MXPA00005065A - Method of detecting prostate specific antigen - Google Patents

Abstract

Detection of secreted Prostate Specific Antigen (PSA) by detecting the presence of an N-terminal activation peptide of PSA in a biological sample is described. The method may be used in screening for or diagnosing disease states associated with increased levels of secreted prostate specific antigen.

Description

METHOD FOR DETECTING SPECIFIC PROSTATE ANTIGEN RELATED REQUEST This application claims the benefit of the provisional application of E.U.A. SN 60 / 044,497, filed on November 24, 1997. This invention was carried out with government support under the National Institutes of Sanitary Concessions HL-49542. The government has certain rights to this invention.

FIELD OF THE INVENTION The present invention relates to methods for detecting Prostate Specific Antigen (PSA) in a subject by detection of an activation peptide that is cut from the proenzyme PSA. Such methods help in the detection, diagnosis and prognosis of diseases, including prostate disease and prostate and breast cancers.

BACKGROUND OF THE INVENTION During 1997 it is expected to diagnose more than 1.3 million new cases of invasive cancer in the United States (Parker et al., CA Cancer J. Clin 47: 5 (1997), this calculation does not include carcinoma in situ (except in the bladder ), nor does it include cancers of basal and squamous cells of the skin). Among women, it is estimated that the three cancers that will be most commonly diagnosed in 1997 will be cancers of the breast, lung and bronchus, and colon and rectum. Breast cancer will only account for 30% of new cancer cases in 1997. Among men, the most common cancers in 1997 will be prostate, lung and bronchus, and colon and rectum, with the prostate being the main cancer site, which represents 43% of new cases of cancer. Prostate cancer accounts for 36% of all cancers in men and 13% of cancer-related deaths in men (surpassed only by lung cancer). It is estimated that approximately 334,500 new cases of prostate cancer and 41,800 deaths related to prostate cancer will occur in the United States in 1997. Rates of prostate cancer incidence have increased over the past 35 years. Parker et al., CA Cancer J. Clin 47: 5 (1997). The test for prostate cancer has traditionally been based on digital rectal examination. Transrectal ultrasound and the measurement of prostate specific antigen (PSA) in the blood have also recently been made available to aid in the diagnosis of prostate cancer. See, for example, Friedman et al., Lancer 337: 1526 (1991); Littrup, Cancer 74 (7 Supl): 2016. However, the cost, relatively low specificity, and invasiveness of rectal imaging techniques prevent their use in routine or large-scale examinations to detect prostate cancer. In addition, the effectiveness of these techniques is not specific and depends on the skill and experience of the person performing the test. Waterhouse and Resnick, J. Urol. 141: 233 (1989); Waterhouse and Resnick, Urology, 36:18 (1990). Benign Prostatic Hypertrophy (BPH) is a condition in men over 50 years of age, and occurs in most men over 80 years of age. BPH and prostate cancer can occur simultaneously in a subject. It is estimated that in 1997 breast cancer would represent 30% of new cancer cases, with approximately 180,200 new cases diagnosed. Parker et al., CA Cancer J. Clin 47: 5 (1997). The diagnosis of breast cancer typically depends on the physical examination of the breast and / or routine mammography, with ultrasound and / or subsequent biopsy as indicated. Due to the known relationship of PSA with diseases such as BPH and prostate and breast cancers, and the high incidence of such diseases, it would be favorable to develop effective and convenient methods to detect the presence of cancer.

BRIEF DESCRIPTION OF THE INVENTION A first aspect of the present invention is a method for detecting a secreted prostate specific antigen (PSA) in a subject, comprising obtaining a biological sample from a subject and detecting the amount of PSA activation peptide in the sample. .

Another aspect of the present invention is a method for detecting in a subject the presence of a condition associated with an increased level of secreted prostate specific antigen (PSA), comprising obtaining a biological sample from a subject, detecting the amount of PSA activation peptide in the sample, and comparison of the amount of detected peptide with a predetermined standard. The detection of a peptide level greater than that of the standard indicates the presence of the condition for which the screening test is being carried out. Another aspect of the invention is a method for detecting prostate disease in a subject, which comprises obtaining a urine sample from the subject, and detecting the presence of the PSA activation peptide in the sample. The presence of the PSA activation peptide in the sample indicates prostate disease. Another aspect of the present invention is a method of A test for determining the presence of a peptide of SEQ ID NO: 1 in a sample, comprising obtaining a test sample, exposing the sample to an antibody specific for a peptide of SEQ ID NO: 1; and detection of antibody binding to peptides present in the sample. The binding of antibodies indicates the presence of peptides of SEQ ID NO: 1 in the sample. Another aspect of the present invention is a method for detecting prostate cancer in a subject, comprising obtaining a biological sample (urine, blood, blood plasma or blood serum) from the subject, and detecting the presence of the activation peptide. of PSA in the sample. The presence of the peptide indicates prostate cancer in the subject. Another aspect of the present invention is an isolated peptide of SEQ ID NO: 1. A further aspect of the present invention is an antibody or antibody fragment that specifically binds to a peptide of SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic drawing of the pro-PSA molecule, where "a.p." indicates the activation peptide and "PSA" indicates the mature active PSA molecule. The arrows indicate the positions of the expected proteolytic cleavage sites. Figure 2A provides SDS-PAGE gels of lysates and cell media from human metastatic prostate adenocarcinoma cells that had been radiolabelled using a pulse-hunting protocol. The cells were hunted for the indicated times and the lysates and medium were collected, and treated with specific antisera of the complete PSA; the gels indicate that the cells produce and secrete pro-PSA.

