Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-04-30
2002-07-23
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007100, C435S007200, C435S960000, C435S975000, C436S063000, C436S064000, C436S811000, C436S813000, C424S009100
Reexamination Certificate
active
06423503
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates generally to prostate-specific antigen (PSA) and specifically to different forms of PSA and their association with prostate cancer and benign prostate disease.
2. Description of the Prior Art
Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
The measurement of serum PSA is a widely used marker for the early detection of human prostate cancer (1-3). Elevated levels of PSA in the blood are symptomatic of prostate disease, which is primarily manifested as either benign prostate hyperplasia (BPH) or prostate cancer (PCa). However, in the range 4-10 ng PSA per ml of serum it is difficult to distinguish BPH from PCa without additional tests such as digital rectal exam and prostate needle biopsy.
Recently, it has been demonstrated that the level of free or non-complexed PSA in serum can improve the discrimination of PCa from BPH (4-6). An elevated ratio of free PSA to total PSA is more highly correlated with BPH. The reasons for the presence of free PSA in serum has therefore become the subject of intensive investigation.
It is generally accepted that the free PSA in serum is enzymatically inactive. PSA is a serine protease capable of complex formation with serum protease inhibitors. Human serum contains high levels of &agr;
1
antichymotrypsin (ACT) and &agr;
2
macroglobulin, both of which have been shown to complex with PSA (7). The majority of PSA in serum that can be detected by immunoassay is in a complex with ACT (4; 6). From 70-95% of the PSA in serum is in a complex with ACT. Studies with PSA purified from seminal plasma have shown that about 30% of the PSA does not form a complex with ACT. This fraction of PSA contains an internal peptide bond cleavage at Lys145 which renders it inactive (4; 6; 7). A more detailed characterization of the inactive forms of PSA from seminal plasma revealed PSA clipped at both Lys145 and Arg85, in addition to a fraction of PSA which was not clipped but which did not form a complex with ACT (8).
The analysis of PSA from sources other than seminal plasma has been more limited. PSA was isolated from BPH nodules (9), where it was found to contain more enzymatically inactive PSA than seminal plasma PSA and to contain additional new clips after Ile1, His54, Phe57 and Lys146. The proenzyme form of PSA (pPSA) has also been reported in the serum of PCa patients (10). Both clipped and proenzyme forms of PSA could therefore be components in the serum of patients with prostate disease.
However, no one has studied the source of variable forms of PSA in serum. The association of both the clipped and proenzyme forms of PSA with different prostate diseases is unknown. Therefore, a need exists for studying various forms of PSA and their association with different prostate tissues and diseases. A need also exists for developing a method to distinguish BPH from PCa.
SUMMARY OF THE INVENTION
It is an object of the present invention to determine the source of variable forms of PSA in serum. It is also an object of the present invention to study any associations of different forms of PSA with different prostate tissue or prostate diseases. It is a further object of the present invention to provide a sensitive method for distinguishing BPH from prostate cancer.
These and other objects and advantages are achieved in the present invention by focusing the study on prostate tissues as the source of variable forms of PSA in serum, since PSA is thought to result from retrograde release of PSA from the prostate into the serum (11). In order to determine what molecular forms of PSA are present in the prostate, the present invention has examined three different types of prostate tissue: 1) non-cancerous peripheral zone tissue (PZ-N); 2) cancerous peripheral zone tissue containing at least 80% tumor (PZ-C); and 3) non-cancerous transition zone tissue (TZ). It is the TZ which becomes hyperplastic in patients with BPH. In contrast, most cancers are found in the peripheral zone (PZ).
From these studies, the present invention has identified two subsets of free PSA which are differentially elevated in prostate TZ and PZ. It is believed that the measurement of these free forms of PSA may help distinguish BPH and PCa in patients with prostate disease.
Accordingly, one aspect of the present invention provides a method for determining different forms of prostate specific antigen (PSA) contained in a sample comprising:
(a) determining the amount of proPSA contained in the sample;
(b) determining the amount of BPSA in the sample; and
(c) mathematically combining the results of step (a) and step (b).
Another aspect of the present invention provides a diagnostic method for determining the presence of BPH or prostate cancer in a sample comprising the steps of:
(a) providing a first agent that specifically binds to proPSA;
(b) providing a second agent that specifically binds to BPSA;
(c) contacting the first agent and the second agent with the sample under a condition that allows the formation of a first binary complex comprising the first agent and the proPSA and a second binary complex comprising the second agent and the BPSA;
(d) detecting or determining the presence or amount of the first and second complexes;
(e) mathematically combining the amount of the first and second complexes, or the amount of proPSA and the amount of BPSA; and
(f) relating the combination to the presence of BPH or prostate cancer in the sample.
A further aspect of the present invention provides a diagnostic kit for determining the presence of BPH or prostate cancer in a sample comprising:
(a) a known amount of a first agent which specifically binds to a proPSA; and
(b) a known amount of a second agent which specifically binds to a BPSA,
wherein the first and the second agents, respectively, comprise a detectable label or binds to a detectable label.
The invention is defined in its fullest scope in the appended claims and is described below in its preferred embodiments.
REFERENCES:
patent: 5501983 (1996-03-01), Lilja et al.
patent: 5858685 (1999-01-01), Stamey et al.
patent: 6300088 (2001-10-01), Enghild et al.
Christensson, Anders et al., “Enzymatic activity of prostate-specific antigen and its reactions with Extracellular serine proteinase inhibitors”, Eur. J. Biochem. 194, 755-763, (1990).
Watt, Kenneth, “Human prostate-specific antigen: Structural and functional similarity with serine Proteases”, Proc. Natl. Acad. Sci. USA, vol. 83, pp. 3166-3170 May 1986.
Chen, Zuxiong, et al., “Prostate Specific Antigen In Benign Prostatic Hyperplasia: Purification And Characterization”, The Journ. Of Urology, vol. 157, 2166-2170, Jun. 1997.
Chu, Larry F., et al., “Different Molecular Forms Of Uncomplexed Prostate Specific Antigen (PSA) Show Similar Immunoreactivities”, The Journ. Of Urology, vol. 161, 2009-2012, Jun., 1999.
Hilz, Helmuth et al., “Molecular Heterogeneity of Free PSA in Sera of Patients with Benign and Malignant Prostate Tumors”, Eur. Urol. 1999; 36:286-292.
Mikolajczyk Stephen D.
Rittenhouse Harry G.
Slawin Kevin
Wang Tang Jang
Wolfert Robert L.
Caputa Anthony C.
Hill D. David
Hogan & Hartson LLP
Hybritech Incorporated
May William H.
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