APPORTER: 545.005W01 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 612-359-3260 (B) TELEFAX: 612-359-3263 (C) TELEX: (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 237 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TI PO DEMO LÉC U LA: peptide (ix) DESCRI PCI ÓN DE SECU ENC IA: SEC IDNO: 1 lie Val Gly Gly Trp Glu Cys Glu Lys His Ser Gln Pro Trp Gln Val 1 5 10 15 Wing Val Tyr Ser His Gly Trp Wing His Cys Gly Gly Val Leu Val His 20 25 30 Pro Gln Trp Val Leu Thr Wing Wing Hxs Cys Leu Lys Lys Asn Ser Gln 35 40 45 Val Trp Leu Gly Arg His Asn Leu Phe Glu Pro Glu Asp Thr Gly Gln 50 55 60 Arg Val Pro Val Ser His Ser Phe Pro His Pro Leu Tyr Asn Met Ser 65 70 75 80 Leu Leu Lys His Gln Ser Leu Arg Pro Asp Glu Asp Ser Ser His Asp 85 90 95 Leu Met Leu Leu Arg Leu Ser Glu Pro Wing Lys lie Thr Asp Val Val 100 105 110 Lys Val Leu Gly Leu Pro Thr Gln Glu Pro Wing Leu Gly Thr Thr Cys 115 120 125 Tyr Wing Ser Gly Trp Gly Ser lie Glu Pro Glu Glu Phe Leu Arg Pro 130 135 140 Arg Ser Leu Gln Cys Val Ser Leu His Leu Leu Ser Asn Asp Met Cys 145 150 1S5 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 Gln Gly He Thr Ser Trp G ^ and Pro Glu 195 200 205 Pro Cys Wing Leu Pro ßlu Lys Pro Wing Val Tyr Thr Lys Val Val His 210 215 220 Tyr Arg Lys Trp He Lys Asp Thr He Wing Wing Asn Pro 225 230 235 (2) INFORMATION FOR SEQ ID NO: 2: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 711 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) SEQUENCE DESCRIPTION : SEQ ID NO: 2: ATT GTG GGA GGC TGG GAG TGT GAG AAG CAT TCC CAA CCC TGG CAG GTG 48 He Val Gly Gly Trp Glu Cys Glu Lys His Ser Gln Pro Trp Gln Val 1 5 10 15 GCT GTG TAC AGT CAT GGA TGG GCA CAC TGT GGG GGT GTC CTG GTG CAC 96 Wing Val Tyr Ser His Gly Trp Wing His Cys Gly Gly Val Leu Val His 20 25 30 CCC CAG TGG GTG CTC ACÁ GCT GCC CAT TGC CTA AAG AAG AAT AGC CAG 144 Pro Gln Trp Val Leu Thr Wing Wing His Cys Leu Lys Lys Asn Ser Gln 35 40 45 GTC TGG CTG GGT CGG CAC AAC CTG TTT GAG CCT GAA GAC ACA GGC CAG 192 Val Trp Leu Gly Arg His Asn Leu Phe Glu Pro Glu Asp Thr Gly Gln 50 55 60 AGG GT C CCT GTC AGC CAC AGC TTC CCA CAC CCG CTC TAC AAT ATG AGC 2 0 Arg Val Pro Val Ser His Ser Phe Pro His Pro Leu Tyr Asp Met Ser 65 70 75 80 CTT CTG AAG CAT CAA AGC CTT AGA CCA GAT GAA GAC TCC AGC CAT GAC 288 Leu Leu Lys His Gln Ser Leu Arg Pro Asp Glu Asp Ser Ser His Asp 85 90 95 CTC ATG CTG CTC CGC CTG TCA GAG CCT GCC AAG ATC ACA GAT GTT GTG 336 Leu Met Leu Leu Arg Leu Ser Glu Pro Ala Lys He Thr Asp Val Val 100 105 110 AAG GTC CTG GGC CTG CCC ACC CAG GAG CCA GCA CTG GGG ACC ACC TGC 384 Lys Val Leu Gly Leu Pro Thr Gln Glu Pro Wing Leu Gly Thr Thr Cys 115 120 125 TAC GCC TCA GGC TGG GGC AGC ATC GAA CCA GAG GAG TTC TTG CGC, CCC 432 Tyr Wing Ser Gly Trp Gly Ser He Glu Pro Glu Glu Phe Leu Arg Pro 130 