Figure 2B provides SDA-PAGE gels as mentioned above for Figure 2A, but treated with specific antisera from the PSA activation peptide. Figure 3 shows graphically the elimination in the plasma of various forms of PSA injected into mice, where the solid circles indicate the injection of radiolabeled a1-ATC; the open circles indicate the injection of radiolabeled «1-AAT PSA complex; "X" indicates the injection of "radiolabelled a1-ACT PSA complex with a 500-fold excess of a PSA complex" 1-ACT not labeled; the closed frames indicate the injection of radiolabeled PSA complex »a1-ACT with a 1,000-fold excess of PSA complex« a1-ACT not labeled; and open frames indicate the injection of radiolabelled PSA * 1-ACT complex with a 2,000-fold excess of PSA complex "a1-ACT unlabeled. The results show that the level of a1-ACT (closed circle) in the blood remains higher with time than the PSA level »a1-ACT (in the absence of excess PSA» a1-ACT, open circles); however, as the level of PSA * a1-ACT complexes in the blood increases, PSA * a1-ACT complexes begin to accumulate in the blood. Figure 4 shows graphically the removal in the plasma of the PSA activation peptide in mice, where the closed circles indicate the injection of the PSA activation peptide labeled with 125 I; open circles indicate the injection of the 125 I-labeled PSA activation peptide together with a 1000-fold excess of the unlabeled activation peptide; and "X" indicates injection of the 125I-labeled PSA activation peptide together with a 2,000-fold excess of the unlabeled activation peptide. The half-life of the activation peptide was not significantly affected by the amount of peptide injected. Figure 5 graphically shows the relative tissue distribution of 125 I-labeled PSA in mice. Figure 6A shows the detection of increasing concentrations of the PSA activation peptide, using antisera to the activation peptide. The concentration of the peptide increased from 0.01 ng of peptide in cavity # 1, to 5.0 ng of peptide in cavity # 9. Figure 6B shows the detection of PSA activation peptide using antisera, in urine samples of eight subjects who had previously been diagnosed with prostate cancer. Figure 6C shows the detection of the activation peptide of PSA using antisera, in blood serum samples from four subjects who had previously been diagnosed with prostate cancer. Figure 6D shows the detection of the PSA activation peptide using antisera, in urine samples from seven control subjects without prostate disease. Figure 6E shows the detection of PSA activation peptide using antisera, in blood serum samples of five control subjects without prostate disease.

DETAILED DESCRIPTION OF THE INVENTION The measurement of the level of prostate specific antigen (PSA) in the blood or serum is an accepted test to detect prostate cancer. This procedure has been used both for initial diagnoses and to monitor the effectiveness of the therapy. Babian et al., Cancer, 69: 1195 (1992); Partin and Oesterling, J. Uroi, 152: 1358-1368 (1994); Brawer et al., J. Urol. 147: 841 (1992); Catalona et al., JAMA 270: 948 (1993); Mettlin et al., Cancer 72: 1701 (1993). PSA is a glycosylated serine protease with an apparent molecular mass of approximately 36kDa. The predicted protein sequence is an amino acid protein 237 with a calculated molecular mass of 26.1 kDa. The difference between the observed and calculated mass is attributed to an N-linked oligosaccharide linked to Asn 5. Belanger et al., Prostate 27: 187 (1995). Analysis of the amino acid sequence indicates that PSA is a member of the serine protease family of chemotrypsin. Serine proteases are synthesized as precursors, and activated by the removal of an N-terminal activation peptide. In certain serine proteases (e.g., elastase and cathepsin G of human neutrophils), the activation peptide is removed prior to secretion of the protease; in other cases (e.g., thrombin and trypsin), the activation peptide is removed extracellularly, after secretion.

