Method of detecting prostate specific antigen

Dectection 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.

FILED OF THE INVENTION
 The present invention relates to methods of detecting Prostate Specific
 Antigen in a subject by detection of an activation peptide that is cleaved
 from the PSA proenzyme. Such methods aid in the detection, diagnosis and
 prognosis of diseases, including prostate disease and breast cancers.
 BACKGROUND OF THE INVENTION
 More than 1.3 million new cases of invasive cancer are expected to be
 diagnosed in the United States during 1997 (Parker et al. CA Cancer J.
 Clin. 47:5 (1997); this estimate does not include carcinoma in situ
 (except in the bladder), nor does it include basal and squamous cell
 cancers of the skin). Among women, it is estimated that the three most
 commonly diagnosed cancers in 1997 will be cancers of the breast, lung and
 bronchus, and colon and rectum. Breast cancer alone will account for 30%
 of new cancer cases in 1997. Among men, the most common cancers in 1997
 will be cancers of the prostate, lung and bronchus, and colon and rectum,
 with prostate being the leading cancer site and accounting for 43% of new
 cancer cases.
 Prostate cancer accounts for 36% of all male cancers and 13% of male
 cancer-related deaths (surpassed only by lung cancer). It is estimated
 that approximately 334,500 new cases of prostate cancer and 41,800
 prostate cancer-related deaths will occur in the United States in 1997.
 Incidence rates of prostate cancer have increased over the past 35 years.
 Parker et al. CA Cancer J. Clin. 47:5 (1997). Screening for prostate
 cancer has traditionally relied on digital rectal screening. Transrectal
 ultrasound and measurement of prostate-specific antigen (PSA) in the blood
 have also recently become available to aid in the diagnosis of prostate
 cancer. See, e.g., Friedman et al., Lancet 337:1526 (1991); Littrup,
 Cancer 74 (7 Suppl):2016. However, the cost, relatively low specificity,
 and invasiveness of rectal imaging techniques precludes these from use in
 routine or large-scale screening for prostate cancer. Additionally, the
 effectiveness of such techniques are non-specific and depend on the skill
 and experience of the examiner. Waterhouse and Resnick, J. Urol. 141:233
 (1989); Waterhouse and Resnick, Urology, 36:18 (1990).
 Benign Prostate Hypertrophy (BPH) is a common condition in men over the age
 of 50, and occurs in the majority of men over the age of 80. BPH and
 prostate cancer may occur simultaneously in a subject.
 It is estimated that in 1997 breast cancer will account for 30 percent of
 new cancer cases, with about 180,200 new cases diagnosed. Parker et al. CA
 Cancer J. Clin. 47:5 (1997). Diagnosis of breast cancer typically depends
 on physical examination of the breast and/or routine mammography, with
 subsequent ultrasound and/or biopsy as indicated.
 Due to the known association of PSA with diseases such as BPH and prostate
 and breast cancers, and the high incidence of such diseases, it would be
 advantageous to develop effective and convenient methods of screening for
 the presence of cancer.
 SUMMARY OF THE INVENTION
 A first aspect of the present invention is a method of screening a subject
 for secreted Prostate Specific Antigen (PSA), comprising obtaining a
 biological sample from a subject and detecting the amount of PSA
 activation peptide in the sample.
 A further aspect of the present invention is a method of screening a
 subject for 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 comparing the amount of peptide detected to a
 pre-determined standard. Detection of a level of peptide greater than that
 of the standard indicates the presence of the condition for which the
 screening is being carried out.
 A further aspect of the present invention is a method of screening a
 subject for prostate disease, comprising obtaining a urine sample from the
 subject, and detecting the presence of PSA activation peptide in the
 sample. The presence of PSA activation peptide in the sample is indicative
 of prostate disease.
 A further aspect of the present invention is an immunoassay method 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 detecting the binding of
 antibody to peptides present in the sample. Binding of antibodies
 indicates the presence of peptides of SEQ ID NO:1 in the sample.
 A further aspect of the present invention is a method of screening for
 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 PSA activation peptide in the sample. The
 presence of the peptide is indicative of prostate cancer in the subject.
 A further 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.

DETAILED DESCRIPTION OF THE INVENTION
 Measurement of the level of serum prostate-specific antigen (PSA) in the
 blood or serum is an accepted screening test for prostate cancer. This
 procedure has been utilized both for initial diagnosis and to monitor the
 effectiveness of therapy. Babian et al., Cancer, 69:1195 (1992); Partin
 and Oesterling, J. Urol., 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 36 kDa. The predicted protein sequence is a 237 amino acid
 protein with a calculated molecular mass of 26.1 kDa. The difference
 between the observed and calculated mass is attributed to one N-linked
 oligosaccharide attached to As.sub.45. Belanger et al., Prostate 27:187
 (1995). Analysis of the amino acid sequence indicates that PSA is a member
 of the chymotrypsin family of serine proteases. Serine proteases are
 synthesized as precursors, and activated by the removal of an N-terminal
 activation peptide. In certain serine proteases (e.g., human neutrophil
 elastase and cathepsin G), the activation peptide is removed before
 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 recombinant pro-PSA seems to support
 the hypothesis that PSA is processed similar to other extracellular
 proteases. Takayama et al., J. Biol. Chem. 272:21582 (1997). See also
 Gauthier et al., Biochim. Biophys. Acta 1174:207 (1993); Lilja, J. Clin.
