JP2003527607A - Lysosomal pepstatin-insensitive proteolytic enzyme as a novel biomarker for breast cancer detection and diagnosis - Google Patents

Abstract

(57) Abstract: The present invention is newly discovered to be associated with breast cancer and is used as a novel biomarker for breast cancer including primary, non-primary, or metastatic breast tumors, neoplasms, and carcinomas Such describes diagnostic and prognostic assays for detecting the presence and activity of the lysosomal pepstatin-insensitive proteolytic enzyme CLN2p in vascular and tissue samples. CLN2p activity was found to be significantly elevated as compared to CLN2p levels in normal sample controls when measured in breast tissue from patients with primary breast carcinoma, whereby CLN2p activity in breast tumors was approximately 2- It was shown to be 7 times higher. The relatively high levels of CLN2p activity in these breast tumors are positively correlated with several known breast cancer biomarkers, such as cathepsin D, estrogen receptor and progesterone receptor. Thus, the present invention provides CLN2p as a novel biomarker for use in detecting, diagnosing and prognosing breast cancer.

Description

Detailed Description of the Invention

The work described herein is partly based on The National Institutes of Health.
tes of Health) grants NS 30147 and CA 58183.

This application claims the benefit of US Provisional Application No. 60 / 188,861 filed March 13, 2000.

FIELD OF THE INVENTION The present invention relates to biomarkers newly found to be associated with breast carcinomas and sensitive methods for detecting and diagnosing breast cancer in patients.

[0004] BACKGROUND lysosomal pepstatin insensitive protease invention (CLN2p) is the result found in the identification of genetic defects in fatal infantile late neural ceroid lipofuscin deposition disease is a pediatric neurodegenerative diseases (LINCL) , DE Sleat et al., "Classical infantile late-stage neuronal ceroid lipofuscinosis and mutations in a lysosomal protein with classical
late-infantile neuronal ceroid lipofuscinosis), Science, 277: 1802-180
5 (1997)). The gene encoding CLN2p is called CLN2. CLN2p has an optimum pH
It is an abundant lysosomal proteolytic enzyme with 3.5 and is extremely stable in frozen tissues or acidified homogenates (MA Junaid et al., “A novel assay for lysosomal pepstatin-insensitive proteolytic enzyme and its late infantile neurotype. A novel assay for lysosoma
l pepstatin-insensitive proteinase and its application for the diagnosis
of late-infantile neuronal ceroid lipofuscinosis) ", Clin. Chim. Acta
, 281: 169-176 (1999)).

Analysis of the substrate cleavage site has been shown that CLN2p cleaves a tripeptide from a peptide with a flexible amino terminus of varying length between 4 and 42 residues, tripeptidyl peptidase 1 (
EC3.4.14.9) (DJ Vines et al., `` Classical late infantile neuronal ceroid lipofuscinosis fibroblasts are deficient in lysosomal tripeptidyl peptidase I (Classical late infantile neuronal). ceroid lipofuscinosis fibr
oblasts are deficient in lysosomal tripeptidyl peptidase I), FEBS Lett
. 443: 131-135 (1999); MA Junaid et al., "Purification and characterization of the purification and characterization of bovine brain lysosomal pepstatin-insensitive proteolytic enzyme, gene product in human infantile late-stage neuronal ceroid lipofuscinosis." of bov
ine brain lysosomal pepstatin-insensitive proteinase, the gene product d
eficient in the human late-infantile neuronal ceroid lipofuscinosis) '',
J. Neurochem., 74: 287-294 (2000)).

In carcinoma metastasis, proteolytic enzymes, especially lysosomal acid proteolytic enzymes, are proteolytically degrading the basement membrane, which separates epithelial cells from stromal cells,
It is believed that it may mediate tumor invasion. Therefore, many laboratories
We are studying the importance of lysosomal enzymes such as as a prognostic marker in breast carcinoma (SM Thorpe et al., "The relationship between high molecular weight (Mr) 52,000 cathepsin D in primary human breast cancer and poor prognosis." (Association between high concentration
of Mr 52,000 chathepsin D and poor prognosis in primary human breast can
cer), Cancer Res., 49: 6008-6014 (1989); AK Tandon et al., "Cathepsin D and prognosis in breast cancer", N.
. Engl. J. Med., 322: 297-302 (1990)).

In some studies, elevated levels of cathepsin D have been reported to be predictive of breast cancer recurrence (JA Foekens et al., "Prognostic Signs of PS2 and Cathepsin D in 710 Human Primary Breast Tumors." Value: Prognostic value of PS2
and cathepsin D in 710 human primary breast tumors: multivariate analysi
S.), J. Clin. Oncol., 11: 899-908 (1993)). However, other researchers have not succeeded in confirming this finding (M. Radvin et al., "Catepsin D in Western blot and immunohistochemistry: no correlation with prognostic signs in induration-negative breast cancer ( Cathepsin D by Western blotting and immunohistochemistry
: failure to confirm correlations with prognosis in node-negative breast
Cancer) ", J. Clin. Oncol., 12: 467-474 (1994). Therefore, there is no consensus in the field regarding the significance and function of cathepsin D and its relationship with breast cancer, which may lead to other uses for the detection, diagnosis, monitoring and / or prognosis of breast cancer, including breast tumors and carcinomas. There remains a need to discover new and reliable markers and indicators for.

Breast cancer is the second leading cause of cancer death in women, according to the American Cancer Society (ACS). 1999
During the year, ACS estimates that there are more than 175,000 cases of invasive breast cancer, resulting in 43,700 deaths. In addition, ACS reports that early detection increases survival rates and treatment options. Current blood markers have been used to diagnose and monitor metastatic breast cancer, but not for early diagnosis of primary breast cancer (K
L. Cheung et al., Cancer Treatment Reviews, 26: 91-102 (2000)). Develop reliable breast cancer detection and diagnostic assays for early detection and involving novel breast cancer biomarkers, thus trying to save lives, improve existing therapies and provide indicators of breast cancer prognosis Is constantly needed.