A recent study of the activation of the recombinant pro-PSA seems to support the hypothesis that PSA is processed in a similar way to other extracellular proteases. Takayama et al., J. Biol. Chem. 272: 21582 (1997). See also Gauthier et al., Biochim. Biophys. Acta 1174: 207 (1993); Lílja, J. Clin. Invest. 76: 1899 (1985); Lundwall and Lilja, FEBS Lett. 214: 317 (1987); Schaller et al., Eur. J. Biochem, 170: 111 (1987). In the blood, the PSA is found in three forms: (i) free PSA; (I) complexes of PSA * a1-antichymotrypsin (PSA »a1-ACT) and (iii) complexes of PSA« a2-macroglobulin (PSA «a2M). Leinonen et al., Clin. Chem 39: 2098 (1993); Stenman et al., Cancer Research 51: 222 (1991). Of these three major serum forms, only free PSA and PSA * a1-ACT are immunodetectable by current commercial assays. PSA complexes »a2M are not recognizable by antisera due to the unique nature of the complex. Barrett and Starkey, Biochem. J. 133-709 (1973); Barrett et al., Biochemical J. 181: 401 (1979). These three forms are considered to represent total PSA in serum, although trace amounts of PSA complexes have been reported for inter-a-trypsin (lal) inhibitor and a1-protease inhibitor (a1PI). Stenman et al., Cancer Res. 51: 222 (1991). Studies of the various forms of serum PSA suggest that the average proportion of PSA »a1-ACT is higher in patients with prostate cancer than in patients with benign prostate hypertrophy (BPH), although there is an overlap between the two groups. Christensson et al., J. Urol. 150: 100 (1993); Stenman et al., Cancer Research 51: 222 (1991). It has been proposed that calculating the ratio of PSA »a1-ACT / total-PSA can provide a way to distinguish between prostate cancer and BPH. Leinonen et al., Clin. Chem. 39: 2098 (1993). Lilja and colleagues reported that PSA »a1-ACT is the main form of circulating PSA (Lilja et al., Clin. Chem. 37: 1618 (1991), Lilja et al., Cancer 70 (1 Suppl): 230 (1992) ); their additional work showed that the PSA ratio «a1-ACT / total-PSA was significantly higher in patients with prostate cancer than in patients with BPH (Christensson et al., J. Uro !. 150: 100 (1993)). The current use of PSA tests is directed towards the detection of the three main forms of PSA in the blood (free PSA, PSA »a1-ACT, and PSA« a2M), as well as PSA complexes with other serine protease inhibitors including lal and a1 PI. However, the use of PSA as a test to diagnose or detect the presence of prostate cancer has several limitations. PSA interacts with many other proteins in the blood. These interactions affect the half-life of PSA in the blood and interfere with detection (and may even prevent it). In addition, the "a2M" PSA complexes are not detected (Lilja et al., Clin.Chem. 37: 1618 (1991), Zhou et al., Clin.Chem. 39: 2483 (1993)). Benign conditions can cause an elevated serum PSA level, resulting in unnecessary additional tests or biopsies; It is also true that some prostate cancers are related to normal serum PSA levels. In addition, as mentioned here, the rapid removal of PSA from circulation in the early stages of disease is a problem not yet recognized with conventional PSA tests. The inventors of the present, while not wishing to consider it a single hypothesis, believe that where prostate cancer is related to a normal concentration of serum PSA, the mechanism of elimination in the PSA plasma has not been exhausted yet. Thus, the secreted PSA is rapidly removed from the bloodstream and a normal serum PSA level is maintained. It is likely that this rate of elimination depends on the general health of the patient, including physical condition, body weight and consumption of alcohol and tobacco. These factors can affect the half-life of forms of PSA in the blood but should not significantly affect the half-life of the activation peptide. As mentioned herein, the removal of the activation peptide is a passive process that does not depend on the reaction of the activation peptide with other peptides, and subsequent endocytosis. PSA is produced by many different tissues in the body and has been shown to be present in low concentrations in breast milk. PSA has also been detected in approximately 30% of breast cancers. Monne et al., Cancer Research 54: 6344 (1994); Yu et al., Clin. Biochem. 27:75 (1994); Yu et al., Cancer Research 55: 2104 (1995); Diamandis et al., Breast Cancer Res. Treatment 32: 301 (1994). Lehrer et al. (Brit. J. Cancer 74: 871 (1996)) reported detection of a PSA fragment in the blood of 18 of 78 women with breast cancer (using PCR to amplify a fragment of the PSA molecule). These authors concluded that their PCR-based test could be used to find circulating cancer cells early in the course of breast cancer to identify patients who require additional treatment. Zarghami and Diamandis (Clin.Chem. 42: 361 (1996)) reported that, using PCR-based assays, PSA mRNA and protein were detected in breast tumor tissue. The inventors of the present invention have demonstrated by sequential analysis of the secreted PSA molecule that the PSA is secreted as an inactive proenzyme or zymogen, with a bound N-terminal activation peptide. It was found that cultured human prostate cancer cells (LNCAP cells) secrete pro-PSA containing an N-terminal activation peptide of 7 residues of sequence Ala-Pro-Leu-lle-Leu-Ser-Arg (SEQ ID NO: 1) (see example 2 in the present). The inventors of the present invention have shown that the activation peptide is cut from pro-PSA outside the cell, where it can be detected, instead of inside the cell where it could possibly be degraded. The inventors of the present believe that the sequence of the PSA activation peptide will be very well conserved; however, it is possible that variants of the PSA activation peptide of SEQ ID NO: 1 may occur. PSA activation peptide variants that arise by conservative amino acid substitution, or that have substantial sequence similar to SEQ ID NO: 1, are also included within the scope of the present invention. In addition, the inventors have demonstrated in an animal model in vivo that when a small amount of PSA is introduced into the bloodstream it rapidly reacts with specific protease inhibitors and is removed from the bloodstream by binding to hepatocyte receptors and subsequent endocytosis (see example 3 herein). The inventors of the present, while not wishing to consider it as a single theory, infer that in the early stages of cancer PSA secretion can be limited, due to the PSA reaction with protease inhibitors, PSA can be rapidly eliminated from the body. bloodstream and therefore can not be detected for diagnostic purposes. The inventors hereby assume that as the cancer progresses and increased levels of PSA are released into the bloodstream, the elimination mechanisms in the plasma are flooded and the PSA complexes begin to accumulate in the blood (see figure 3) allowing the detection of PSA in the blood for diagnostic purposes. The inventors of the present have discovered that (i) PSA is secreted as a proenzyme (pro-PSA) that contains an N-terminal activation peptide; (ii) that the PSA in the blood does not react with pro-PSA antisera and has therefore been activated; (iii) the plasma elimination kinetics of PSA suggest that PSA accumulates in the blood when the elimination mechanism has been saturated; (V) the removal in the plasma of the activation peptide indicates filtration outside the body by the kidney; and (v) the activation peptide can be detected in urine and blood. The inventors of the present invention identified the N-terminal activation peptide sequence of PSA produced by cultured human prostate cancer cells (LNCAP cells) such as Ala-Pro-Leu-lle-Leu-Ser-Arg (SEQ ID NO: 1). ). The results herein indicate that the PSA activation peptide (e.g., peptide of SEQ ID NO: 1) in urine or blood is a reliable indicator of PSA secreted in a subject. The activation peptide is cut off from PSA during pro-PSA activation and apparently does not interact with other proteins, but is removed from the blood by simple renal filtration. The activation peptide is consequently easy to detect by testing its presence in urine by any suitable method. Because the presence of the PSA activation peptide indicates the presence of secreted PSA, screening for the presence of the activation peptide in urine provides a method to detect cancers associated with increased levels of secreted PSA, including prostate cancers and cancers of breast. Therefore, the inventors of the present have determined that the PSA activation peptide can be detected in biological samples of subjects, and that the level of the PSA activation peptide is an indicator of secreted PSA. Accordingly, detection of the PSA activation peptide indicates PSA secretion. Accordingly, the detection and / or quantitative measurement of the PSA activation peptide is useful in detecting diseases related to increases in PSA. These conditions include BPH, prostate cancer and breast cancer. In a preferred embodiment of the present invention, the condition by which the screening test is performed is breast cancer, and the sample being examined is urine or blood. In another preferred embodiment of the present invention, the condition by which the screening test is carried out is prostate cancer, and the sample being examined is urine or blood. As used herein, an "increased level" of the PSA activation peptide refers to a level that increases above a predetermined standard, or that increases above the level in a control sample. The predetermined standard can be based on the detection of PSA activation peptide in healthy subjects, and can be zero or undetectable. Alternatively, when testing a subject, the predetermined standard can be based on a previous test result from the same subject, i.e., the test results can be monitored over time to detect changes in the activation peptide. PSA. It is known that the PSA test is useful in detecting metastatic or persistent disease in patients undergoing medical or surgical treatment of prostate cancer, where the persistent elevation of (or increases in) PSA levels following treatment indicates recurrent or residual disease. . The level of the PSA activation peptide that is considered as an indicator of disease may differ among objective diseases by which the detection method is used; the level of the PSA activation peptide can be measured as an amount per unit of test sample, or as a percentage of the total protein in a test sample. The diagnosis or level of the PSA activation peptide indicator for a particular disease state can be determined using routine clinical test methods known in the art. Any number of protocols can be used to develop information to be used in carrying out the diagnostic methods of the present invention.; The methods and guidelines for developing suitable study protocols are known to those skilled in the art. The methods described herein may be employed with subjects suspected of having a disease state associated with increased PSA levels, including but not limited to BPH, prostate cancer, and breast cancer. The methods herein can be used both to monitor subjects who have been previously diagnosed with the target condition, to monitor subjects receiving treatment for the target condition, or to examine subjects who have not been previously diagnosed. the objective condition, including asymptomatic subjects. Subjects include humans as well as mammalian veterinary subjects, and include both male and female subjects. The methods described herein are particularly suitable for detecting prostate cancer and for aiding in the diagnosis and prognosis of prostate cancer. As used herein, methods of detection and diagnosis do not mean that the methods are 100% specific or sensitive in indicating the presence of the objective disease state; rather, a screening test or positive diagnosis indicates that the subject is at increased risk (compared to the general population) of having the target condition. In a sampling of multiple subjects, the positive test results will correlate with the presence of the objective condition. The specificity and sensitivity of the methods herein may vary depending on the condition being examined or monitored, the biological sample being examined, the general health of the subject being examined, and other factors, as will be apparent to the experts in the art. In a particular embodiment of the present invention, the subject has previously been diagnosed with a disease associated with elevated PSA levels (such as prostate or breast cancer) and may have already received treatment for said disease. The methods herein are suitable for monitoring the recurrence or progression of the disease or the success of the treatment thereof; in such cases, the levels of the PSA activation peptide in a subject can be compared with time. Prostate cancer is a well-recognized disease entity. As used herein, the term prostate cancer includes any histological type of cancer that appears in prostate tissue. The most common tumor that appears in the prostate is adenocarcinoma. Adenoid cystic carcinomas, carcinosarcomas, and sarcomas, as well as other histological types of cancer, can also occur in the prostate. Breast cancer is a well-recognized disease entity. As used here, the term breast cancer includes any histological type of cancer that appears in the breast tissue. Breast cancers appear more commonly in the epithelium; Other histological types of mammary carcinoma have been described. Benign Prostate Hypertrophy (BPH) is a common condition in men older than 50 years, and occurs in most men over 80 years. The treatment of BPH includes drug therapy to reduce the volume of the prostate and surgical resection of the prostate. Increased serum PSA concentrations are reported in BPH. Samples taken from subjects for use in the methods described herein are usually biological fluids such as urine, blood (including whole blood, blood serum and blood plasma), ascites fluid, cyst fluid (such as breast cyst fluid). ) or other bodily fluids that may contain the PSA activation peptide. When performing the test for prostate cancer, urine is a preferred test sample. The methods for obtaining samples to be tested will be carried out according to techniques known in the art, and may depend on the condition for which the test is being performed and the condition of the subject. The samples may go through additional conventional preparation steps before detection of the PSA activation peptide, as will be apparent to those skilled in the art. For example, samples may go through the addition of preservatives, concentration steps, filtration, etc.