 Invest. 76:1899 (1985); Lundwall and Lilja, FEBS Lett. 214:317 (1987);
 Schaller et al., Eur. J. Biochem. 170:111 (1987).
 In blood, PSA is found in three forms: (i) free-PSA; (ii)
 PSA.cndot..alpha.1-antichymotrypsin complexes (PSA.cndot..alpha.1-ACT) and
 (iii) PSA.cndot..alpha.2-macroglobulin complexes (PSA.cndot..alpha.2M).
 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.cndot..alpha.1-ACT are immunodetectable by current commercial
 assays. PSA.cndot..alpha.2M complexes are not recognizable by the antisera
 because of 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, even though
 trace amounts of PSA complexes to intera-trypsin inhibitor (I.alpha.I) and
 .alpha.1-protease inhibitor (.alpha.1PI) have been reported. Stenman et
 al., Cancer Res. 51:222 (1991).
 Studies of the various PSA forms in serum suggest that the mean proportion
 of PSA.cndot..alpha.1-ACT is higher in patients with prostate cancer than
 in patients with benign prostate hypertrophy (BPH), although an overlap
 occurs 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.cndot..alpha.1-ACT/total-PSA may provide
 a way to discriminate prostate cancer and BPH. Leinonen et al., Clin.
 Chem. 39:2098 (1993). Lilja and coworkers reported that
 PSA.cndot..alpha.1-ACT is the major form of circulating PSA (Lilja et al.,
 Clin. Chem. 37:1618 (1991); Lilja et al., Cancer 70(1 Suppl):230 (1992));
 their further work demonstrated that the ratio of
 PSA.cndot..alpha.1-ACT/total-PSA was significantly higher in patients with
 prostate cancer than in patients with BPH (Christensson et al., J. Urol.
 150:100 (1993)).
 Current use of PSA testing is directed toward detecting the three major PSA
 forms in the blood (free-PSA, PSA.cndot..alpha.1-ACT, and
 PSA.cndot..alpha.2M), as well as complexes of PSA with other serine
 protease inhibitors including I.alpha.I and .alpha.1PI. However, the use
 of PSA as a screening or diagnostic test for the presence of prostate
 cancer suffers from several limitations. PSA interacts with several other
 proteins in the blood. These interactions affect the half-life of PSA in
 the blood and interfere (and may even prevent) detection. Additionally,
 PSA.cndot..alpha.2M complexes are not detected (Lilja et al., Clin. Chem.
 37:1618 (1991), Zhou et al., Clin. Chem. 39:2483 (1993)). Benign
 conditions may cause an elevated serum PSA level, resulting in unnecessary
 biopsies or additional testing; it is also true that some prostate cancers
 are associated with normal serum PSA concentrations.
 Further, as discussed herein, the rapid removal of PSA from the circulation
 in the early stages of disease is a heretofore unrecognized problem with
 conventional PSA testing. While not wishing to be held to a single
 hypothesis, the present inventors believe that where prostate cancer is
 associated with a normal serum PSA concentration, the plasma elimination
 mechanism of PSA has not yet been exhausted, so that secreted PSA is
 quickly removed from the bloodstream and a normal serum PSA level is
 maintained. This clearance rate is likely to depend on the overall health
 of the patient, including physical condition, body weight, and alcohol and
 tobacco consumption. These factors may affect the half-life of PSA forms
 in the blood but should not significantly affect the half-life of the
 activation peptide. As discussed herein, the removal of the activation
 peptide is a passive process not dependent 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 about 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
 detecting 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
 requiring additional treatment. Zarghami and Diamandis (Clin. Chem. 42:361
 (1996)) reported that, using PCR-based tests, PSA mRNA and protein were
 detected in breast tumor tissue.
 The present inventors have shown by sequence analysis of the secreted PSA
 molecule that PSA is secreted as an inactive pro-enzyme or zymogen, with
 an attached N-terminal activation peptide. Cultured human prostate cancer
 cells (LNCAP cells) were found to secrete pro-PSA containing a 7-residue
 N-terminal activation peptide of sequence Ala-Pro-Leu-Ile-Leu-Ser-Arg (SEQ
 ID NO:1) (see Example 2 herein). The present inventors have shown that the
 activation peptide is cleaved from pro-PSA outside of the cell, where it
 can be detected, rather than inside the cell where it would likely be
 degraded. The present inventors believe that the sequence of the PSA
 activation peptide will be highly conserved, however it is possible that
 variants of the PSA activation peptide of SEQ ID NO:1 may occur. PSA
 activation peptide variants arising by conservative amino acid
 substitution, or having substantial sequence similarity to SEQ ID NO:1,
 are also encompassed within the scope of the present invention.