CLN2p is an acidic lysosomal proteolytic enzyme that has been found to be nearly as abundant as cathepsin D in brain and other tissues. For example, human brain (grey matter) cathepsin D activity has been reported to be 1069.6 ± 54.4 nmole / hr / mg protein (DE Sleat et al., Biochem. J., 334: 547-551 (1998)). On the other hand, human brain CLN2 protease activity is reported to be 917 ± 43 nmole / hour / mg protein (MA Junaid and RK Pullarkat, NeuroSci. Lett., 264: 157-160.
(1999)). In addition, CLN2p activity is very high in actively cell-dividing tissues / organs such as spleen and testis. Due to its abundance, CLN2p may play a crucial role in metabolism. Table 1 presents exemplary data showing the distribution and activity of CLN2p in various rat tissues.

[0010]

[Table 1]

In some cancers, proteolytic enzymes are extra important because of their claimed role in the metastatic process. Disease-free or overall survival
Much effort has been expended in exploring the potential for increased proteolytic enzymes during metastasis as prognostic markers to predict overall survival. These studies rely on semi-quantitative immunoassays that measure levels of lysosomal acid proteases such as cathepsin D and cathepsin B (MK Schwartz, "Tissue cathepsins as tumor markers").
, Clin. Chim. Acta, 237: 67-78 (1995)). However, there are many limitations regarding the use of semi-quantitative immunoassays. For example, in the evaluation of estrogen / progesterone receptors by immunohistochemistry, only staining of the cell nuclei is considered positive. Unfortunately, the antigenicity of these receptors may not be preserved during the use of certain techniques for immobilization. Therefore, false negative results are possible.

The present inventors have shown that CLN2p acid proteolytic enzyme is present and active in tumors of breast cancer, especially in patients with primary breast cancer, and the amount of CLN2p activity is important for prognosis of breast cancer patients. It has been newly discovered that it can be correlated with other factors such as known estrogen receptor, epidermal growth factor receptor, and progesterone receptor levels. In addition, the present invention overcomes the limitations of the semi-quantitative assay method described above by using a quantitative measurement of CLN2p enzyme levels and / or enzyme activity levels in breast cancer, ie, in cancer cells and tumor cells of the breast. ing.

The present invention provides the initial recognition and demonstration of CLN2p as a diagnostic and / or prognostic marker for breast carcinomas, including breast tumors and cancers at various stages of progression. The present invention advantageously also provides the detection of CLN2p as a biomarker with breast cancer blood by use of the assay methods described herein. The assay according to the invention and its results are specific for breast cancer, for example in lung cancer, because CLN2p activity is similar to that found in normal tissues (normal lung tissue and lung). The mean CLN2p activity +/- SD for tumor tissue is 1418 +/- 505 nmol / hr / mg protein and 1649 +/- 200 nmol / hr / mg protein, respectively).

SUMMARY OF THE INVENTION It is an object of the present invention to provide newly discovered enzyme biomarkers associated with breast carcinomas and active in cells of breast carcinomas including breast tumors and breast cancers. According to the invention, this enzyme is the pepstatin-insensitive lysosomal tripeptidyl peptidase CLN2p, which has hitherto only been associated with the diagnosis of late infantile neuronal ceroid lipofuscinosis, a rare childhood neurodegenerative disease. Was disclosed.

Another object of the present invention is to provide a method for the detection and diagnosis of breast carcinomas, including breast tumors and breast cancers, which cells of breast carcinomas or biological tissues or vascular fluid samples such as Assaying the presence and / or enzyme activity of CLN2p in breast tissue, tissue extracts, cell lysates, blood, serum or plasma and the like. According to the present invention, comparative results are obtained by assaying normal samples, ie normal breast tissue from non-cancerous individuals, preferably from non-cancerous individuals. An increased CLN2p level or amount in a test sample and / or an increased CLN2p enzyme activity level compared to the value determined for a normal control can be used for detection and diagnosis of breast cancer in a patient. In addition, the increased CLN2p activity (ie, enzymatic activity) in the test sample compared to the CLN2p activity of the normal control can also be used for prognostic indication of breast cancer in patients. In addition, the CLN2p level and activity assays of the invention can be used in screening assays, eg, screening breast tissue and blood samples of patients for indicators of cancer. In addition, the assay method of the present invention can be used for early diagnosis of primary breast cancer as well as detection and diagnosis of non-primary breast cancer or metastatic breast cancer.

Another object of the invention is to provide such antibodies, especially monoclonal antibodies, or immunoreactive portions thereof, which are immunoreactive with CLN2p, as well as immunoassays,
Especially the use of such antibodies in enzyme linked immunosorbent assays (ELISA) to detect and measure CLN2p in breast carcinomas.

Yet another object of the present invention is to provide a sensitive and reliable enzyme assay for the diagnosis of breast cancer in an individual, including the stages exhibited by the individual, and further reliable and specific. It is to provide a biomarker.

Yet another object of the present invention is such a treatment or regimen for breast carcinoma for use in diagnosing the progression of a breast carcinoma in a patient or in an individual undergoing various treatments or therapies. To provide a sensitive and reliable assay for measuring or monitoring the performance of, and further a reliable and specific biomarker.

Another object of the invention may be carried out using a vascular fluid sample, preferably a blood, serum or plasma sample, taken from a patient or individual to be tested for or treated for breast carcinoma. It is to provide such a simple assay method.

Yet another object of the present invention is the use as a diagnostic and / or prognostic biomarker or in a panel of biomarkers for the detection and monitoring of breast carcinomas including various breast tumors and cancer types. CLN2p, which can
Alone or in combination with the status of other cancer-related molecules, in particular cathepsin D levels, estrogen receptor levels, and / or progesterone receptor levels,
To provide an assay for diagnostic and / or prognostic indications.

Further objects and advantages afforded by the present invention will be apparent from the detailed description that follows.

[0022] The invention relates to the detection of breast carcinoma, diagnosis and / or newly discovered assays pepstatin insensitive lysosomal tripeptidyl peptidase CLN2p is associated as a biomarker for prognosis and methods I will provide a. Prior to the present invention, CLN2p was only disclosed and considered to be associated with the diagnosis of the rare childhood neurodegenerative disease, late infantile neuronal ceroid lipofuscinosis.

The new finding was first discovered by the present invention that CLN2p is highly active and has detectable activity in a significant number of breast carcinomas tested. According to the present invention, CL
N2p and its proteolytic enzyme activity have been found to be reliable biomarkers for the detection and diagnosis of breast cancer, tumors and carcinomas. Proteolytic enzymes may play a prominent role in tumor progression by facilitating the detachment of tumor cells from the basement membrane and their spread to other tissues, so that in breast tissue or vascular fluid samples Determination of high levels of CLN2p and / or high CLN2p activity is indicative of breast cancer in a patient.