The levels of the PSA activation peptide can be determined as an amount of peptide per sample volume, or as a percentage of the total protein in the sample. Antibodies that can be used to carry out the present invention include antibodies that specifically bind to a peptide of SEQ ID NO: 1 and fragments of said antibodies, which fragments specifically bind to a peptide of SEQ ID NO: 1. Such antibodies and antibody fragments can be produced by a variety of techniques, as mentioned below. The term "antibodies" as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. Of these, IgM and IgG are preferred. The antibodies used in the methods herein can be obtained according to known techniques, and can be monoclonal or polyclonal, and can be of any species of origin, including (for example) mouse, rat, rabbit, horse or human, or they can be chimeric antibodies. See example, M. Walker et al., Molec. Immunol. 26: 403 (1989). The antibodies can be recombinant monoclonal antibodies produced according to the methods described in Reading, U.S. Pat. No. 4,474,893, or Cabilly et al., Patent of E.U.A. No. 4,816,567. Antibodies can also be chemically constructed by specific antibodies made in accordance with the method described in Segel et al., U.S. Pat. No. 4,676,980. Antibody fragments included within the scope of the present invention include, for example, Fab, F (ab ') 2 and Fv fragments, and corresponding fragments obtained from antibodies other than IgG. Such fragments can be produced by known techniques. Polyclonal antibodies used to carry out the methods of the present invention will be produced by immunizing a suitable animal (eg, rabbit, goat, etc.) with the target antigen, collecting the animal's immune serum, and separating the polyclonal antibodies from the immune serum, according to known procedures. Monoclonal antibodies used in the methods herein may be produced in a hybridoma cell line according to the technique of Kohler and Milstein, Nature 265: 495 (1975) and other techniques known in the art. The monoclonal Fab fragments can be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, for example, Huse, Science 246: 1275 (1989). The methods described herein detect the presence of PSA activation peptide, which, as determined by the inventors herein, indicates the presence of activated PSA in the subject to whom the test is being conducted. Any suitable method can be used to detect the PSA activation peptide, as would be apparent to one skilled in the art. Preferred detection methods are immunoassay formats, which may be homogeneous assays or heterogeneous assays. In a homogeneous assay the immunological reaction usually comprises antibody to the PSA activation peptide, a labeled analyte (labeled PSA activation peptide) and the test sample of interest. The signal arising from the label is modified, directly or indirectly, by the binding of the antibody to the labeled analyte. Both the immunological reaction and the detection of the degree of the immunological reaction are carried out in a homogeneous solution. The immunochemical labels that may be employed include free radicals, radioisotopes, fluorescent dyes, enzymes, coenzymes, etc. In a heterogeneous assay approach the reagents are usually the test sample, antibody specific for the PSA activation peptide and means for producing a detectable signal, as mentioned above. The antibody is generally immobilized on a support (e.g., a sphere, plate or slide) and contacted with the test sample in the liquid phase. The support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal that is related to the presence of the PSA activation peptide. The means to produce a detectable signal include the use of radioactive labels, fluorescent labels, enzyme labels, etc .; as will be apparent to those skilled in the art. Examples of suitable immunoassays include radioimmunoassays, immunofluorescence methods, enzyme linked immunoassays, and the like. Those skilled in the art will be familiar with numerous specific immunoassay formats and variations thereof that will be useful in carrying out the methods of the present invention. The antibodies described herein can be attached to a solid support suitable for a diagnostic assay (e.g., spheres, plates, slides or cavities formed from materials such as latex or polystyrene) according to known techniques. Antibodies can bind to detectable elements such as radiolabels (eg, 35S, 125l, 1311), enzyme labels (eg, horseradish peroxidase, alkaline phosphatase), and fluorescent labels (eg, fluorescein) according to techniques known. The diagnostic equipment for carrying out the methods of the present invention can be produced in a number of ways. In one embodiment, the diagnostic kit comprises (a) an antibody or antibody fragment that specifically binds to the PSA activation peptide (eg, peptide of SEQ ID NO: 1) attached to a solid support and to a second antibody or antibody fragment bound to a detectable element. The equipment may also include auxiliary reagents such as pH regulating agents and protein stabilizing agents.; and may include (where necessary) other members of the detectable signal producing system of which the detectable element is a part (e.g., enzyme substrates); agents for reducing basic interference in a test, control reagents, apparatus for performing a test, and the like, as will be apparent to those skilled in the art. A second embodiment of a test kit of the present invention comprises an antibody or antibody fragment specific for the PSA activation peptide, and a specific binding partner for the antibody bound to a detectable group. As described above, auxiliary agents may also be included. The test equipment can be packed in a suitable manner, typically with all the elements in a single container along with a sheet or printed instructions to carry out the test. The step for detecting the presence of PSA in the blood or serum of the test subject can be carried out simultaneously with the methods of the present invention, as an additional indication of whether or not the subject suffers from the disease state being evaluated. . Various methods for detecting PSA are known in the art; see, for example, the patent of E.U.A. 5,672,480 to Dowell et al .; patent of E.U.A. 5,658,730 to McGill et al .; patent of E.U.A. No. 5,654,161 to Tewari; patent of E.U.A. 5,599,677 to Dowell et al .; and 5,501, 983 of Lilja et al. (It is intended that the descriptions of all of the US patent mentioned herein are incorporated herein in their entirety). The methods herein may be used in conjunction with other diagnostic or screening tests designed to detect the objective condition. The use of the phrase "substantial sequence homology" in the specification and claims herein means that DNA, RNA or amino acid sequences having minor sequence variations of the actual sequences described and claimed herein are considered equivalent to the sequences of the present invention. In this sense, 'small unimportant sequence variations' mean that 'homologous' sequences (i.e., the sequences having substantial sequence similarity to the DNA, RNA or proteins described and claimed herein) will be functionally equivalent to the sequences described and claimed in the present invention. Functionally equivalent sequences will function in substantially the same way to produce substantially the same compositions as the nucleic acid and amino acid compositions described and claimed herein. The use of the word "isolated" in the present means that the DNA, RNA, polypeptides or proteins have been separated from their cellular environments in vivo by human intervention. Sequences with "substantial sequence similarity" refer to nucleotide sequences that share at least 90% identity with the nucleic acids of the invention; and the amino acid sequences that commonly share at least between 70%, 80%, 85%, 90% or even 95% amino acid identity with polypeptides of the invention. It is recognized, however, that polypeptides or nucleic acids containing lower levels of similarity to those described above, that arise as splice variants or generated by conservative amino acid substitutions, or by substitution of degenerate codons, also fall within the scope of of the present invention.