135 140 AGG AGT CTG CAG TGT GTG AGC CTC CAT CTC CTG TCC AAT GAC ATG TGT 480 Arg Ser Leu Gln Cys Val Ser Leu His Leu Leu Ser Asn Asp Met Cys 145 150 155 160 GCT AGA GCT CTT GAG AAG GTG AC GAG TTC ATG TTG TGT GCT GGG 528 Wing Arg Wing Tyr Ser Glu Lys Val Thr Glu Phe Met Leu Cys Wing Gly 165 170 175 CTC TGG ACÁ GGT GGT AAA GAC ACT TGT GGG GGT GAT TCT GGG GGT CCA 576 Leu Trp Thr Gly Gly Lys Asp Thr Cys Gly Gly Asp Ser Gly Gly Pro 180 185 190 CTT GTC TGT AAT GGT GTG CTT CAA GGT ATC ACA TCA TGG GGC CCT GAG 624 Leu Val Cys Asn Gly Val Leu Gln Gly He Thr Ser Trp Gly Pro Glu 195 200 205 CCA TGT GCC CTG CCT GAA AAG CCT GCT GTG TAC ACC AAG GTG GTG CAT 672 Pro Cys Wing Leu Pro Glu Lys Pro Wing Val Tyr Thr Lys Val Val His 210 215 220 TAC CGG AAG TGG ATC AAG GAC ACC ATC GCA GCC AAC CCC 711 Tyr Arg Lys Trp He Lys Asp Thr He Ala Wing Asn Pro 225 230 235 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 261 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Met Trp Asp Leu Val Leu Ser He Wing Leu Ser Val Gly Cys Thr Gly 1 5 10 15 Wing Val Pro Leu He Gln Ser Arg He Val Gly Gly Trp Glu Cys Glu 20 25 30 Lys His Ser Gln Pro Trp Gln Val Wing Val Tyr Ser His Gly Trp Wing 35 40 45 His Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Wing Ala 50 55 60 His Cye Leu Lys Lys Asn Ser Gln Val Trp Leu Gly Arg His Asn Leu 65 70 75 80 Phe Glu Pro Qlu Asp Thr Gly Gln Arg Val Pro Val Ser His Ser Phe 85 90 95 Pro His Pro Leu Tyr Asn Met Ser Leu Leu Lys His Gln Ser Leu Arg 100 105 110 Pro Asp Glu Asp Ser Ser His Asp Leu Met Leu Leu Arg Leu Ser Glu 115 120 125 Pro Wing Lys He Thr Asp Val Val Lys Val Leu Gly Leu Pro Thr Gln 130 135 140 Glu Pro Wing Leu Gly Thr Thr Cys Tyr Wing Ser Gly Trp Gly Ser He 145 150 155 160 Glu Pro Glu Glu Phe Leu Arg Pro Arg Ser Leu Gln Cys Val Ser Leu 165 '170 175 His Leu Leu As As Asp Met Cys Ala Arg Ala Tyr Ser Glu Lys Val 180 185 190 Thr Glu Phe Met Leu Cys Ala Gly Leu Trp Ti ? r Gly Gly Lys Asp Thr 195 200 205 Cys Gly Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gln 210 215 220 Gly He Thr Ser Trp Gly Pro Glu Pro Cys Ala Leu Pro Glu Lys Pro 225 230 235 240r Thr Lys Val Val His Tyr Arg Lys Trp He Lys Asp Thr 245 250 255 He Wing Wing Asn Pro 260 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 832 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: double (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (ix) 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 He Wing Leu Ser Val Gly 1 5 10 TGC ACT GGT GCC GTG CCC CTC ATC CAG TCT CGG ATT GTG GGA GGC TGG 96 Cys Thr Gly Ala Val Pro Leu He Gln Ser Arg He Val Gly Gly Trp 15 20 25 GAG TGT GAG AAG CAT TCC CAA CCC TGG CAG GTG GCT GTG TAC AGT CAT 144 Glu Cys Glu Lys His Ser Gln Pro Trp Gln Val Wing Val Tyr Seir His 35 40 45 GGA TGG GCA CAC TGT GGG GGT GTC CTG GTG