CLN2p activity levels were higher than the highest levels seen in normal tissues in all but one of the breast tumor specimens tested (Example 2).
It shows that LN2p is advantageous as a diagnostic marker for breast cancer. In addition, high CLN2
The p-activity level is specific to breast cancer tissue. For example, CLN2p activity in lung cancer tissue was unaffected when compared to CLN2p activity in normal lung tissue.

The significance of the prognostic manifestations of CLN2p activity for patients already diagnosed with breast cancer is another aspect of the invention, which, as will be appreciated by those of skill in the art, relates to direct comparison with clinical outcome. is doing. Cathepsin D, another proteolytic enzyme in breast cancer
There are regrets in the published study that attempted to define the role of the prognostic features of. This may be due, in part, to a poor understanding of the role of cathepsin D in stromal components of tumors, such as inflammatory cells infiltrating different forms of cathepsin D (MD Johnson et al., Cancer Res., 53: 873-877 (1993)). The major secretory form of cathepsin D in estrogen receptor (ER) -positive MCF-7 cells is the molecular weight
It is a 52 kDa precursor protein that is proteolytically cleaved into a single polypeptide 48 kDa form and ultimately processed into a mature enzyme of two subunits (S. Yonezawa et al., J. Biol. Chem. ., 263: 6504-16511 (1988)). Cathepsin D
The 52kDa form is inactive, while the mature 2 subunit 48kDa form of the enzyme is proteolytically active. In contrast, CLN2p is only active as a single polypeptide with a molecular weight of 46 kDa.

Findings on Cathepsin D (B. Westley and HA Rochefort, Cell, 20: 353-3
62 (1980)), we show that intracellular levels of CLN2p activity in basal secretion or CLN2p activity were independent of whether ER-positive MCF-7 cells were previously exposed to the hormone β-estradiol. In, I found that there is no difference. These results demonstrate that increased levels of CLN2p and cathepsin D are regulated by an independent and unrelated mechanism in breast cancer.

Many early studies have been associated with an increase in cathepsin D following estrogen exposure, but it has been pointed out that cathepsin D proteolytic enzyme is increased even in some ER-negative breast carcinoma cell lines. In addition, elevated serum levels of cathepsin D (ie, 10 ± 8 pmole / ml) have been reported in 60% of patients with metastatic breast cancer, although cathepsin D was higher in women with primary breast disease than in healthy women. Was not significantly increased (ie, 5 ± 2 pmole / ml and 4 ± 2 pmole / ml, respectively) (JP
Brouillet et al., Cancer, 79: 2132-2136 (1997)).

As agreed by those skilled in the relevant arts (eg GM Clark, B
Reast Cancer Res. Treat., 30: 117-126 (1994)), there is still a strong need for new and reliable biomarkers of diagnostic and prognostic indicators for breast cancer. According to the invention, CLN2p has the desirable property of many clinically useful biomarkers: it has a high level of activity in almost all breast tumors; Associated with some, but not all, previously established prognostic factors for breast cancer; this enzymatic activity is stable in long-term stored frozen samples and Quantitative biochemical assays as described in the text can be used to accurately determine the activity level of CLN2p proteolytic enzyme in the sample being tested.

As described herein, CLN2 proteolytic enzyme has high levels of activity in cells and tissue samples from breast cancer patients, including patients with primary breast cancer, as well as measurable levels in plasma samples. It was also discovered by the present inventors that they also have CLN2p enzymatic activity (mean activity +/- SD for normal samples is 19.9 +/- 1.9 nmol / m
l / hour. ). In addition, the assay method for detecting CLN2p enzyme activity in samples from breast cancer patients according to the present invention is sensitive and reliable. For example, as little as about 2-10 μg, preferably 5-10 μg tissue protein, or about 5-10 μl plasma sample is required. Therefore, this proteolytic enzyme assay requires only about 2-10 μg of tissue protein, so that the diagnostic method according to the invention is well suited for samples obtained by needle biopsy.

Accordingly, one aspect of the invention is a cell and tissue sample, including cells and tissue samples from individuals with breast cancer or carcinoma, including various breast neoplasms, cancers and tumors, as well as primary disease samples. CLN2 as an assayable biomarker in Escherichia coli
Regarding p. In this aspect, the invention relates to CLN2p, or CL in a specimen or sample.
To determine the enzyme level and activity of N2p functional protease fragments,
Substrate-specific enzyme assays performed on breast tissue specimens or samples, eg, specimens obtained from breast biopsies or aspiration are provided (Examples 1 and 2).

Another aspect of the invention provides a simple blood based assay for primary breast cancer as well as breast cancer including non-primary breast cancer and metastatic breast cancer. CLN2p is known to have measurable activity in normal plasma samples (19.9 +/- 1.9 nmol / ml
/Time). Furthermore, the present invention shows that increased CLN2p activity in breast tumors or cancers, including primary breast tumors and cancers, is readily detected due to the newly found high activity levels of this enzyme in breast cancer tissue samples. It is shown. Thus, increased CLN2p activity in breast cancer tissues, including primary breast cancer tissues, may be clinically associated with increased activity of this biomarker in vascular fluid samples such as blood, serum, or plasma for assay purposes. it can. Since increased CLN2p activity in breast tumors may lead to increased enzyme activity in plasma, analysis of CLN2p activity in plasma indicated
A simple blood-based biomarker for breast cancer is provided. To assay for CLN2p activity in serum or plasma, vascular fluids derived from blood samples taken from such individuals tested are assayed in a manner similar to that described for breast cancer tissue or cell samples. (Eg, Example 1, materials and methods,
And Example 4).

It will be appreciated by those skilled in the art that vascular fluids, ie blood, serum or plasma, have a physiological (ie near neutral) pH. Since the CLN2p enzyme is most stable in an acidic environment, the blood sample to be tested as a result should have approximately 1 or 2 hours to stabilize CLN2p before performing the enzyme detection and activity assays of the invention. It is preferably processed within. For treatment of blood samples, preferably in the presence of an anticoagulant such as EDTA (K ₃ EDTA) or heparin, blood samples are collected. The sample is centrifuged at low speed to separate plasma from cellular components and the plasma is immediately frozen at about -20 ° C until used for analysis.