The present invention is explained by the examples described below.

EXAMPLE 1 Materials and methods Reagents The detection reagents in ECL Western Blotting were obtained from Amersham (Arlington Heights, IL). RPMI 1640 medium, RPMI 1640 amines selection kit, Dulbecco's phosphate buffered saline solution, Earls balanced salt solution, and penicillin and streptomycin were obtained from Gibco (Grand Island, NY). The epidermis growth factor and L-glutamine were obtained from Sigma (St. Louis, MO). The PSA antiserum was obtained from DAKO Corporation (Carpintería, CA). Human metastatic prostate adenocarcinoma cells (LNCaP) were obtained from the North American Type Culture Deposit (Rockville, MD). The radiochemical compounds were obtained from (DuPont / NEN). ACT was purified according to the above described (Salveson et al., J. Biol. Chem. 260: 2432 (1985)). Urine samples were obtained from the Urology Clinic of the Duke University Medical Center. Human prostate tissues (normal, benign, hypertrophic, malignant) were obtained from the Duke University Medical Center. A pathologist confirmed the histology of each tissue.

Purification of PSA All steps were carried out at 4 ° C. Approximately 100 g of prostate tissue (Vitris Tempest) were homogenized in 300 ml of 0.05 M Tris-CI, 0.1 M NaCl, 0.01 M EDTA, at pH 7.4. The homogenate was filtered through several layers of cloth or fabric and clarified by centrifugation. Subsequently, the supernatant was digested with 0.1 mg / ml RNase, 0.2 mg / ml DNase and 0.005 M MgCI for 4 hours at 4 ° C. After 4 hours of incubation, the supernatant was dialyzed overnight against 0.01 M HEPES, at pH 8. The next day, the sample was clarified by centrifugation and applied to a column of Q-Sepharose FF (Pharmacia) (2.5 x 20 cm ) equilibrated in 0.01 M HEPES, at pH 8. The loaded column was thoroughly washed in an equilibrium regulator and subsequently developed with a linear gradient (total volume of 2 liters), from 0 M NaCl to 0.4 M NaCl. Fractions were taken of 4 ml and were tested for PSA detection by Western blotting. The active fractions were deposited and concentrated by ultrafiltration (Amicon) and applied to a S-200 HR (Pharmacia) gel filtration column (2.5 x 150cm) equilibrated in 50 mM HEPES, 150 mM NaCl. Fractions of 4 ml were taken and tested for PSA detection by Western blotting. Fractions containing PSA were deposited and dialysed in 10 mM HEPES at pH 8 and separated into a MONO-Q 5/5 HR (Pharmacia) connected to an FPLC system also distributed by Pharmacia. The column was equilibrated in 10 mM HEPES and developed using a linear gradient between 0 M NaCl and 400 mM NaCl.

Preparation of antiserum A peptide, Ala-Pro-Leu-lle-Leu-Ser-Arg-Cys (SEQ ID NO: 2), was synthesized, corresponding to an N-terminal activation peptide of PSA (Bio Synthesis, Lewisville, TX ). Cys is not part of the activation peptide but was added to facilitate coupling to ovalbumin using N-hydroxysuccinimide ester of m-maleimidobenzoic acid (Liu et al., Biochemistry 18: 690 (1979); Kitagawa and Aikawa, J. Biochem 79: 233 (1976)). Another peptide, Ala-Pro-Leu-lle-Leu-Ser-Arg (SEQ ID NO: 1) was synthesized using the MAP (multiple antigen peptide) technique (Bio Synthesis, Lewisville, TX). Rabbit antiserum was obtained from the activation peptide-ovalbumin conjugates and the MAP activation peptide, in rabbits using a standard protocol known in the art.