 In addition, the present inventors have shown in an in vivo animal model
 that when a small amount of PSA is introduced into the blood stream it
 quickly reacts with specific protease inhibitors and is removed from blood
 circulation by binding to hepatocyte receptors and subsequent endocytosis
 (see Example 3 herein). While not wishing to be held to a single theory,
 the present inventors infer that in early stages of cancer PSA secretion
 may be limited and, due to the reaction of PSA with protease inhibitors,
 PSA may be rapidly eliminated from the blood stream and thus undetectable
 for diagnostic purposes. The present inventors surmise that as cancer
 progresses and increased levels of PSA are released into the blood stream,
 the plasma elimination mechanisms are overwhelmed and PSA complexes begin
 to accumulate in the blood (see FIG. 3), allowing detection of PSA in the
 blood for diagnostic purposes.
 The present inventors have discovered that (i) PSA is secreted as a
 proenzyme (pro-PSA) containing an N-terminal activation peptide; (ii) that
 PSA in the blood does not react with pro-PSA antisera and therefor has
 been activated; (iii) the plasma elimination kinetics of PSA suggest that
 PSA accumulates in the blood when the clearance mechanism has been
 saturated; (iv) plasma elimination of the activation peptide indicates
 filtration out of the blood by the kidney; and (v) the activation peptide
 is detectable in urine and in blood.
 The present inventors identified the sequence of the N-terminal activation
 peptide of PSA produced by cultured human prostate cancer cells (LNCAP
 cells) as Ala-Pro-Leu-Ile-Leu-Ser-Arg (SEQ ID NO:1). The present results
 indicate that the presence of the PSA activation peptide (e.g., peptide of
 SEQ ID NO:1) in urine or blood is a reliable indicator of secreted PSA in
 a subject. The activation peptide is cleaved from PSA during activation of
 pro-PSA and apparently does not interact with other proteins, but is
 cleared from the blood by simple renal filtration. The activation peptide
 is consequently easy to detect by assaying for its presence in the 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 of screening for cancers
 associated with increased levels of secreted PSA, including prostate
 cancers and breast cancers.
 Thus, the present inventors have determined that PSA activation peptide can
 be detected in biologic samples from subjects, and that the level of PSA
 activation peptide is an indicator of secreted PSA. Accordingly, detection
 of PSA activation peptide is indicative of PSA secretion. Detection and or
 quantitative measurement of PSA activation peptide is therefore useful in
 screening for or detecting diseases associated with increases in PSA. Such
 conditions include BPH, prostate cancer and breast cancer. In a preferred
 embodiment of the present invention, the condition for which the screening
 test is carried out is breast cancer, and the sample being screened is
 urine or blood. In a further preferred embodiment of the present
 invention, the condition for which the screening test is carried out is
 prostate cancer, and the sample being screened is urine or blood.
 As used herein, an "increased level" of PSA activation peptide refers to a
 level that is increased over a predetermined standard, or increased over
 the level in a control sample. The pre-determined standard may be based on
 the detection of PSA activation peptide in healthy subjects, and may be
 zero or undetectable. Alternatively, in testing a subject, the
 pre-determined standard may be based on an earlier test result of the same
 subject, i.e., the test results may be followed over time to detect
 changes in PSA activation peptide. PSA testing is known to be useful in
 detecting metastatic or persistent disease in patients following medical
 or surgical treatment of prostate cancer, where persistent elevation of
 (or increases in) PSA levels following treatment indicates recurrent or
 residual disease. The level of PSA activation peptide that is considered
 as an indicator of disease may differ among the target diseases for which
 the screening method is used; the level of PSA activation peptide may be
 measured as an amount per unit of test sample, or as a percentage of the
 total protein in a test sample. The diagnostic or indicator level of PSA
 activation peptide for a particular disease state may be determined using
 routine clinical testing methods known in the art. Any number of protocols
 can be used to develop data for use in performing the diagnostic methods
 of the present invention; the methods and guidelines for developing
 suitable study protocols are known to those in the art.
 The methods disclosed 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 present
 methods may be employed both to monitor subjects who have been previously
 diagnosed with the target condition, to monitor subjects undergoing
 treatment for the target condition, or to screen subjects who have not
 been previously diagnosed with the target condition, including
 asymptomatic subjects. Subjects include humans as well as mammalian
 veterinary subjects, and include both male and female subjects. The
 methods disclosed herein are particularly suited for screening for
 prostate cancer, and for aiding in the diagnosis and prognosis of prostate
 cancer.
 As used herein, methods of screening and diagnosis do not mean that the
 methods are 100% specific or sensitive in indicating the presence of the
 target disease state; rather, a positive screening or diagnostic test
 indicates that the subject is at an increased risk (compared to the
 general population) of being afflicted with the target condition. In a
 sampling of multiple subjects, positive test results will be correlated
 with the presence of the target condition. The specificity and sensitivity
 of the present methods may vary depending on the condition being screened
 or monitored, the biological sample being screened, the general health of
 the subject being screened, and other factors, as will be apparent to
 those skilled in the art.
 In a particular embodiment of the present invention, the subject has
 previously been diagnosed as having a disease associated with elevated PSA
 levels (such as prostate or breast cancer), and may have already undergone
 treatment for such disease. The present methods are suitable to monitor
 the recurrence or progression of the disease or the success of the
 treatment thereof; in such cases, the levels of PSA activation peptide in
 a subject may be compared over time.