In another aspect, in order to stabilize CLN2p, the blood sample is acidified immediately after collection to a pH of about 3.0-5.5, preferably 3.5-5.0, and more preferably 3.5-4.0.
Can be A preferred plasma pH lowering agent is a pH 3.5 ammonium formate buffer. One of ordinary skill in the art will recognize that acidification of a blood sample may result in precipitation of serum albumin in the sample. Therefore, removal of albumin precipitated after acidification, otherwise removal of the unprecipitated portion of the blood sample for analysis,
It is preferably carried out prior to carrying out the CLN2p detection or activity assay on a fresh blood sample acidified according to the invention.

According to the invention, detection of increased levels of CLN2p or CLN2p enzyme activity in a sample is diagnostic of breast cancer. CLN2p activity ranges from 112 to 343 nmol / hr / mg protein in normal breast tissue, ie, breast tissue obtained from individuals with no detectable breast cancer, carcinoma or tumor or who are not precancerous. However, in breast tumor or cancer tissues (ie, from patients with breast tumors, cancers, or carcinomas), CLN2p activity is generally in the range of 496-5787 nmol / hr / mg protein.

By comparison, CLN2p activity in breast tumor or cancer tissue is at least about 2 to 17 times higher than CLN2p activity in normal tissue. Thus, relatively high CLN2p activity in breast tissue is an indicator of cancer and is directly associated with cancer status. The amount or level of CLN2p in breast tumor or cancer tissue is generally proportional to the increased activity level of the enzyme detected in that tissue. Therefore, the amount or level of CLN2p is expected to be at least about 2 to 17 times higher in breast cancer tissue relative to normal non-cancerous tissue. A normal CL2Np activity value means a value determined from tissues obtained from non-cancerous individuals during reduced surgical procedures, rather than from tissues that appear normal in patients with breast tumors or cancer. Such normal tissue controls were used because apparently "normal" appearing tissue from breast cancer patients is often found to have relatively high CLN2p activity. Thus, changes in breast cancer patients may be associated with increased CLN2p in accordance with the invention even if such differences cannot be detected by histochemical means.
It can be detected by activity.

Changes in CLN2p levels during monitoring can facilitate diagnosis and detection of breast cancer, which can lead to a course of treatment or therapy, or to assess the outcome of treatment or therapy in patients with breast cancer. To do. Therefore, by monitoring CLN2p levels, abnormal levels of the enzyme, or elevated enzyme activity, can be an indicator of a cancerous condition that can be reproducibly detected, resulting in appropriate treatment protocols. Can be determined and implemented. In addition, relatively high or persistently high levels of CLN2p activity in breast cancer patients as compared to normal CLN2p values can be used to indicate a poor prognosis and / or an increased chance of recurrence of breast cancer.

Another aspect of the invention is an assay method using an antibody for the detection of CLN2p, particularly in a cell or tissue sample obtained from a breast carcinoma, tumor or cancer, or in a vascular fluid sample of a patient (eg, Antibodies, or immunogenic fragments or portions thereof, which are immunoreactive with the CLN2p enzyme for use in (ELISA). Both polyclonal antibodies (PAbs) and monoclonal antibodies (MAbs) specific for CLN2p, or immunogenic fragments or parts thereof, are encompassed by the present invention. CLN2p isolated and / or purified from a natural source (eg MA Junaid et al., J. Neurochemistry, 74
: 287-294 (2000)) or a CLN2p enzyme synthetically or recombinantly prepared,
Alternatively, those peptides can be used as immunogens to induce specific reactions and inject them into host animals to produce anti-CLN2p antibodies. Immunogenic fragments or portions of CLN2p can be prepared using methods and protocols practiced in the art by the use of proteolytic enzymes or by the synthesis of peptides bearing epitopes that elicit immune responses and produce antibodies. Obtainable.

In another embodiment, the present invention contemplates a method of producing an antibody immunoreactive with CLN2p or an immunoreactive fragment or portion thereof, comprising the steps of: Immunizing an animal with CLN2p as an immunogen, isolating serum from the animal, wherein the serum comprises an antibody that is immunoreactive with an epitope on the CLN2p immunogen, and Optionally purifying the anti-CLN2p antibody using immunoglobulin purification techniques practiced in the art. Alternatively, the spleen of an immunized animal, preferably a mouse, can be used in commonly practiced hybridoma generation techniques, as described herein,
And a monoclonal anti-CLN2p antibody can be produced and, if desired, isolated for use.

As used herein, “antibody” or “antibodies” refer to the immunogen CL
By intact molecules as well as capable of binding the epitopic determinants of N2p are meant fragments thereof, such as Fab, F (ab) ₂ and Fv. As will be appreciated by one of skill in the art, the immunogen can optionally be conjugated to a carrier protein to increase immunogenicity, especially when small peptides or CLN2p fragments are used. Commonly used carriers that are chemically coupled to peptides routinely include serum albumin; ie, bovine, sheep, goat, or fish serum albumin; thyroglobulin; and keyhole limpet hemocyanin. The subsequently bound immunogenic carrier is a recipient animal (e.g., mouse, rat, sheep, goat or rabbit).
Used in the immunization of.

The term “antigenic determinant” means a fragment of a molecule (ie, an epitope) that makes contact with a particular antibody. When isolated and / or purified CLN2p is used to immunize a host animal, many regions of the enzyme will be bound to defined regions on the enzyme or to antibodies that specifically bind to the three-dimensional structure. Capable of inducing production;
These regions or structures are called antigenic determinants or epitopes. An antigenic determinant can compete with the intact antigen (ie, the immunogen used to elicit an immune response) for binding to the antibody.

These antibodies can be elicited in an animal host by immunization with an immunogenic component derived from CLN2p or can be formed by in vitro immunization (sensitization) of immune cells. Again, these antibodies encode the appropriate antibody
It can also be made in recombinant systems transformed, transfected, infected or transduced with DNA. Alternatively, these antibodies can be constructed by biochemical reconstitution of purified heavy and light chains. Antibodies encompassed by the present invention include hybrid antibodies, chimeric antibodies, humanized antibodies (see, eg, CJ Queen et al., US Pat. No. 5,585,089) and monovalent antibodies. Using such antibodies, for example, CLN2p or immunogenic fragments or portions thereof, etc., by chromatography on an antibody-bound solid phase matrix or support (E. Harlow and
D. Lane, “Using Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory) ", Cold Spring Harbor, NY
(1989)) or by an immunoassay, and can be detected in a test sample. C
Antibodies that specifically recognize and bind LN2p, but not similar enzymes or proteins, are preferred.