Metabolic labeling and pulse-hunting analysis Human metastatic prostate adenocarcinoma cells (LNCaP) maintained in an RPMI 1640 medium (RPMI) supplemented with 10% fetal bovine serum, epidermal growth factor (5 mg / 500 ml), L-glutamine (150 mg / 500 ml) and 1% penicillin Streptomycin in 5% CO2. For standard biosynthetic radiolabelling, cells were cultured in 50 mm tissue culture plates to an 80% confluence. The cells were washed twice with balanced saline solution of Earls, and subsequently incubated for 30 minutes in RPMI without fetal bovine serum and without the amino acids that would be used for the subsequent metabolic labeling. After adding [35S] Met, the cells were incubated for 5 minutes (pulse period). In the case of the immunoprecipitated proteins that were to be subjected to radio-frequency analysis, [35 S] Met was added together with [3 H] lie, [3 H] Leu or [3 H] Val. At the end of the labeling period, the cells were washed in a timely manner on two occasions with serum-free RPMI and were hunted with a complete "cold" medium for several periods of time.

Lysis and immunoprecipitation The conditioned medium was collected and frozen. The cell lysates were prepared by three rapid freeze-thaw cycles in a salty regulator with 0.5% Triton X-100 and a proteinase inhibitor cocktail. Before immunoprecipitation, the lysate and conditioned medium samples were rinsed by the addition of a pre-immune serum and subsequently a G-protein Sepharose 4 FF (Pharmacia). The supernatants were incubated overnight with the relevant specific antiserum. The next day, G-protein Sepharose 4 FF was added and immunoprecipitates were collected by gentle centrifugation. Subsequently, the immunoprecipitates were repeatedly washed and the ligated proteins were released from the G-protein Sepharose 4 FF by boiling in an SDS sample regulator or by 100 mM glycine HCl (pH 2.7), before SDS-PAGE.

Protein sequence analysis and amino acid analysis Proteins and peptides were analyzed by automated Edman degradation in an Applied Biosystems 477A sequencer, with on-line PTH analysis using an Applied Biosystems 120A HPLC system. The proteins and peptides were hydrolysed in 6N HCl and the composition was determined using a Beckman 6300 amino acid analyzer. Both instruments were operated in accordance with the manufacturer's specifications.

Radio-frequency analysis This analysis was carried out in accordance with that described above (Salvesen and Enghild, Biochemistry 29: 5304 (1990), Thogersen and Enghild, J. Biol. Chem. 270: 18700 (1995)). In summary, after immunoprecipitation and SDS-PAGE, the double-labeled proteins [35S] and [3H] were electrotransferred to immobilon membranes (Matsudaira, J. Biol. Chem. 262: 10035 (1987)). The proteins were identified by autoradiography and the bands of interest were cut and analyzed by automated Edman degradation. The amino acids of anilinothiazolinone (ATZ) released after each cycle were collected and counted for detection of radioactivity [35S] and [3H]. In the experiments intended for radiosequence analysis, metabolic labeling was carried out using the appropriate radioactive amino acids expected within the first 20 m-teminal residues of the mature proteins. Subsequent radiosequence analyzes of the radioactive ATZ-amino acid release bands in the anticipated Edman degradation cycle, served to identify the protein band.

Polyacrylamide gel electrophoresis The supernatants from the immunoprecipitates treated with SDS were recovered by centrifugation and runs in sodium dodecylsulfate (SDS) -electrophoresis in polyacrylamide gel (PAGE), in gradient gels of 5 to 15% (Bury, J Chromatography 213: 491 (1981)). The gels were stained, destained, dried and subjected to imaging on a Phosphorlmager apparatus (Molecular Dynamics 41 OA). Immunoprecipitates with radio-frequency analysis were transferred to membranes in PROBLOTT ™. After electrophoresis, the PROBLOTT ™ membranes were dried and exposed directly to an X-ray film overnight at -70 ° C.

Radiolabelling of proteins and peptides Proteins were radioiodinated using N-chlorobenzensulfonamide (Markwell, Anal. Biochem. 125: 427 (1982)) immobilized on polystyrene spheres in accordance with the instructions provided by the manufacturer (IODO-BEADS® Pierce) . The activation peptide was labeled using a 125I-Bolton-Hunter reagent (Bolton and Hunter, Biochem. J. 133: 529 (1973)), in accordance with the manufacturer's instructions (DuPont / NEN).

Plasma elimination studies This procedure has been described in detail in other documents. Imber and Pizzo, J. Biol. Chem 256: 8134 (1981); Enghild et al., J. Biol. Chem. 26920159 (1994); Christensen et al., J. Biol. Chem. 270: 14859 (1995). Briefly, approximately 1.0 μg of radioiodinated protein or peptide was grafted to the lateral flow of CD-1 mice. Samples of 25 μl were taken at time intervals, by puncturing the retro-optic region of the eyes of these mice. The initial point of time, taken between 5 and 10 seconds after the injection, was considered as representing 100% of the radioactivity in circulation. Each preparation was studied at least in triplicate. After the plasma elimination experiments, the mice were perfused and their organs extracted, which were counted with a counter-? Western blotting The membranes were developed using a Western blotting ECL equipment distributed by Amersham ™. In summary, after transfer to the PVDF membranes, they were blocked for 1 hour in 20 mM Tris-Cl, 137 mM NaCl, at pH 7.6, with 0.1% Tween (TBS-T regulator) and 5% of the reagents blockers supplied. The membrane was washed in TBS-T regulator before adding the primary antibody (1/2000 dilution). After 1 hour of incubation, the membrane was washed in TBS-T regulator and the second antibody labeled with horseradish peroxidase (1 / 20,000 dilution) was added. The membranes were incubated for 1 hour and washed with TBS-T regulator, and developed using the reagent provided.

Enzyme Linked Immunosorbent Assay (ELISA) Plates with 96 Costar RIA / EIA cavities (Costar, Cambridge, MA) were incubated for 2 hours at 23 ° C, with increasing amounts of samples to be tested, in a total volume of 50 μl in PBS , at pH 7.3. The cavities with known concentrations of activation peptide and β2-microglobulin were analyzed simultaneously for comparison. The coated plates were washed and blocked with PBS containing 5% CARNATION® nonfat dry milk and 0.05% Tween 80 (blocking buffer) for 2 hours at 23 ° C. Subsequently, the plates were incubated with 100 μl of activation peptide antiserum diluted in blocking buffer, overnight, at 4 ° C. The next day, the plates were washed briefly and incubated for 2 hours using 100 μl (1/2000 dilution) of anti-rabbit IgG coupled to alkaline phosphatase. They were washed with blocking buffer and PBS, and subsequently the substrate p-nitrophenyl phosphate (1 mg / ml in 0.1 M glycine, 1 mM MgCl, 1 mM ZnCl 2, at pH 10.4) was added. The alkaline phosphatase activity was kinetically monitored at 37 ° C using a THERMOmax microplate reader (Molecular Devices, Menlo Park, CA).