 Prostate cancer is a well recognized disease entity. As used herein, the
 term prostate cancer includes any histological type of cancer arising from
 prostate tissue. The most common tumor arising in the prostate is
 adenocarcinoma. Adenoid cystic carcinomas, carcinosarcomas, and sarcomas,
 as well as other histological types of cancers, may also occur in the
 prostate.
 Breast cancer is a well recognized disease entity. As used herein, the term
 breast cancer includes any histologic type of cancer arising from breast
 tissue. Breast cancers most commonly arise from epithelium; other
 histologic types of mammary carcinoma have been described.
 Benign Prostate Hypertrophy (BPH) is a common condition in men over the age
 of 50, and occurs in the majority of men over the age of 80. Treatment of
 BPH includes drug therapy to decrease prostate volume and surgical
 resection of the prostate. Increased serum concentrations of PSA are
 reported in BPH.
 Samples taken from subjects for use in the methods disclosed herein are
 generally 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 body fluids that would contain the PSA activation
 peptide. In testing for prostate cancer, urine is a preferred test sample.
 Methods of obtaining samples to be tested will be carried out according to
 techniques known in the art, and may depend on the condition being
 screened for and the condition of the subject. Samples may undergo
 additional conventional preparation steps prior to the detection of the
 PSA activation peptide, as will be apparent to those skilled in the art.
 For example, samples may undergo the addition of preservatives,
 concentrating steps, filtration, etc.
 The levels of PSA activation peptide may be determined as an amount of
 peptide per volume of sample, or as a percentage of the total protein in
 the sample.
 Antibodies which may be used to carry out the present invention include
 antibodies which bind specifically to a peptide of SEQ ID NO:1 and
 fragments of such antibodies, which fragments bind specifically to a
 peptide of SEQ ID NO:1. Such antibodies and antibody fragments may be
 produced by a variety of techniques, as discussed 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 present methods may be
 obtained in accordance with known techniques, and may be monoclonal or
 polyclonal, and may be of any species of origin, including (for example)
 mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See,
 e.g., M. Walker et al., Molec. Immunol. 26:403 (1989). The antibodies may
 be recombinant monoclonal antibodies produced according to the methods
 disclosed in Reading, U.S. Pat. No. 4,474,893, or Cabilly et al., U.S.
 Pat. No. 4,816,567. The antibodies may also be chemically constructed by
 specific antibodies made according to the method disclosed 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').sub.2, and Fv
 fragments, and the 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 may be produced by immunizing a suitable animal (e.g., rabbit,
 goat, etc.) with the target antigen, collecting immune serum from the
 animal, and separating the polyclonal antibodies from the immune serum, in
 accordance with known procedures.
 Monoclonal antibodies used in the present methods 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. Monoclonal
 Fab fragments may be produced in Escherichia coli by recombinant
 techniques known to those skilled in the art. See, e.g., Huse, Science
 246:1275 (1989).
 The methods disclosed herein detect the presence of PSA activation peptide,
 which the present inventors have determined is indicative of the presence
 of activated PSA in the subject being tested. Any suitable method of
 detecting PSA activation peptide may be used, as would be apparent to one
 skilled in the art. Preferred detection methods are immunoassay formats,
 which may be homogeneous assays of heterogeneous assays. In a homogeneous
 assay the immunological reaction usually involves antibody against 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 detection of the
 extent of the immunological reaction are carried out in a homogeneous
 solution. 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 PSA activation peptide, and means for
 producing a detectable signal, as discussed above. The antibody is
 generally immobilized on a support (such as a bead, plate or slide) and
 contacted with the test sample in 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.
 Means for producing a detectable signal include the use of radioactive
 labels, fluorescent labels, enzyme labels, and so forth, as will be
 apparent to one 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 which will be
 useful for carrying out the methods of the present invention.
 Antibodies described herein may be conjugated to a solid support suitable
 for a diagnostic assay (e.g., beads, plates, slides or wells formed from
 materials such as latex or polystyrene) in accordance with known
 techniques. Antibodies may be conjugated to detectable elements such as
 radiolabels (e.g., .sup.35 S, .sup.125 I, .sup.131 I), enzyme labels
 (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent
 labels (e.g., fluorescein) in accordance with known techniques.
 Diagnostic kits for carrying out the methods of the present invention may
 be produced in a number of ways. In one embodiment, the diagnostic kit
 comprises (a) an antibody or antibody fragment that specifically binds to
 PSA activation peptide (e.g., peptide of SEQ ID NO:1) conjugated to a
 solid support and a second such antibody or antibody fragment conjugated
 to a detectable element. The kit may also include ancillary reagents such
 as buffering 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 background interference in a test, control reagents,
 apparatus for conducting 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
 conjugated to a detectable group. Ancillary agents as described above may
 likewise be included. The test kit may be packaged in any suitable manner,
 typically with all elements in a single container along with a sheet or
 printed instructions for carrying out the test.
 The step of detecting the presence of PSA in the blood or serum of the test
 subject may be carried out concurrently with the methods of the present
 invention, as a further indication of whether or not the subject is
 afflicted with the disease state being assessed. Various methods of
 detecting PSA are known in the art; see e.g., U.S. Pat. No. 5,672,480 to
 Dowell et al.; U.S. Pat. No. 5,658,730 to McGill et al.; U.S. Pat. No.