CAC CCC CAG TGG GTG CTC 192 Gly Trp Wing His Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu 50 55 60 ACÁ GCT GCC CAT TGC CTA AAG AAG AAT AGC CAG GTC TGG CTG GGT CGG 240 Thr Wing Wing His Cys Leu Lys Lys Asn Ser Gln Val Trp Leu Gly Arg 65 70 75 CAC AAC CTG TTT GAG CCT GAA GAC ACA GGC CAG AGG GTC CCT GTC AGC 288 His Asn Leu Phe Glu Pro Glu Asp Thr Gly Gln Arg Val Pro Val Ser 80 85 90 CAC AGC TTC CCA CAC CCG CTC TAC AAT ATG AGC CTT CTG AAG CAT CAA 336 His Ser Phe Pro His Pro Leu Tyr Asn Met Ser Leu Leu Leys His Gln 95 100 105 AGC CTT AGA CCA GAT GAA GAC TCC AGC CAT GAC CTC ATG CTG CTC CGC 384 Ser Leu Arg Pro Asp Glu Asp Ser Ser His Asp Leu Met Leu Leu Arg 110 115 120 125 CTG TCA GAG CCT GCC AAG ATC ACA GAT GTT GTG AAG GTC CTG GGC CTG 432 Leu Ser Glu Pro Ala Lys He Thr Asp Val Val Lys Val Leu Gly Leu 130 135 140 CCC ACC ACC GAG CCA GCA CTG GGG ACC ACC TGC TAC GCC TCA GGC TGG 480 Pro Thr Gln Glu Pro Wing Leu Gly Thr Thr Cys Tyr Wing Ser Gly Trp 145 150 155 GGC AGC ATC GAA CCA GAG GAG TTC TTG CGC CCC AGG AGT CTT CAG TGT 528 Gly Ser He Glu Pro Glu Glu Phe Leu Arg Pro Arg Ser Leu Gln Cys 160 165 170 GTG AGC CTC CAT CTC CTG TCC AAT GAC ATG TGT GCT AGA GCT TAC TCT 576 Val Ser Leu His Leu Leu Ser Asn Asp Met Cys Ala Arg Ala Tyr Ser 175 180 185 GAG AAG GTG ACÁ GAG TTC ATG TTG TGT GCT GGG CTC TGG ACA GGT GGT 62 Glu Lys Val Thr Glu Phe Met Leu Cys Ala Gly Leu Trp Thr Gly Gly 190 195 200 205 AAA GAC ACT TGT GGG GGT GAT TCT GGG GGT CCA CTT GTC TGT AAT GGT 672 Lys Asp Thr Cys Gly Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly 210 215 220 GTG CTT CAA GGT ATC ACA TCA TGG GGC CCT GAG CCA TGT GCC CTG CCT 720 Val Leu Gln Gly He Thr Ser Trp Gly Pro Glu Pro Cys Ala Leu Pro 225 230 235 GAA AAG CCT GCT GTG TAC ACC AAG GTG GTG CAT TAC CGG AAG TGG ATC 768 Glu Lys Pro Wing Val Tyr Thr Lys Val Val His Tyr Arg Lys Trp He 240 245 250 AAG GAC ACC ATC GCA GCC AAC CCC TGAGTGCCCC TGTCCCACCC CTACCTCTAG_822_Lys Asp Thr He Ala Wing Asn Pro 255 260 TAAACTGCAG 832 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 244 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Wing Ala His 35 40 45 Cys Leu Lys Lys Asn Ser Gln Val Trp Leu Gly Arg His Asn Leu Phe 50 55 60 Glu Pro Glu Asp Thr Gly Gln Arg Val Pro Val Ser His Ser Phe Pro 65 70 75 80 His Pro Leu Tyr Asn Met Be Leu Leu Lys His Gln Ser Leu Arg Pro 85 90 95 Asp Glu Asp Ser Ser His Asp Leu Met Leu Leu Arg Leu Ser Glu Pro 100 105 110 Wing Lys He Thr Asp Val Val Lys Val Leu Gly Leu Pro Thr Gln Glu 115 120 125 Pro Wing Leu Gly Thr Thr Cys Tyr Wing Ser Gly Trp Gly Ser He Glu 130 135 140 Pro Glu Glu Phe Leu Arg Pro Arg Ser Leu Gln Cys Val Ser Leu His 145 150 155 160 Leu Leu Be Asn Asp Met Cys Wing Arg Wing Tyr Ser Glu Lys Val Thr 165 170 175 Glu Phe Met Leu Cys Wing Gly Leu Trp Thr Gly Gly Lys Asp Thr