EXAMPLE 2 Results: Biosynthesis and PSA processing The cDNA encoding PSA suggests an N-terminal activation peptide of 7 amino acids. Lundwall and Lilja, FEBS Lett. 214: 317 (1987). However, the putative activation peptide had not previously been detected in the purified PSA. Schaller et al. Eur. J. Biochem. 170: 111 (1987); Zhang et al., Clin. Chem. 41: 1567 (1995); Sensabaugh and Blake, J. Urol. 144: 1523 (1990); Watt et al., Proc. Natl. Acad. Sci. 83: 3166 (1986). Consequently, it was not clear whether the activation peptide had been removed intracellularly prior to secretion, as is the case with granulated serine proteases (Young et al., Cell 47: 183 (1986); Lobe et al., Science 232: 858 (1986), Sinha et al., Proc. Natl. Acad. Sci. 84.2228 (1987), Wilde et al., J. Biol. Chem. 265: 2038 (1990), Salvesen and Enghild, Biochemistry 29: 5304 (1990). )), or after secretion as with most serine proteases. Figure 1 is a schematic diagram of the PSA, characterized in that the arrows indicate the positions of the expected proteolytic cleavage sites. The box represents mature active PSA (sequence not illustrated), and a.p. refers to the activation peptide.

To investigate post-translational PSA processing, the present inventors used radiolabelling and biosynthetic radiosequencing techniques (described in Example 1) to characterize both intracellular and secreted PSA. These analyzes were performed using biosynthetically radiolabelled LNCaP cells and a polyclonal PSA antibody (Figure 2A), as well as a specific peptide antiserum against the activation peptide (Figure 2B). The cells were radiolabeled by a pulse-hunting protocol as described above and the lysates and medium were harvested and treated with antiserum specific to the entire PSA and activation peptide. The samples were analyzed by reduced SDA-PAGE. After electrophoresis, the gel was dried and subjected to imaging on a Phosphorlmager apparatus. As illustrated, the cells produce and secrete pro-PSA. These results indicate that the PSA does not go through any N-terminal processing event. This was confirmed by radiosequence analysis of both intracellular and secreted PSA (data not shown). These studies determine that PSA is secreted as an inactive proenzyme containing an N-terminal activation peptide of 7 residues with sequence and Ala-Pro-Leu-lle-Leu-Ser-Arg- (SEQ ID NO: 1). The activation of pro-PSA is an extracellular event.

EXAMPLE 3 Elimination of a1 -ACT PSA complexes and tissue distribution of a1 -ACT in mice The clearance rate of 125 I-α-CACT was compared with the clearance rate of the 125 I-ARACT »PSA complexes in mice, using techniques as described above. As illustrated in Figure 3, when comparing the ai-ACT injected separately (black circles) and the α-ACT in complex together with the PSA (white circles), it can be seen that the average life of the PSA complex ACT was reduced significantly with respect to the native a? -ACT. To simulate a situation in which more PSA is secreted, a 125-PSA complex was injected «a? -ACT with an excess of 500 times (X), 1000 times (black squares) and 2000 times (white squares), of PSA complexes «AarACT" cold ". These experiments showed that the PSAar ACT complex is initially cleared from the blood, and as the level of PSA complex to ACT increases in the blood the clearance mechanism becomes saturated and the ai-ACT PSA complexes begin to accumulate in the blood. The studies showed that 125l-a? -ACT »PSA complexes were removed from the circulation of the mouse with a half-life of approximately 20 minutes; the half-life of 125 l-rACT was estimated at several hours. To investigate whether the accumulation of complexes 125l-arACT «PSA in the blood was caused by a saturation of the clearance mechanism, complexes 125l-to -ACT« PSA were co-injected together with an excessively large amount of complexes to? -ACT «PSA not marked. The half-life increased from around 20 minutes to several hours. These experiments show that the rate of clearance is significantly affected by the level of complex α -ACT * PSA in the bloodstream, and indicate that the accumulation of a? -ACT * PSA in the blood is due to saturation of the clearance mechanism. The removal of the activation peptide in the plasma was studied by injecting a 125I-labeled activation peptide (Ala-Pro-Leu-lle-Leu-Ser-Arg; SEQ ID NO: 1) into the lateral flow of a mouse. Removal of 125l-labeled activation peptide in the plasma was continued for one hour (black circles, Figure 4); the half-life of the peptide was less than 2 minutes. The activation peptide labeled with 125 I was also injected with an excess of 1000 times (white circles) and 2000 times (X) of unlabeled activation peptide. The rate of clearance was not significantly affected by the level of activation peptide in the bloodstream. After the elimination experiments in the plasma, the organs of the test mice were examined to determine if there was radioactivity, by means of a counter-? The predominance of ai-ACT in the bladder and kidneys indicates that the peptide was eliminated from the bloodstream by renal filtration (Figure 5).

The above results indicate that at the initial levels, PSA *? -ACT complexes rapidly clear up from the bloodstream of mammals; however, by introducing more "cn-ACT" PSA complexes into the bloodstream, the clearance mechanism becomes saturated, resulting in an excess of PSA * to? -ACT complexes in the bloodstream.

EXAMPLE 4 Detection of PSA activation peptide in biological samples The results obtained indicate that the activation peptide PSA that is cut from PSA is cleared from the bloodstream by renal filtration, and is present in the urine and blood. To determine whether the PSA activation peptide is measurable in the urine or serum of patients with benign prostatic hypertrophy or with prostate cancer, the activation peptide of SEQ ID NO: 1 was synthesized and a peptide antiserum was prepared polyclonal in rabbits. The specific character of the antiserum was verified by ELISA, against the synthetic activation peptide, which was provided in amounts between 0.01 and 5.0 ng of peptide (Figure 6A, increasing concentration of activation peptide, from point 1 to point 9). The antiserum produced a sensitive dose-dependent reaction. Serum and urine samples were collected from control patients (without prostate disease) and from patients with prostate cancer, and were tested for the presence of the activation peptide as indicated above. The activation peptide was detected in the urine (FIG. 6B) and in the serum (FIG. 6C) of the cancer patients, but not in the urine (FIG. 6D) or in the serum (FIG. 6E) of the control patients. The results indicate that the detection of PSA activation peptide in the urine or serum of subjects is feasible and indicates the presence of PSA.