 5,654,161 to Tewari; U.S. Pat. No. 5,599,677 to Dowell et al.; and U.S.
 Pat. No. 5,501,983 to Lilja et al. (The disclosures of all U.S. Patent
 cited herein are intended to be incorporated herein in their entirety).
 The present methods may be used in conjunction with other diagnostic or
 screening tests designed to detect the target condition.
 Use of the phrase "substantial sequence homology" in the present
 specification and claims means that DNA, RNA or amino acid sequences which
 have slight and non-consequential sequence variations from the actual
 sequences disclosed and claimed herein are considered to be equivalent to
 the sequences of the present invention. In this regard, `slight and
 non-consequential sequence variations` mean that `homologous` sequences
 (i.e., the sequences that have substantial sequence similarity with the
 DNA, RNA, or proteins disclosed and claimed herein) will be functionally
 equivalent to the sequences disclosed and claimed in the present
 invention. Functionally equivalent sequences will function in
 substantially the same manner to produce substantially the same
 compositions as the nucleic acid and amino acid compositions disclosed and
 claimed herein.
 Use of the phrase "isolated" in the present specification and claims means
 that DNA, RNA, polypeptides or proteins have been separated from their in
 vivo cellular environments through the efforts of human beings.
 Sequences having "substantial sequence similarity" refer to nucleotide
 sequences that share at least about 90% identity with invention nucleic
 acids; and amino acid sequences that typically share at least about 70%,
 80%, 85%, 90% or even 95% amino acid identity with invention polypeptides.
 It is recognized, however, that polypeptides or nucleic acids containing
 less than the above-described levels of similarity arising as splice
 variants or generated by conservative amino acid substitutions, or by
 substitution of degenerate codons, are also encompassed within the scope
 of the present invention.
 The present invention is explained below in the Examples set forth below.
 EXAMPLE 1
 Materials and Methods
 Reagents--ECL Western blotting detection reagents were from Amersham
 (Arlington Heights, Ill.). RPMI Medium 1640, RPMI 1640 select amine kit,
 Dulbecco's phosphate buffered saline, Earls's balanced salt solution, and
 penicillin Streptomycin were from Gibco (Grand Island, N.Y.). Epidermal
 growth factor, L-glutamine were from Sigma (St. Louis, Mo.). PSA anti sera
 was from DAKO Corporation (Carpinteria, Calif.). Human metastatic prostate
 adenocarcinoma (LNCaP) cells were obtained from American Type Culture
 Collection (Rockville, Md.). Radiochemicals were from (DuPont/NEN).
 .alpha..sub.1 -ACT was purified as previously described (Salveson et al.,
 J. Biol. Chem. 260:2432 (1985)). Urine samples were collected from the
 Duke University Medical Center Urology Clinic. Human prostatic tissues
 (normal, benign, hypertrophic, malignant) were obtained from Duke
 University Medical Center. Histology of each tissue was confirmed by a
 pathologist.
 Purification of PSA--All steps were performed at 4.degree. C. Approximately
 100 g of prostate tissue was homogenized (Vitris Tempest) in 300 ml of
 0.05 M Tris-Cl, 0.1 M NaCl, 0.01 M EDTA, pH 7.4. The homogenate was
 filtered through several layers of cheesecloth and cleared by
 centrifugation. The supernatant was subsequently digested with 0.1 mg/ml
 RNAse, 0.2 mg/ml DNAse and 0.005 M MgCl for 4 hours at 4.degree. C.
 Following a 4 hour incubation the supernatant was dialyzed overnight
 against 0.01 M HEPES, pH 8. The next day the sample was clarified by
 centrifugation and applied to a Q-Sepharose FF (Pharmacia) column
 (2.5.times.20 cm) equilibrated in 0.01 M HEPES, pH 8. The charged column
 was washed extensively in equilibration buffer and then developed with a
 linear gradient (total volume of 2 liter) from 0 M NaCl to 0.4M NaCl.
 Fractions of 4 ml were collected and tested for PSA by western blotting.
 The active fractions were pooled and concentrated by ultrafiltration
 (Amicon) and applied to a S-200 HR gelfiltration (Pharmacia) column
 (2.5.times.150 cm) equilibrated in 50 mM HEPES, 150 mM NaCl. Fractions of
 4 ml were collected and assayed for PSA by western blotting. The PSA
 containing fractions were pooled and dialyzed into 10 mM HEPES, pH 8 and
 separated on a MONO-Q 5/5 HR (Pharmacia) connected to a Pharmacia FPLC
 system. The column was equilibrated in 10 mM HEPES and developed using a
 linear gradient from 0 M NaCl to 400 mM NaCl.