Cys 180 185 190 Gly Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gln Gly 195 200 205 He Thr Ser Trp Gly Pro Glu Pro Cys Wing Leu Pro Glu Lys Pro Wing 210 215 220 Val Tyr Thr Lys Val Val His Tyr Arg Lys Trp He Lys Asp Thr He 225 230 235 240 Ala Ala Asn Pro (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 766 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: double (D) TOPOLOGY: linear (¡) i) TYPE OF MOLECULE: cDNA (ix) ASPECT: (A) NAME / KEY: CDS (B) LOCATION: 1. 732 (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GTG ccc CTC ATC CAG TCT CGG ATT GTG GGA GGC TGG GAG TGT GAG AAG 48 Val Pro Leu He Gln Ser Arg He Val Gly Gly Trp Glu Cys Glu Lys 1 5 10 15 CAT TCC CAA CCC TGG CAG GTG GCT GTG TAC AGT CAT GGA TGG GCA CAC 96 His Ser Gln Pro Trp Gln Val Wing Val Tyr Ser His Gly Trp Wing His 20 25 30 TGT GGG GGT GTC CTG GTG CAC CCC CAG TGG GTG CTC ACÁ GCT GCC CAT 144 Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala Wing His 35 40 45 TGC CTA AAG AAG AAT AGC CAG GTC TGG CTG GGT CGG CAC AAC CTG TT 192 Cys Leu Lys Lys Asn Ser Gln Val Trp Leu Gly Arg His Asn Leu Phe 50 55 60 GAG CCT GAA GAC ACA GGC CAG AGG GTC CCT GTC AGC CAC AGC TC CCA 240 Glu Pro Glu Asp Thr Gly Gln Arg Val Pro Val Ser His Ser Phe Pro 65 70 75 80 CAC CCG CTC TAC AAT ATG AGC CTT CTG AAG CAT CAA AGC CTT AGA CCA 288 His Pro Leu Tyr Asn Met Ser Leu Leu Lys His Gln Ser Leu Arg Pro 85 90 95 GAT GAA GAC TCC AGC CAT GAC CTC ATG CTG CTC CGC CTG TCA GAG CCT 336 Asp Glu Asp Ser Ser His Asp Leu Met Leu Leu Arg Leu Ser Glu Pro 100 105 110 GCC AAG ATC ACA GAT GTT GTG AAG GTC CTG GGC CTG CCC ACC CAG GAG 384 Ala Lys He Thr Asp Val Val Lys Val Leu Gly Leu Pro Thr Gln Glu 115 120 125 CCA GCA CTG GGG ACC ACC TGC TAC GCC TCA GGC TGG GGC AGC ATC GAA 432 Pro Ala Leu Gly Thr Thr Cys Tyr Wing Ser Gly Trp Gly Ser He Glu 130 135 140 CCA GAG GAG TTC TTG CGC CCC AGG AGT CTG CAG TGT AGG CTC CAT 480 Pro Glu Glu Phe Leu Arg Pro Arg Ser Leu Gln Cys Val Ser Leu His 145 150 155 160 CTC CTG TCC AAT GAC ATG TGT GCT AGA GCT TAC TCT GAG AAG GTG ACÁ 528 Leu Leu Be Asn Asp Met Cys Wing Arg Wing Tyr Ser Glu Lys Val Thr 165 170 175 GAG TC ATG TTG TGT-GCT GGG CTC TGG ACA GGT GGT AAA GAC ACT TGT 576 Glu Phe Met Leu Cys Wing Gly Leu 'Trp Thr Gly Gly Lys Asp Thr Cys 180 185 190 GGG GGT GAT TCT GGG GGT CCA CTT GTC TGT AAT GGT GTG CTT CAA GGT 624 Gly Gly Asp Ser Gly Gly Pro Leu Val Cys AßT? Gly Val Leu Gln Gly 195 200 205 ATC ACÁ TCA TGG GGC CCT GAG CCA TGT GCC CTG CCT GAA AAG CCT GCT 672 He Thr Ser Trp Gly Pro Glu Pro Cys Wing Leu Pro Glu Lys Pro Wing 210 215 220 GTG TAC ACC AAG GTG GTG CAT TAC CGG AAG TGG ATC AAG GAC ACC ATC 720 Val Tyr Thr Lys Val Val His Tyr Arg Lys Trp He Lys Asp Thr He 225 230 235 240 GCC GCC AAC CCC TGAGTGCCCC TGTCCCACCC CTACCTCTAG TAAA 766 Ala Ala Asn Pro (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 237 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 7: He Val Gly Gly Trp Glu Clu Glu Lys His Ser Gln Pro Trp Gln Val 1 5 10 15 Leu Val Ala Ser Arg Gly Arg Ala Val Cys Gly Gly Val Leu Val His 20 25 30 Pro Gln Trp Val Leu Thr Ala Wing His Cys He Arg Asn Lys Ser Val 35 40 45 He Leu Leu Gly Arg His Ser Leu Phe His Pro Glu Asp Thr Gly Gln 50 55 60 Val Phe Gln Val Ser His Ser Phe Pro His Pro Leu Tyr Asp Met Ser 65 70 75 80 Leu Leu Lys Asn Arg Phe Leu Arg Pro Gly Asp Asp Ser Ser His Asp 85 90 95 Leu Leu Leu Arg Leu Ser Glu Pro Wing Glu Leu Thr Asp Wing Val 100 105 110 Lys Val Met Asp Leu Pro Thr Gln Glu Pro Ala Leu Gly Thr Thr Cys 115 120 125 Tyr Wing Ser Gly Trp .Gly Ser He Glu Pro Glu Glu Phe Leu Thr Pro 130 135 140 Lys Lys Leu Gln Cys Val Asp Leu His Val He Ser As Asp Val Cys 145 150 155 160 Wing Gln Val His Pro Gln Lys Val Thr Lys Phe Met Leu Cys Wing Gly 165 170 175 Arg Trp Thr Gly Gly Lys Ser Thr Cys Ser Gly Asp Ser Gly Gly Pro 180 185 190 Leu Val Cys Asn Gly Val Leu Gln Gly He Thr Ser Trp Gly Ser Glu 195 200 205 Pro Cys Ala Leu Pro Glu Arg Pro Ser Leu Tyr Thr Lys Val Val His 210 215 220 Tyr Arg Lys Trp He Lys Asp Thr He Val Wing Asn Pro 225 230 235 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 237 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 8: He Val Gly Gly Trp Glu Cys Glu Lys His Ser Gln Pro Trp Gln Val 1 5 10 15 Wing Val Tyr Ser His Gly Trp Wing His Cys Gly Gly Val Leu Val His 20 25 30 Pro Gln Trp Val Leu Thr Wing Wing His Cys Leu Lys Lys Asn Ser Gln 35 40 45 Val Trp Leu Gly Arg His Asn Leu Phe Glu Pro Glu Asp Thr Gly Gln 50 55 60 Arg Val Pro Val Ser His Ser Phe Pro His Pro Leu Tyr Asn Met Ser 65 70 75 80 Leu Leu Lys His Gln Ser Leu Arg Pro Asp Glu Asp Ser Ser His Asp 85 90 95 Leu Met Leu Leu Arg Leu Ser Glu Pro Wing Lys He Thr Asp Val Val 100"105 110 Lys Val Leu Gly Leu P'ro Thr Gln Glu Pro Wing Leu Gly Thr Thr Cys 115 120 125 Tyr Wing Ser Gly Trp Gly Ser He Glu Pro Glu Glu Phe Leu Arg Pro 130 135 140 Arg Ser Leu Gln 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 Gln Gly He 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 He Lys Asp Thr He Ala Wing Asn Pro 225 230 235 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: cDNA (x) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ACGCGGATCC AGCATGTGGG ACCTGGTTCT CT 32 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 10: ACAGCTGCAG TTTACTAGAG GTAGGGGTGG GAC 33 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ATATGGATCC ATATGTCAGC ATGTGGGACC TGGTTCTCTC CA 42 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 12: ATATGGATCC TCAGGGGTTG GCTGCGATGG T 31 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TCATCTCTGT ATCC 14 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 base