EXAMPLE 5 Biotinylation of PSA activation peptide A PSA activation peptide containing a Cys terminal Cys residue (tional Ala-Pro-Leu-Ile-Leu-Ser-Arg-Cys; SEQ ID NO: 2) was biotinylated using N- (6- [biotinamido ] hexyl) -3 '- (2'-pyridylthio) propionamide (Pierce). This reagent can be used in a standard ELISA test as follows: 1) coat a 96-well ELISA plate with antiactivation peptide antiserum and residual block binding sites (PBS with 0.05% Tween 20 and 0.25% albumin of bovine serum (BSA)); 2) ng a fixed concentration of biotinylated PSA activation peptide and increasing the amounts of the sample (eg, urine, blood, blood serum) to a series of cavities; 3) wash the cavities before ng avidin and biotinylated horseradish peroxidase; 4) after incubation, wash the cavities and detect the residual biotinylated PSA activation peptides in a plate reader at 450 nm using the horseradish peroxidase substrate TURBO 3,3 ', 5,5'-tetramethylbenzidine (TMB) (Pierce) The above examples are by way of illustration but not limitation. The invention is described by the following claims, with their equivalents included therein.

Claims (40)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for examining a subject for the purpose of detecting secreted prostate specific antigen (PSA), which includes: (a) obtaining a biological sample from a subject; (b) detecting the amount of PSA activation peptide in this sample; the detection of PSA activation peptide in this sample indicates the presence of PSA secreted in this subject.

2. A method according to claim 1, further characterized in that the amino acid sequence of this PSA activation peptide is SEQ ID NO: 1.

3. A method according to claim 1, further characterized in that it includes comparing the amount of detected peptide with a predetermined standard.

4. A method according to claim 1, further characterized in that the biological sample is urine.

5. A method according to claim 1, further characterized in that the biological sample is selected from a group consisting of blood, blood plasma or blood serum.

6. A method according to claim 1, further characterized in that the aforementioned detection step is carried out by an immunoassay.

7. A method for examining a subject for the purpose of detecting the presence of a condition related to an increase in the level of secreted prostate specific antigen (PSA), which includes: (a) obtaining a biological sample from a subject; (b) detecting the amount of PSA activation peptide in that sample; and (c) comparing that amount of detected peptide with a predetermined standard, characterized in that the detection of a peptide level higher than the standard indicates the presence of this state.

8. A method according to claim 7, further characterized in that the amino acid sequence of the aforementioned PSA activation peptide is SEQ ID NO: 1.

9. A method according to claim 7, further characterized in that the biological sample is urine.

10. A method according to claim 7, further characterized in that the biological sample is blood, blood plasma or blood serum.

11. A method according to claim 7, further characterized in that the condition is prostate cancer.

12. A method according to claim 11, further characterized in that the subject has not been diagnosed with prostate cancer before.

13. - A method according to claim 7, further characterized in that the aforementioned condition is benign prostatic hypertrophy.

14. A method according to claim 13, further characterized in that the subject has not been diagnosed with benign prostatic hypertrophy before.

15. A method according to claim 7, further characterized in that the aforementioned condition is breast cancer.

16. A method according to claim 15, further characterized in that the subject has not been diagnosed with breast cancer before.

17. A method for examining a subject for the purpose of detecting a prostate disease that includes: (a) obtaining a urine sample from this subject; (b) detecting the presence of PSA activation peptide in this sample; wherein the presence of a peptide of SEQ ID NO: 1 in this sample indicates that this subject is diseased of the prostate.

18. A method according to claim 17, further characterized in that the amino acid sequence of the aforementioned PSA activation peptide is SEQ ID NO: 1.

19. A method according to claim 17, further characterized in that it includes the step of comparing the level of detected peptide with a predetermined standard, wherein the prostate disease is indicated when the level of detected peptide is greater than the predetermined standard.

20. A method according to claim 17, further characterized in that the detection step is carried out by immunoassay.

21. A method according to claim 20, further characterized in that the immunoassay is selected from radioimmunoassay and enzyme linked immunoassay.

22. A method according to claim 17, further characterized in that the aforementioned biological sample is urine.

23. A method according to claim 17, further characterized in that the biological sample is blood, blood plasma or blood serum.

24. A method according to claim 17, further characterized in that the subject has not yet been diagnosed with prostate disease.

25. A method according to claim 17, further characterized in that the disease of the prostate is benign prostatic hypertrophy.

26. A method according to claim 17, further characterized in that the disease of the prostate is prostate cancer.

27. - An immunoassay method for determining the presence of a peptide of SEQ ID NO: 1 in a sample, which includes: a) obtaining a sample for the test; b) exposing this sample to an antibody specific for a peptide of SEQ ID NO: 1; and c) detecting the binding of this antibody with the peptides present in the aforementioned sample; characterized in that the binding of antibodies indicates the presence of peptides of SEQ ID NO: 1 in the sample.

28. A method according to claim 27, further characterized in that the sample is urine.

29. A method according to claim 27, further characterized in that the sample is blood or blood serum.

30. A method for examining a subject for the purpose of detecting prostate cancer, which includes: a) obtaining a biological sample from this subject, and this sample is selected from a group consisting of urine, blood, blood plasma and blood serum; and b) detecting the presence of a prostate-specific antigen (PSA) activation peptide in this sample; wherein the presence of the PSA activation peptide in the sample is indicative of prostate cancer in this subject.

31. A method according to claim 30, further characterized in that it includes comparing the detected level of PSA activation peptide with a predetermined standard, wherein a level higher than the predetermined standard is indicative of prostate cancer.

32. - A method according to claim 30, further characterized in that the amino acid sequence of the aforementioned PSA activation peptide is SEQ ID NO: 1.

33. A method according to claim 30, further characterized in that the detection step is carried out by immunoassay.

34. A method according to claim 33, further characterized in that the immunoassay is selected from radioimmunoassay and enzyme-linked immunoassay.

35. A method according to claim 30, further characterized in that the biological sample is urine.

36. A method according to claim 30, further characterized in that the subject has not been previously diagnosed with prostate disease. 37.- A peptide isolated from SEQ ID NO: 1. 38.- An antibody or antibody fragment that specifically binds to a peptide of SEQ ID NO: 1. 39.- An antibody according to claim 38, further characterized in that this antibody is labeled for radioimmunoassay or enzyme-linked immunoassay. 40.- An antibody according to claim 38, further characterized in that it is bound to a solid support.