These antibodies are used as in vitro diagnostic agents in standard immunoassay protocols to test (or screen) for the presence of CLN2p in biological samples, particularly breast tissue samples and vascular fluid samples. can do. Preferably, the assay using these antibodies to detect the presence of CLN2p in a sample is such that an immune complex between the antibody and the CLN2p antigen that is suspected to be present in the sample is allowed to form. Contacting the sample with at least one antibody under conditions. The formation of this immune complex, if present, which is indicative of the presence of CLN2p in the sample, is then detected and measured by suitable means.

These assays encompassed by the present invention include, for example, a direct format (where the labeled primary antibody reacts with the antigen, eg CLN2p), an indirect format (where the labeled secondary antibody is
Reacts with primary antibody bound to antigen), competitive method (addition of labeled antigen, etc.)
), Or a sandwich format (both labeled and unlabeled antibodies are used), as well as other formats described in the art. In one such assay, a biological sample is contacted with an antibody of the invention and a labeled secondary antibody is used to bind these antibodies and between the antibody-bound CLN2p. The presence of CLN2p as a complex is detected.

Such assays include radioimmunoassay (RIA), ELISA, indirect immunofluorescence assay, western blot assay, immunohistochemical assay, chemiluminescent assay, immunoprecipitation assay, dot blot assay. Methods, slot blot assays, etc., but are not limited thereto. These antibodies are
It can be labeled or unlabeled, depending on the type of assay used. Labels that can be combined with these antibodies include those known in the art, including enzymes (e.g. horseradish peroxidase or glucose oxidase), radioisotopes, fluorogens (e.g. fluorescein isothiocyanate (FITC)). , Fluorescein isocyanate (FIC), 5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TR
(ITC), lissamine, etc.), chromogenic substrates, cofactors, biotin / avidin, chemiluminescent compounds, phosphorescent compounds, gold colloids, colored particles and magnetic particles, but are not limited thereto.

As will be appreciated by those of skill in the art, where the basic indicator group is an enzyme, additional reagents (eg, substrates) are required for visible signal formation. Radioactive elements of various types, eg ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ⁵¹ Cr (γ-emitter); ³² P, ³ H, ³⁵ S (β-emitter)
, And ¹¹ C, ¹⁴ C, ¹⁵ O, or ¹³ N (positron emitters) can also be used as detectable labels. The labeled complex can be visually detected by a spectrophotometer, or other detector, depending on the grouping or indicating group.

Modification of antibodies can be carried out by modifying carrier proteins or peptides, or by known insoluble supports or matrices such as polystyrene or polyvinyl chloride microtiter plates, wells or tubes; glass tubes or glass beads; and paper. , To cellulose and cellulose derivatives, and to chromatographic supports such as silica by any known means. Other suitable solid supports or matrices include, by way of non-limiting example, the following: cross-linked dextran (Pharmacia, Piscataway, NJ); agarose, polystyrene beads (diameter about 1-5μ; eg Abbott Laboratories, Illinois. ); And cross-linked polyacrylamide; nitrocellulose or nylon based webs such as sheets, pieces, paddles or rods.

One method of using the antibodies of the invention to detect CLN2p in a sample associated with breast cancer is to use a solid support or matrix to which the antibody recognizes all or part of CLN2p. Immunoassay, including contacting the support with a sample or an aliquot of the sample and adsorbing onto a solid support or matrix.
CLN2p is detected by a radioactively or non-radioactively labeled detection molecule (ie a suitable antibody) which specifically binds to CLN2p bound to the CLN2p antibody.

Another method of detecting CLN2p in a sample associated with breast cancer is with an antibody that specifically recognizes and binds CLN2p or an immunogenic fragment or portion thereof of said sample,
Incubation under conditions that allow the antibody to bind to the enzyme, followed by the addition of detectable antibodies, such as those that bind to various epitopes on CLN2p and are bound to a solid support. Or by the addition of an antibody such that it binds to an antibody already bound to CLN2p, wherein the added antibody provides a labeled and selectable marker. ing.
The added selectable antibody, or bindable fragment thereof, may be bound to a solid support, or they may in turn be bound by another detectable antibody bound to the support. There is a possibility that Such immunoassays and variations thereof are known and practiced in the art.

As a preferred but non-limiting example, the present invention is for detecting, diagnosing and / or quantifying the presence of CLN2p in breast tissue or fluid samples of individuals undergoing testing either in vivo or in vitro. As a particularly useful and practical method using an antibody, an ELISA method (Engvall et al., Immunochemistry, 8: 871-4 (1971); “Basic and Clinical Immunochemistry”, Chapter 22, Fourth Edition) , D.
P. Stites et al., Lange Medical Publications, Los Altos, CA., 1982; and DJ Reen, Methods Mol. Biol., 32: 461-6 (1994)). ELISA protocols using various antibodies are known and practiced in the art and are suitable for use in the present invention.

The invention thus encompasses diagnostic systems, in particular diagnostic kits, such as those used in an ELISA format for detecting CLN2p in biological tissue / cell samples or vascular fluids. For example, a suitable diagnostic system uses anti-CLN2p specific antibodies as individually packaged immunological reagents for assaying the presence or amount of CLN2p or a fragment thereof in a sample in a suitable amount of at least one assay. Including. Also included are instructions for use of the packaged reagents. The instructions for use generally include a description of the reagent concentration, or at least one assay parameter, such as the relative amounts of the reagent and sample to be mixed, the retention time of the reagent / sample mixture, temperature, buffer conditions and the like. Preferred diagnostic systems further include, for example, CLN2p or a fragment thereof capable of binding, and a detectable label or indicator for signaling complex formation of the anti-CLN2p antibody of the invention.