pairs ( B) TYPE: nucleic acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GAGTAAGCTC TA 12 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GATGACTCCA GCCACGACCT 20 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 16: CACAGACACC CCATCCTATC 20 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 17: GAGGGTTGTG TACAGTCATG GAT 23 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 18: ACACACTGAA GACTCCTGGG GCG 23 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids (B) ) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (ix) 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 15 Ala His Cys (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: peptide (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 20: His Cys Leu Lys Lys Asn Ser Gln Val Trp Leu Gly Arg His Asn Leu 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 21: His Leu Leu Ser Asn Asp Met Cys Ala Arg Ala Tyr Ser Glu Lys 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 55 amino acids (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 22: Wing Val Tyr Ser His Gly Trp Wing His Cys Gly Gly Val Leu Val His 1 5 10 15 Pro Gln Trp Val Leu Thr Wing Wing His Cys Leu Lys Lys Asn Ser Gln 20 25 30 Val Trp Leu Gly Arg His Asn Leu Phe Glu Pro Glu Asp Thr Gly Gln 40 45 Arg Val Pro Val Ser His Ser 50 55 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 711 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) ) MOLECULE TYPE: cDNA (ix) DESCRIPTION OF SEQUENCE: SEQ ID NO: 23: ATTGTssOAG GCTGGGAGTG CGAGAAGCAT TCCCAACCCT GGCAGGTGCT TGTGGCCTCT 60 CGTGGCAGGG CAGTCTGCGG CGGTGTTCTG GTGCACCCCC AGTGGGTCCT CACAGCTGCC 120 CACTGCATCA GGAACAAAAG CGTGATCTTG CTGGGTCGGC ACAGCCTGTT TCATCCTGAA 180 GACACAGGCC AGGTATTTCA GGTCAGCCAC AGCTTCCCAC ACCCGCTCTA CGATATGAGC 240 CTCCTGAAGA ATCGATTCCT CAGGCCAGGT GATGACTCCA GCCACGACCT CATGCTGCTC 300 CGCCTGTCAG AGCCTGCCGA GCTCACGGAT GCTGTGAAGG TCATGGACCT GCCCACCCAG 360 GAGCCAGCAC TGGGGACCAC CTGCTACGCC TCAGGCTGGG GCAGCATTGA ACCAGAGGAG 420 TTCTTGACCC CAAAGAAACT TCAGTGTGTG GACCTCCATG TTATTTCCAA TGACGTGTGT 480 GCGCAAGTTC ACCCTCAGAA GGTGACCAAG TTCATGCTGT GTGCTGGACG CTGGACAGGG 540 GGCAAAAGCA CCTGCTCGGG TGATTCTGGG GGCCCACTTG TCTGTAATGG TGTGCTTCAA 600 GGTATCACGT CATGGGGCAG TGAAC CATGT GCCCTGCCCG AAAGGCCTTC CCTGTACACC 660 AAGGTGGTGC ATTACCGGAA GTGGATCAAG GACACCATCG TGGCCAACCC C 711 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 24: Leu Lys Asn Ser Gln Val Trp Leu Gly Arg His Asn Leu Phe Glu 1 5 10 15 Pro Glu Asp Thr Gly Gln Arg Val 20 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 25: Cys Ala Leu Pro Glu Lys Pro Ala Val Tyr Thr Lys Val Val His Tyr 1 10 15 Arg Lys Trp lie Lys Asp Thr He Ala Wing 20 20 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 26: Gln Val Ala Val Tyr Ser His Gly Trp Ala His Cys 1 5 10

