Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
1999-03-12
2001-09-04
Caputa, Anthony C. (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C530S350000, C530S828000, C435S007100, C436S063000, C436S064000
Reexamination Certificate
active
06284873
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates generally to the prostate and specifically to a novel complex of hK2 and PI-6 formed in the prostate, and methods of using the novel complex.
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.
Three members of the human kallikrein family have been identified so far, designated hK1, hK2 and hK3 (1). All are serine proteases with high sequence identity. Two of these kallikreins, hK2 and hK3, are found almost exclusively in the prostate (extensively reviewed in (2). hK3, known more commonly as prostate-specific antigen, PSA, is a widely used serum marker for prostate. More recently, hK2 has become the focus of investigations into its possible role as a prostate cancer marker, as well as possible roles in prostate cancer biology (2).
hK2 is similar to PSA in many respects such as prostate tissue localization (3; 4), 80% sequence identity (5; 6), and regulation by androgens (7; 8). From a biochemical perspective, hK2 is different from PSA in that it shows a strong trypsin-like activity while PSA has weak chymotrypsin-like activity. However, unlike true kallikreins, hK2 shows little or no kininogenase-like activity (9; 10) and so does not appear to function primarily as a prostatically-expressed kininogenase.
The physiological roles for hK2 have not been established, though several activities have been described. hK2 has been shown to activate the zymogen form of PSA (pPSA) (11-13), and the zymogen of hK2 (autoactivation) (14). In this respect hK2 is unique from the other human tissue kallikreins, hK1 and PSA. The activation of pPSA by hK2 is particularly interesting since it suggests a possible physiological role for hK2 in the regulation of PSA activity.
Immunohistochemical studies using hK2-specific monoclonal antibodies have shown hK2 to be more highly expressed in prostate carcinoma than in normal tissues (15). This is the inverse of PSA which tends to be lower in more poorly differentiated cancer epithelium than in normal tissues.
hK2 has been shown to activate urokinase-type plasminogen activator (uPA) by cleavage of the single-chain uPA at Lys
158
to generate the two-chain active form of uPA (12; 16). It has been recently reported that hK2 rapidly complexes with PAI-1 in vitro and that hK2 also inactivates about 6 moles of PAI-1 during complex formation (17). It is therefore possible that the elevated levels of hK2 in prostate cancer play a biological role either by the activation of urokinase or by the inactivation of PAI-1, the primary inhibitor of urokinase.
Physiologically hK2 has been identified as a complex with ACT in serum (18)and PCI in seminal plasma (19). However, it was not previously known whether hK2 forms any tissue specific complex in the prostate. The physiological and biological role of hK2 in the prostate was also not known. Therefore, a need exists to study the role of hK2 in the prostate, particularly in prostate cancer.
SUMMARY OF THE INVENTION
The present invention is based on the discovery of a complex in prostate tissue extracts consisting of hK2 and a serine protease inhibitor known as protease inhibitor-6 (P-6). PI-6 was first reported in placental tissue where it was called placental thrombin inhibitor due to its ability to form in vitro complex with thrombin (20). It has also been called CAP, cytoplasmic antiprotease, since it has been shown to be cytoplasmically localized (21). PI-6 is expressed in epithelial and endothelial cells and has been described in a number of human tissues and cells including kidney, heart, skeletal muscle and platelets (22-24). It has not been reported in the prostate. In all cases so far PI-6 appears to be cytoplasmically localized.
The discovery of hK2-PI6 complex in the prostate is unique from the previous reports of hK2-ACT and hK2-PCI in at least two major respects: 1) PI-6 itself is a novel and relatively newly described member of the serine protease inhibitor family which has not been reported in prostate tissues; and 2) PI-6 is thought to be intracellular, which suggests either a novel pathway for the formation with the putatively extracellular hK2, or an altered pathway, possibly due to the oncogenic process.
While not wanting to be bound by the theory, it is believed that the finding of prostatic PI-6 in complex with hK2 suggests that PI-6 may play certain roles in the prostate. PI-6 represents a tissue-specific inhibitor of hK2 which may have biological significant in the regulation of hK2, and may play a role in prostate cancer biology.
It is a discovery of the present invention that the hK2-PI6 complex is elevated in prostate tumor compared to benign or normal prostate tissue. It is also a discovery of the present invention that the level of PI-6 is elevated in prostate tumor. Therefore, both the complex and the level of hK2 or PI-6 alone may be a novel marker for prostate cancer.
Accordingly, one aspect of the present invention provides an isolated and substantially pure complex consisting of protease inhibitor-6 (PI-6) and human kallikrein 2 (hK2). The hK2 contained in the complex may be an intact hK2 or a fragment of hK2. The complex of the present invention exists at an elevated level in a prostate tumor tissue.
Another aspect of the present invention provides an antibody that is specifically immunoreactive with a complex consisting of protease inhibitor-6 (PI-6) and human kallikrein 2 (hK2). The antibody may be a polyclonal antibody or a monoclonal antibody.
A further aspect of the present invention provides a method of detecting or determining in a sample a complex consisting of protease inhibitor-6 (PI-6) and human kallikrein 2 (hK2). The method comprises the steps of.
(a) contacting an amount of an agent which specifically binds to the complex to be detected with the sample under a condition that allows the formation of a ternary complex comprising the agent and the hK2-PI6 complex; and
(b) detecting or determining the presence or amount of the ternary complex.
In preferred embodiments, the agent comprises an antibody. The sample to be detected may be a mammalian tissue sample, particularly a prostate tissue sample, or a sample of human physiological fluid including, but not limited, to serum, seminal plasma, urine, and blood.
Yet another aspect of the present invention provides an immunoassay for detecting hK2-PI6 complexes in a biological fluid sample. The immunoassay comprises the steps of:
(a) providing a first antibody that recognizes one epitope of the hK2-PI6 complex, and a second antibody that recognizes another epitope of the hK2-PI6 complex,
(b) contacting the first antibody with the sample under a condition that allows the first antibody to bind to the complex to form a first complex that comprises the first antibody and the hK2-PI6 complex,
(c) contacting the second antibody with the first complex under a condition that allows the second antibody to bind to the hK2-PI6 complex to form a second complex that comprises the first antibody, the second antibody and the hK2-PI6 complex, and
(d) detecting and determining the second complex as a measurement of the hK2-P16 complex contained in the sample.
According to embodiments of the present invention, the first and second antibodies are respectively an antibody that is specifically immunoreactive for the hK2-PI6 complex. Alternatively, one of the first antibody and second antibodies is an antibody against hK2, and another of the first and second antibodies is an antibody against PI-6.
A further aspect of the present invention provides a diagnostic kit for detecting or determining in a sample a complex consisting of PI-6 and hK2. The kit comprises a known amount of an agent which specifically binds to
Mikolajczyk Stephen D.
Saedi Mohammad S.
Caputa Anthony C.
Hampson Gary T.
Holleran Anne L.
Hybritech Incorporated
May William H.
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