In an immunoassay for use in the present invention, the monoclonal or polyclonal anti-CLN2p antibody is either directly or indirectly in a quantity of C in the sample.
It is used as an immunochemical reagent for forming an immune reaction product that is related to the amount of LN2p. Similarly, the amount of CLN2p in the sample either directly or indirectly
To determine the amount of CLN2p, or a detectable fragment thereof, in a biological fluid sample using CLN2p, or a polypeptide fragment thereof, as a reagent to form a quantity-related product. An immunoassay has been devised. Such assays include detection assays, diagnostic assays, screening assays and prognostic assays.

Hybridomas producing monoclonal antibodies against the immunogenic components of CLN2p can be produced by well known techniques. Hybridomas can be made by fusing immortalized cell lines with B lymphocytes that produce the desired antibody. Alternatively, non-fusion techniques for making immortalized antibody-producing cell lines are possible and within the scope of the invention (see Casali et al., Science, 234: 476 (1986)). Immortalized cell lines are typically transformed into mammalian cells, particularly melanoma cells of rodent, bovine or human origin. Most often, rat or mouse melanoma cell lines are used as a convenient and workable substance.

Hybridomas can be selected using standard techniques such as HAT (hypoxanthine-aminopterin-thymidine) selection. Hybridomas secreting the desired monoclonal antibody can be labeled with standard immunoassays such as immunoblotting, ELISA,
It can be selected by assaying the medium of the cells by radioimmunoassay (RIA), or an equivalent assay. Antibodies immunoreactive with CLN2p can be recovered and isolated from the culture medium using standard protein purification techniques. (Tij
issen, 1985, "Practice and Theory of Enzyme Immunoassay
Enzyme Immunoassays) ", Elsevier, Amsterdam).

In another embodiment, the invention encompasses an assay method for detecting CLN2p enzyme activity levels in a tissue or vascular fluid sample (eg, plasma or serum) to be tested. Such assays for breast tissue samples include the preparation of tissue extracts and homogenates, and supernatants derived therefrom, using techniques practiced in the relevant arts. The level of CLN2p activity is determined by the amount of hydrolysis of the substrate as produced by CLN2p in the test sample (i.e., CBZ-Arg-Gly-Phe-Phe-Leu-AFC, as described in Examples 1 and 2). The amount of hydrolysis of the peptide substrate to the product Leu-AFC) is determined. The results of this test sample are compared to the enzyme activity level from a normal sample. The results of CLN2p activity analysis are quantified using high performance liquid chromatography (HPLC) as described in Examples 1 and 2.

Another aspect of the present invention is the use of CLN2p alone or in the status of other cancer associated molecules, in particular cathepsin D levels, estrogen receptor (ER) levels, and / or progesterone receptor (PgR) levels. Detection assay such that it can be used as a diagnostic and / or prognostic biomarker (s) for the detection and monitoring of breast carcinomas, including various breast tumors and cancer types Method, diagnostic assay or prognostic assay. Cathepsins D, ER and P
CLN2p levels assayed in combination with a panel of gR levels provide a biomarker assay for diagnostic or prognostic indications for outcome of breast carcinoma and possible disease.

Other binding assays known to those of skill in the art for detecting CLN2p in a sample are encompassed by the present invention. Such assays include immunoassays using the antibodies described above, and many other binding assays, such as target proteins in a sample (e.g., CLN2p) that are specific to the target protein. Assays that can be reacted with a binding molecule that is capable of chemically binding. The binding molecule can be, for example, a ligand-receptor pair (ie, a molecule pair capable of specific binding interaction), an antibody-antigen, an enzyme-substrate, a nucleic acid-nucleic acid, a protein-nucleic acid, or any known in the art. Other specific binding pairs and their members can be included. The binding molecule can be designed to have an enhanced affinity for the target protein. The binding molecule can be linked or linked to a detectable label or indicator for detection by means known in the art. Means for attaching labels or indicators to binding molecules are well known in the art; commercial kits for labeling proteins and the like can also be used.

EXAMPLES The following examples set forth herein are meant to illustrate and exemplify various aspects of practicing the invention, but are not intended to limit the invention in any way. Absent.

Example 1 Materials and Methods Human breast carcinoma tissue was used as the National Breast Cancer Tissue Resource.
issue Resource) (Baylor College of Medicine, Houston, TX) or the National Cancer Institute Cooperat
ive Human Tissue Network) (Philadelphia, PA).
Tumor specimens from 220 patients with primary breast carcinoma and normal tissues from 8 non-cancerous subjects who underwent debulking surgery for megamammosis with no evidence of neoplasia or precancerous status were presented. Used in the tests described in the specification. Results of CLN2p activity in breast tumors were compared to CLN2p activity in normal breast tissue obtained as described above. "Normal-looking" breast tissue from a breast cancer patient or from a patient whose cancerous sample is being tested is a normal reference because such "normal-looking" tissue generally has relatively high CLN2p activity. Not suitable for getting value. In contrast, CLN2p activity in normal lung tissue was similar to CLN2p activity detected in cancerous lung tissue, unlike significantly higher levels of CLN2p activity in tumors versus normal breast tissue.

To measure CLN2p activity in a tissue source, tissues (50-100 mg) were treated with 50 mM containing 0.15 M NaCl and 0.1% Triton X-100 in various rates of tissue tearers. It was homogenized in 2 ml of ammonium formate buffer (pH 3.5). 14,000 xg
CLN2p was determined using the supernatant obtained after centrifugation at 10 min. These extracts were frozen and stored at -20 ° C when not used immediately.

Substrate Preparation Tetrapeptide substrate GFFL-7-amino-4-trifluoromethylcoumarin (purity> 99
%) And measurement of CLN2p activity in tissue extracts by HPLC is virtually as described by MA Junaid et al., Clin. Chim. Acta, 281: 169-176 (1999); Janide (M.
A. Junaid) et al., J. Neurochem., 74: 287-294 (2000); and Janide (MA).
Junaid) et al., Neurosci. Lett., 264: 157-160 (1999).

Synthetic peptide CBZ-Arg-Gly-Phe-Phe-Leu Aminotrifluoromethylcoumarin (AF
C) was purchased from Enzyme Systems, Products (Livermore, CA). All other chemicals are Sigma Chemical (Sigma
Chemical) (St. Louis, MO). Reversed-phase C18 high performance liquid chromatography (HPLC) columns are available from Rainin Instruments (
Purchased from Woburn, WA).