 Preparation of antisera--A peptide, Ala-Pro-Leu-Ile-Leu-Ser-Arg-Cys (SEQ ID
 NO:2), corresponding to an N-terminal activation peptide of PSA, was
 synthesized (Bio Synthesis, Lewisville, Tex.). The Cys is not part of the
 activation peptide but was added to facilitate coupling to ovalbumin by
 using m-Maleimidobenzoic acid-N-Hydroxysuccinimide Ester (Liu et al.,
 Biochemistry 18:690 (1979); Kitagawa and Aikawa, J. Biochem. 79:233
 (1976)). Another peptide, Ala-Pro-Leu-Ile-Leu-Ser-Arg (SEQ ID NO:1) was
 synthesized using the MAP (Multiple Antigen Peptide) technique (Bio
 Synthesis, Lewisville, Tex.). Rabbit antisera to the activation
 peptide-ovalburin conjugates and the MAP activation peptide were raised in
 rabbits using a standard protocol known in the art.
 Metabolic Labeling and Pulse-Chase Analysis--Human metastatic prostate
 adenocarcinoma (LNCaP) cells were maintained in RPMI Medium 1640 (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% CO.sub.2. For standard biosynthetic radiolabeling, cells were grown in
 50 mm tissue culture plates until 80% confluent. The cells were washed
 twice with Earls's balanced salt solution, and then incubated for 30
 minutes in RPMI without fetal bovine serum and lacking the amino acids
 intended for subsequent use in metabolic labeling. After the addition of
 [.sup.35 S] Met, the cells were incubated for 5 minutes (pulse period). If
 the immunoprecipitated proteins were destined for radiosequence analysis
 [.sup.35 S] Met was added together with [.sup.3 H] Ile, [.sup.3 H] Leu or
 [.sup.3 H] Val. At the end of the labeling period, cells were promptly
 rinsed twice with serum free RPMI and chased with "cold" complete medium
 for various periods of time.
 Lysis and Immunoprecipitation--The conditioned medium was collected and
 frozen. Cell lysates were prepared by three rapid freeze-thaw cycles in
 high salt buffer containing 0.5% Triton X-100 and a proteinase inhibitor
 cocktail. Prior to immunoprecipitation, the samples of lysates and
 conditioned medium were cleared by the addition of a pre-immune serum
 followed by the addition of protein-G Sepharose 4 FF (Pharmacia). The
 supernatants were incubated overnight with the relevant specific
 antiserum. The next day protein-G Sepharose 4 FF was added and
 immunoprecipitates were collected by gentle centrifugation. The
 immunoprecipitates were then washed several times and bound proteins were
 released from the protein G Sepharose 4 FF by boiling in SDS sample buffer
 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. Proteins and peptides were hydrolyzed in 6N HCl and the
 composition was determined using a Beckman 6300 amino acid analyzer. Both
 instruments were operated as recommended by the manufacturer.
 Radiosequence Analysis--These analysis were performed as previously
 described (Salvesen and Enghild, Biochemistry 29:5304 (1990); Thogersen
 and Enghild, J. Biol. Chem. 270:18700 (1995)). Briefly, following
 immunoprecipitation and SDS-PAGE the [.sup.35 S] and [.sup.3 H] double
 labeled proteins were electrotransferred to immobilon membranes
 (Matsudaira, J. Biol. Chem. 262:10035 (1987)). The proteins were
 identified by autoradiography and bands of interest were excised and
 analyzed by automated Edman degradation. The anilinothiazolinone (ATZ)
 amino acids released after each cycle were collected and counted for
 [.sup.35 S] and [.sup.3 H] radioactivity. In the experiments destined for
 radiosequence analysis the metabolic labeling was performed using
 appropriate radioactive amino acids expected within the first 20
 N-terminal residues of the mature proteins. Subsequent radiosequence
 analysis of the bands and release of radioactive ATZ-amino acid in the
 anticipated cycle of Edman degradation provided identification of the
 protein band.
 Polyacrylamide Gel Electrophoresis--The supernatants from SDS-treated
 immunoprecipitates were recovered by centrifugation and run in sodium
 dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in 5-15%
 gradient gels (Bury, J. Chromatography 213:491 (1981)). The gels were
 stained, destained, dried, and subjected to imaging on a PhosphorImager
 (Molecular Dynamics 410A). Immunoprecipitates for radiosequence analysis
 were transferred to PROBLOTT.TM. membranes. Following electrophoresis the
 PROBLOTT.TM. membranes were dried and exposed directly to X-ray film
 overnight at -70.degree. C.
 Protein and peptide radiolabeling--Proteins were radioiodinated using
 N-chloro-benzenesulfonamide (Markwell, Anal. Biochem. 125:427 (1982))
 immobilized on polystyrene beads according to the instructions provided by
 the manufacturer (IODO-BEADS.RTM. Pierce). The activation peptide was
 labeled using .sup.125 I-Bolton-Hunter reagent (Bolton and Hunter,
 Biochem. J. 133:529 (1973)) as suggested by the manufacturer (DuPont/NEN).
 Plasma Elimination Studies--This procedure has been described in detail
 elsewhere. 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). In brief, approximately 1.0 .mu.g of radioiodinated
 protein or peptide was injected into the lateral tail vein of CD-1 mice.
 Blood samples of 25 .mu.l were collected at timed intervals via
 retroorbital puncture. The initial time point, taken 5-10 seconds after
 injection, was considered to represent 100% radioactivity in circulation.
 Each preparation was studied at least in triplicate. Following the plasma
 elimination experiments, the mice were perfused and the organs removed and
 counted in a .gamma.-counter.