Claims (42)

1. - A diagnostic method for detecting hK2 DNA comprising: (a) contacting an amount of DNA obtained through reverse transcription of RNA from a human physiological sample comprising cells suspected of containing hK2 RNA, with an amount of at least two oligonucleotides under conditions effective to amplify the DNA through polymerase chain reaction in order to produce an amount of amplified hK2 DNA, wherein at least one oligonucleotide is a specific oligonucleotide in hK2, and wherein the sample is from a human being at risk for, or suffering from, an indication associated with aberrant spatial hK2 expression; and (b) detecting the presence of amplified hK2 DNA.

2. A method for detecting metastatic prostate cancer in a human being, comprising: (a) contacting a quantity of DNA obtained through reverse transcription of RNA from a human physiological sample comprising cells suspected of containing RNA hK2, with an amount of at least two oligonucleotides under conditions effective to amplify the DNA through polymerase chain reaction in order to produce an amount of amplified hK2 DNA, wherein at least one oligonucleotide is a specific oligonucleotide in hK2; and (b) detecting the presence of amplified hK2 DNA, wherein the presence of hK2 DNA is indicative of the presence of metastatic prostate cancer in a human.

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

4. The method according to claim 3, wherein the tissue is selected from the group consisting of prostate capsule, seminal vesicle, bone marrow and lymph node.

5. The method according to claim 3, wherein the tissue is non-prostatic tissue.

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

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

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

9. The method according to claim 1 or 2, wherein the amplified hK2 DNA is subjected to agarose gel electrophoresis before detection.

10. The method according to claim 1 or 2, characterized in that it further comprises quantifying the amount of amplified hK2 DNA.

11. The method according to claim 1 or 2, characterized in that it further comprises: (c) contacting a second quantity of DNA obtained through reverse transcription of RNA from the human physiological sample with an amount of at least two oligonucleotides under conditions effective to amplify the specific antigen in prostate (PSA) DNA, but not hK2 DNA, through polymerase chain reaction in order to produce amplified PSA DNA; and (d) detecting the presence of amplified PSA DNA.

12. A diagnostic method for detecting hK2 RNA comprising: (a) extracting RNA from a physiological sample obtained from a human being; (b) reverse transcription of extracted RNA to produce DNA; (c) contacting the DNA with an amount of at least two oligonucleotides under conditions effective to amplify the DNA through polymerase reaction in order to produce an amplified amount of hK2 DNA, wherein at least one oligonucleotide is a specific oligonucleotide in hK2; and (d) detecting the presence of amplified hK2 DNA, wherein the presence of amplified hK2 DNA is indicative of metastatic prostate cancer in a human.

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

14. - The method according to claim 12, wherein the sample is a sample of physiological fluid.

15. The method according to claim 12, where the human being has had a radical prostatectomy, and where the presence of hK2 DNA is indicative of the presence of persistent prostate cancer in humans.

16. A method to verify the progression of prostate cancer, comprising: (a) contacting a quantity of DNA derived by reverse transcription of RNA from a physiological sample of a human being suffering from prostate cancer with a amount of at least two oligonucleotides under conditions effective to amplify the DNA through the polymer chain reaction in order to produce an amount of amplified hK2 DNA, wherein at least one oligonucleotide is a specific oligonucleotide in hK2; (b) detecting or determining the amount of amplified hK2 DNA; (c) repeating steps (a) and (b) at a later point in time; and (d) comparing the result of step (b) with the result of step (c), wherein an increase in the amount of hK2 DNA is indicative of the progression of prostate cancer in said human being.