The reaction was completed at room temperature with the peptide CBZ-Arg-Gly-Phe-Phe-Leu-AFC (2 mg) in 0.25 mL of 50% acetonitrile with 100 μg trypsin in 100 mM Tris-HCl (pH 7.4). Processed until. The progress of the reaction was confirmed by analyzing an aliquot of the reaction mixture by reverse phase HPLC. The formed product Gly-Phe-Phe-Leu-AFC was analyzed by reverse phase HPLC.
And evaporated to dryness under nitrogen (purity> 99%). These HPLC conditions are:
The same as described in the "HPLC analysis" section.

CLN2p Assay An aliquot of the supernatant containing 5-10 μg protein was added to 7 nmol (280 μM) in 20 mM ammonium formate buffer (pH 3.5) containing 0.2 mM pepstatin-A and 0.5 mM E-64.
M) Incubated with the peptide substrate Gly-Phe-Phe-Leu-AFC (dissolved in 40% dimethylformamide). The final volume of this assay mixture was 25 μL.
Incubation was carried out at 37 ° C. for 5-10 minutes, after which the reaction was stopped by adding 100 μL ice-cold acetone. The supernatant obtained after centrifugation at 12,000 xg for 2 minutes was
Dried under nitrogen, resuspended in 50 μL 60% acetonitrile and 10 μL used for HPLC analysis.

HPLC analysis This reaction product was subjected to Microsorb-MV C1 in an isocratic solvent system composed of 55% acetonitrile and 0.1% trifluoroacetic acid.
It was analyzed by reverse-phase HPLC with 8 columns (0.46x10 cm, 3 μm, 100Å) at a flow rate of 0.5 mL / min. The eluate was detected at 340 nm in a variable wavelength ultraviolet (UV) detector and the peaks were integrated using a Spectra-Physics SP 4290 integrator. The total length and chart speed of this detector are 0.001 cm / min and 0.5 c, respectively.
Set to m / min. CLN2p activity was calculated based on the amount of Leu-AFC formed from the corresponding peak area obtained by injection of a known amount of Leu-AFC.

The protein concentration in the extract was determined according to the method of Lowry et al. (OH Lowry et al., J. Biol. Chem., 193: 265-275 (1951)) using bovine serum albumin as a standard. Determined by law. CLN2p activity in the extract is expressed as nmol / hr / mg protein, and estrogen receptor (ER), progesterone receptor (PgR) and epidermal growth factor receptor (EGFR) levels, cathepsin D activity, S phase fraction And associated with DNA ploidy status.

The standard multipoint dextran-coated charcoal dust assay was modified as described above for incorporation of ¹²⁵ I-labeled estradiol and [ ³ H] R5020 in a single assay, ER and Enables simultaneous measurement of both PgR (LG
Dressler et al., Cancer, 61: 420-427 (1988)). Levels greater than or equal to 3 fmol / mg protein for ER were considered positive and levels greater than or equal to 5 fmol / mg protein for PgR were considered positive.

EGFR was measured by radioligand binding assay using a fixed concentration of radiolabeled EGF and a variable concentration of unlabeled EGF. Malignant tissue was disrupted and homogenized, followed by ultracentrifugation at approximately 108,000 xg for 1 hour at 4 ° C. After removing fat and cytosol, the membrane pellet was rehomogenized and briefly centrifuged. This sample was added to labeled EGF and incubated. Combined EGF from free EGF
Was achieved by a polyethylene glycol gradient and bound fractions were quantified with a gamma counter. Data after subtraction of non-specific binding were analyzed by Scatchard analysis and reported in fmol / mg membrane protein. 10 fmol
Levels greater than or equal to / mg protein were considered positive.

Cathepsin D levels in the tumor cytosol were measured using a modified immunoradiometric (IRMA) method of the double antibody method. Cytosol was prepared in 10 mM Tris buffer containing 1.5 mM EDTA, 5 mM dithiothreitol and 10 mM molybdate. The cytosolic protein concentration was adjusted to approximately 1-2 mg / mL and then diluted 60-fold. Three reference cytosols prepared from pooled, non-aggregated breast tumor tissue were included in each assay along with the controls provided by the manufacturer. Immunoradiometry (CIS-US, Bedford, MA) used D7E3 monoclonal antibody as the capture antibody and radiolabeled M1G8 monoclonal antibody as the detection antibody. The bound radioactivity was directly proportional to the level of cathepsin D in the specimen. Levels of cathepsin greater than 50 pmol / mg cytosolic protein were considered high.

DNA analysis was performed on breast tumors or cancer cells. DNA ploidy and S phase fractions were determined by flow cytometry as previously described (LG Dressl
er et al., Cancer, 61: 420-427 (1988), and CR Wenger et al., Breast Cancer Res.
Treat., 28: 9-20 (1993)). Polyploidy is the amount of DNA in normal diploid cells of the amount of DNA in the tumor
Means ratio to quantity. S phase by flow cytometry measures all cells actively synthesizing DNA. S-phase fractions are estimated using the MODFIT program (Verity Software House, Topsham, ME) using single-cut debris stripping and S-trap trapezoids for S-phase components. did. An S phase fraction greater than or equal to 6.7 for diploid tumors or greater than or equal to 11.0 for aneuploid tumors was considered high.

Statistical Analysis CLN2p activity in normal and breast carcinoma tissues was expressed as mean ± SD and compared at the 5% significance level by two-sample Student's t-test. The relationship between the other biomarkers for CLN2p activity and breast carcinoma has been described by Spearman's rank correlation coefficient (r _s) or Wilcoxon rank sum test.

Example 2 HPLC analysis of CLN2p activity in breast tumor tissue and normal non-cancerous breast tissue from patients with breast carcinoma was performed. The results are shown in Figures 1A and 1B. Less than 5% of the substrate is L in normal tissue when the same amount of protein is incubated.
-More than 90% of the substrate was hydrolyzed in cancerous tissue, whereas it was hydrolyzed to AFC. In the patient samples shown in FIGS. 1A and 1B, these values correspond to CLN2p-specific activity of approximately 300 nmol / hr / mg protein and 3000 nmol / hr / mg protein in normal and tumor tissues, respectively.