 Western blotting--Membranes were developed using the ECL Western blotting
 kit from Amersham.TM.. Briefly, following transfer to PVDF membranes the
 membranes were blocked for 1 hour in 20 mM Tris-Cl, 137 mM NaCl, pH 7.6
 containing 0.1% Tween (TBS-T buffer) and 5% of the supplied blocking
 reagents. The membrane was washed in TBS-T buffer before the primary
 antibody was added (1/2000 dilution). Following a 1 hour incubation the
 membrane was washed in TBS-T buffer and the horse radish peroxidase
 labeled second antibody was added (1/20,000 dilution). The membranes were
 incubated for 1 hour and washed with TBS-T buffer, and developed using the
 supplied reagent.
 Enzyme-Linked Immunosorbent Assay (ELISA)--Costar 96-well RIA/EIA plates
 (Costar, Cambridge, Mass.) were incubated for 2 hours at 23.degree. C.
 with increasing amounts of sample to be tested, in a total volume of 50
 .mu.l in PBS, pH 7.3. Wells containing known concentrations of activation
 peptide and .beta..sub.2 -microglobulin were simultaneously analyzed for
 comparison. Coated plates were washed and blocked with PBS containing 5%
 CARNATION.RTM. non-fat dry milk and 0.05% Tween 80 (blocking buffer) for 2
 hours at 23.degree. C. Plates were then incubated with 100 .mu.l of
 activation peptide antisera diluted in blocking buffer overnight at
 4.degree. C. The next day the plates were briefly washed and incubated for
 2 hours using 100 .mu.l (1/2000 dilution) of alkaline phosphatase-coupled
 anti-(rabbit IgG). After washing with blocking buffer and PBS, the
 substrate p-nitrophenyl phosphate (1 mg/ml in 0.1 M glycine, 1 mM
 MgCl.sub.2, 1 mM ZnCI.sub.2, pH 10.4) was added. Alkaline phosphatase
 activity was followed kinetically at 37.degree. C. using a THERMOmax
 microplate reader (Molecular Devices, Menlo Park, Calif.).
 EXAMPLE 2
 Results: Biosynthesis and Processing of PSA
 The cDNA encoding PSA suggests an N-terminal 7 amino acid activation
 peptide. Lundwall and Lilja, FEBS Lett. 214:317 (1987). However, the
 putative activation peptide had not previously been detected in 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). It was consequently not
 clear whether the activation peptide was removed intracellularly before
 the secretion as seen with granule 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 seen with most serine proteases. FIG. 1 provides a schematic
 diagram of PSA, where the arrows indicate the positions of the expected
 proteolytic cleavage sites. The solid bar represents the mature active PSA
 (sequence not shown), and a.p. indicates the activation peptide.
 To investigate the post-translational processing of PSA, the present
 inventors utilized biosynthetic radiolabeling and radiosequencing
 techniques (described in Example 1) to characterize both intracellular and
 secreted PSA. These analyses were performed using biosynthetically
 radiolabelled LNCaP cells and a polyclonal PSA antibody (FIG. 2A) and a
 peptide antisera specific against the activation peptide (FIG. 2B). The
 cells were radiolabelled using a pulse-chase protocol as described above,
 and the lysates and medium were collected and treated with specific
 antisera to the whole PSA and to the activation peptide. The samples were
 analyzed by reduced SDS-PAGE. Following electrophoresis, the gel was dried
 and subjected to imaging on a Phosphorlmager. As shown, the cells produce
 and secrete pro-PSA.
 The present results indicate that PSA does not undergo any N-terminal
 processing event. This was confirmed by radiosequence analysis of both
 intracellular and secreted PSA (data not shown). These studies establish
 that PSA is secreted a s an inactive pro-enzyme containing a 7-residue
 N-terminal activation peptide of sequence Ala-Pro-Leu-Ile-Leu-Ser-Arg-
 (SEQ ID NO:1). The activation of pro-PSA is an extracellular event.
 EXAMPLE 3
 Elimination of PSA.cndot..alpha.1-ACT Complexes and Tissue Distribution of
 .alpha.1-ACT in Mouse
 The clearance rate of .sup.125 I-.alpha..sub.1 -ACT was compared to the
 clearance rate of .sup.125 I-.alpha..sub.1 -ACT.cndot.PSA complexes in
 mice, using techniques as described above. As shown in FIG. 3, comparing
 .alpha..sub.1 -ACT injected alone (solid circles) and .alpha..sub.1 -ACT
 in complex with PSA (open circles) shows that the half life of the
 PSA.cndot..alpha.1-ACT complex was significantly reduced compared to
 native .alpha.1-ACT. To mimic a situation in which more PSA is sereted,
 .sup.125 I-PSA.cndot..alpha.1-ACT complex was injected with a 500-fold
 (X), 1000-fold (solid squares) and 2000-fold (open squares) excess of
 `cold` PSA.cndot..alpha.1-ACT complexes. These experiments showed that
 PSA.cndot..alpha.1-ACT complex is initially removed from the blood, and
 that as the level of PSA.cndot..alpha.1-ACT complex increases in the blood
 the clearance mechanism becomes saturated and PSA.cndot..alpha.1-ACT
 complexes begin to accumulate in the blood.