17. A method for physiologically verifying prostate cancer, comprising: (a) contacting a quantity of DNA obtained through reverse transcription of RNA from a physiological sample of a human being suffering from prostate cancer with a amount of at least two oligonucleotides under conditions effective to amplify the DNA through polymerase chain reaction in order to produce an amount of amplified hK2 DNA, wherein at least one oligonucleotide is a specific oligonucleotide in hK2; Y (b) detecting or determining the presence or amount of the amplified hK2 DNA, wherein the presence or amount of the amplified hK2 DNA is indicative of the disease state of the prostate cancer.

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

19. The method according to claim 16, wherein the sample of step (a) is obtained before the human being undergoes hormone therapy.

20. The method according to claim 19, wherein the hormone therapy is androgen therapy.

21. The method according to claim 20, wherein androgen therapy is provocative androgen therapy.

22. The method according to claim 16 or 17, wherein the sample is a non-prostatic tissue sample.

23. The method according to claim 16 or 17, wherein the sample is a sample of physiological fluid.

24. - The method according to claim 23, wherein the fluid is whole blood.

25. A diagnostic kit for detecting hK2 RNA in a physiological sample suspected of containing hK2 RNA, comprising the package containing (a) a known amount of a specific first oligonucleotide in hK2, wherein the oligonucleotide consists of at least about 7-50 nucleotides, wherein the oligonucleotide has at least 80% identity SEQ ID NO: 4, and wherein the oligonucleotide comprises SEQ ID NO: 17; and (b) a known amount of a second specific oligonucleotide in hK2, wherein the oligonucleotide consists of at least about 7-50 nucleotides, wherein the oligonucleotide has at least about 80% identity with a nucleotide sequence that is complementary to SEQ ID NO: 4, and wherein the oligonucleotide comprises SEQ ID NO: 14 or SEQ ID NO: 18.

26. The diagnostic equipment according to claim 25, wherein the second oligonucleotide comprises SEQ ID NO: 14.

27. The diagnostic equipment according to claim 25, wherein the first oligonucleotide comprises SEQ ID NO: 17.

28. The diagnostic equipment according to claim 25, wherein the second oligonucleotide comprises SEQ ID NO: 18.

29. - An isolated, purified peptide comprising SEQ ID NO: 22, a biologically active subunit thereof, or a biologically active variant thereof.

30. - An isolated, purified peptide comprising SEQ ID NO: 26, a biologically active subunit thereof, or a biologically active variant thereof.

31. A purified antibody that specifically reacts with a protein or polypeptide comprising the peptide of claim 29 or 30. The antibody according to claim 31, wherein the antibody is a monoclonal antibody. 33.- A hybridoma cell line that produces the antibody of claim

32. 34.- A polyclonal antibody preparation comprising the antibody of claim 31. 35.- A specific oligonucleotide in hK2 consisting of at least about 7-50 nucleotides, said oligonucleotide has at least about 80% identity or aspect complementary to a nucleotide sequence having SEQ ID NO: 4, and said oligonucleotide comprises SEQ ID NO: 17, SEQ ID NO: 14 or SEQ ID NO: 18 36.- The oligonucleotide according to claim 35, characterized in that it comprises SEQ ID NO: 14. 37. The oligonucleotide according to claim 35, characterized in that it comprises SEQ ID NO: 17. 38.- The oligonucleotide according to claim 35, characterized in that it comprises SEQ ID NO: 18. 39.- A method for detecting or determining the presence of metastatic prostate cancer in a sample of human non-prostate tissue, comprising: (a) mixing an amount of an agent, which binds to a polypeptide hK2 and which does not binds to hK3, with the cells of the human tissue sample in order to form a binary complex comprising the agent and the cells; and (b) 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. 40.- The method according to claim 39, wherein the agent is an antibody. 41. The method according to claim 39, wherein the antibody is a member of a population of polyclonal antibodies. 42. The method according to claim 39, wherein the antibody is a monoclonal antibody.