 Thestuie shwed that the .sup.125 I-.alpha..sub.1 -ACT.cndot.PSA complexes
 were removed from the mouse circulation with a half-life of approximately
 20 minutes; the half life of .sup.125 I-.alpha..sub.1 -ACT was estimated
 to be several hours. To investigate if the accumulation of .alpha..sub.1
 -ACT.cndot.PSA complexes in the blood were caused by a saturation of the
 clearance mechanism, .sup.125 I-.alpha..sub.1 -ACT.cndot.PSA complexes
 were coinjected with a large excess of unlabeled .alpha..sub.1
 -ACT.cndot.PSA complexes. The half life increased from approximately 20
 minutes to several hours. These experiments show that clearance rate is
 significantly affected by the level of .alpha..sub.1 -ACT.cndot.PSA
 complex in the blood stream and indicate that the accumulation of
 .alpha..sub.1 -ACT.cndot.PSA in the blood is caused by a saturation of the
 clearance mechanism.
 Plasma elimination of the activation peptide was studied by injecting
 .sup.125 I labeled activation peptide (Ala-Pro-Leu-Ile-Leu-Ser-Arg; SEQ ID
 NO:1) into the lateral tail vein of a mouse. Plasma elimination of
 .sup.125 I labeled activation peptide was followed for 1 hour (solid
 circles, FIG. 4); the half-life of the peptide was less than 2 minutes.
 The .sup.125 I labeled activation peptide was also injected with a
 1000-fold (open circles) and 2000-fold (X) excess of un-labelled
 activation peptide. The clearance rate was not significantly affected by
 the level of activation peptide in the blood stream.
 Following the plasma elimination experiments the organs of the test mice
 were examined for radioactivity using a .gamma.-counter. The predominance
 of .alpha..sub.1 -ACT in bladder and kidney indicates that the peptide is
 removed from the blood stream by renal filtration (FIG. 5).
 The above results indicate that at initial levels, PSA.cndot..alpha.1-ACT
 complexes are cleared rapidly from the bloodstream of mammals; however, as
 more PSA.cndot..alpha.1-ACT complexes are introduced into the bloodstream,
 the clearance mechanism is saturated, leading to an excess of
 PSA.cndot..alpha.1-ACT complexes in the bloodstream.
 EXAMPLE 4
 Detection of PSA Activation Peptide in Biological Samples
 The results provided above indicate that the PSA activation peptide that is
 cleaved from PSA is cleared from the bloodstream by renal filtration, and
 is present in urine and in the blood. To test whether PSA activation
 peptide is measurable in the urine or serum of patients with benign
 prostatic hypertrophy or prostate cancer, the activation peptide of SEQ ID
 NO:1 was synthesized and a polyclonal peptide antisera was prepared in
 rabbits. The specificity of the antisera was verified by ELISA against the
 synthetic activation peptide, which was provided in amounts ranging from
 0.01-5.0 ng of peptide (FIG. 6A; increasing concentration of activation
 peptide from dot 1 to dot 9). The antisera produced a dose-dependent
 sensitive reaction.
 Serum and urine samples from control patients (no prostate disease) and
 patients with prostate cancer were obtained and tested for the presence of
 the activation peptide as discussed above. The activation peptide was
 detected in the urine (FIG. 6B) and serum (FIG. 6C) of the cancer
 patients, but not in the urine (FIG. 6D) or serum (FIG. 6E) of controls.
 These results indicate that 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
 PSA Activation peptide containing an added C-terminal Cys residue
 (Ala-Pro-Leu-Ile-Leu-Ser-Arg-Cys; SEQ ID NO:2) was biotinylated using
 N-(6-[biotinamido])hexyl)-3'-(2'-pyridyldithio)propionamide(Pierce). This
 reagent can be used in a standard ELISA assay as follows:
 1) coat a 96-well ELISA plate with anti-activation peptide antiserum and
 block residual binding sites (PBS containing 0.05% Tween 20 and 0.25%
 bovine serum albumin (BSA));
 2) add a fixed concentration of biotinylated PSA activation peptide and
 increasing amounts of sample (e.g., urine, blood, blood serum) to a series
 of wells,
 3) wash the wells before adding avidin and biotinylated horseradish
 peroxidase;
 4) after incubation, wash the wells and detect residual biotinylated PSA
 activation peptide in a plate reader at 450 nm using the horseradish
 peroxidase substrate TURBO 3,3',5,5'-Tetramethyl Benzidine (TMB)(Pierce).
 The foregoing examples are illustrative of the present invention, and are
 not to be construed as limiting thereof. The invention is described by the
 following claims, with equivalents of the claims to be included therein.
 SEQUENCE LISTING
 &lt;100&gt; GENERAL INFORMATION:
 &lt;160&gt; NUMBER OF SEQ ID NOS: 2
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 1
 &lt;211&gt; LENGTH: 7
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Homo sapiens
 &lt;400&gt; SEQUENCE: 1
 Ala Pro Leu Ile Leu Ser Arg
 1 5
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 2
 &lt;211&gt; LENGTH: 8
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Homo sapiens
 &lt;400&gt; SEQUENCE: 2
 Ala Pro Leu Ile Leu Ser Arg Cys
 1 5