FIG. 2 shows CL in breast tissue of tumors of normal subjects and patients with primary breast carcinoma.
A scatter plot of N2p activity is shown. CLN2p levels are 112-343 nm for normal tissues
ol / h / mg protein (n = 8) and 165-2787 nmol / h / mg protein for tumor tissue (n = 220).

These results indicate that breast tumors have at least 7-fold higher CLN2p activity (1813 ± 834, mean ± SD, P <0.001) when compared to normal subject tissues (251 ± 66, mean ± SD).
It has shown that it has. Of the total 220 tumor tissues analyzed, all but one sample had CLN2p activity greater than the highest normal value determined. This is CLN2p
It has been suggested that activity is a clinically useful diagnostic test for breast carcinoma. It is not known why one tumor tissue had such low CLN2p activity. Interestingly, this one particular sample (CLN2p activity 164 nmol / hr / mg protein) also appeared abnormal in the other methods, which also had low cathepsin D levels, negative ER, PgR and EGFR. It had levels, diploid DNA ploidy status and a low S phase fraction. Without wishing to be bound by theory or to be limited to one atypical sample, a simple anomalous patient sample in which simple biological variations or mutations show low CLN2p values May contribute to the outliers obtained for.

Example 3 CLN2p activity was compared to other therapeutic biomarkers measured in breast cancer specimens (see Example 1). The relationship of these factors, shown as dichotomous variables, is shown in Table 2.

A description of the test for estrogen receptor (ER) / progesterone receptor (PgR) describes that the presence and level of hormone receptors in tumor cells (eg breast tumor cells) is associated with response to hormone therapy and clinical outcomes. It has been shown to be strongly correlated (H. Battifora, Appl. Immunohistochem., 2 (3): 143-145 (1994); SM.
Veronese et al., Appl. Immunohistochem., B3: 85-90 (1995); I. de Mascarel et al., App
l. Immunohistochem., 3: 222-231 (1995); LP Pertshuk et al., Cancer, 77: 2514-2.
519 (1996); N. Weidner, Calif. Seminars in Pathology, 10-14 (1996); C. Coh
En et al., Abstr. Annual Meeting of the USCAP, 181A (1997); CT Taylor, Cancer
r, 77 (12): 2419-2422 (1996)). Overexpression of epidermal growth factor receptor (EGFR) is associated with poor prognosis (N. Weidner et al., Breast Cancer Res. Treatment, 29: 97-107 (1
994); J. Baselga et al., Breast Cancer Res. Treatment, 29: 127-138 (1994)).

[0076]

Table 2 Comparison of various levels of CLN2p activity with other biomarkers in 200 samples from patients with primary breast carcinoma

CLN2p activity was positively correlated with cathepsin D, ER and PgR (P <0.0001). EG
No positive or negative correlation was observed for FR, DNA ploidy status or S phase fractions. Similar results were observed when biomarker measurements for prognostic indications were presented as continuous variables (Table 3).

[0078]

Table 3 Comparison of CLN2p activity with other biomarkers in 200 samples from patients with primary breast carcinoma

[0079] As can be seen from Table 3, the cytoplasm and CLN2p activity was determined by immunohistochemical staining of stromal tumor cells, cathepsin _{D (r s = 0.45, p} <0.0001); tumor cell nuclei immunohistochemistry It was significantly correlated with ER (r _s = 0.31, p <0.0001) and PgR levels (r _s = 0.43, p <0.0001) as determined by dynamic staining. However, EGFR levels or S
No significant correlation with the phase fraction was observed. Multivariate regression analysis with both continuous and dichotomous explanations showed that cathepsin D and PgR showed CLN2 in breast cancer tissues.
It was shown to be the strongest predictor of p activity (r ² = 0.24 and 0.2, respectively).
0). In summary, the results of Example 3 indicate that CLN2p alone or cathepsin D, ER
And / or in combination with PgR status suggests an attractive prognostic biomarker assay for breast carcinomas, including breast cancer and tumors.

This example demonstrates that CLN2p activity level, cathepsin D activity level, and ER and / or
Or analysis of PgR levels to be used as a reliable biomarker in a biomarker panel for breast cancer detection and / or prognostic indications to provide a reliable indication of the outcome of a patient's breast cancer disease or condition Makes it clear that you can.

Example 4 This example illustrates the use of the assay method of the present invention to determine CLN2p in blood samples, especially plasma samples.

For plasma samples, freshly collected heparinized blood was spun at low speed (
~ 1000 xg), plasma components were separated from cellular components. Plasma was stored frozen at -20 ° C if not used immediately for analysis. Prior to performing this assay, plasma samples should be
The contaminated platelets were removed by centrifugation at 2,000 xg for 5 minutes. For CLN2p activity determination, 5 μl of plasma was taken in 20 mM ammonium formate buffer (pH 3.5) containing 0.2 mM pepstatin-A and 0.5 mM E-64 (inhibitor of enzyme with active thiol group, Sigma). Incubated with 280 μM peptide substrate Gly-Phe-Phe-Leu-AFC. The final volume of this assay mixture was 25 μl. Incubation is 3
It was carried out for 10 minutes at 7 ° C., after which the reaction was stopped by adding 100 μl of ice-cold acetone. The supernatant obtained after centrifugation at 12,000 xg for 2 minutes was dried under nitrogen and resuspended in 50 μl of 60% acetonitrile and as described in Example 1.
25 μl was used for HPLC analysis.

Results from 8 normal plasma samples obtained using the above protocol showed that CLN2p activity in these samples was 19.9 +/- 11.9 nmol / hr / ml plasma. Plasma samples from breast cancer patients, using the above protocol and assayed for CL2Np activity as described herein, show about 10% more activity than normal plasma controls, as shown in Table 4.
It showed a 10-fold greater CLN2p activity. Thus, plasma provides a suitable source of vascular fluid from which CLN2p activity data and results can be obtained.

[0084]

[Table 4] CLN2 protease activity in plasma * Mean ± standard deviation

The contents of all patents, patent applications, published articles, books, reference manuals and abstracts cited herein are incorporated by reference in order to more fully describe the state of the art to which this invention belongs. Are incorporated herein by reference in their entirety.

Various changes may be made in the above description which do not depart from the scope and spirit of the invention, and therefore all the scope of the claims included in the description or limited to the appended claims The content is intended to be construed as an illustration and illustration of the present invention. Many modifications and variations of the present invention are possible in light of the above disclosure.

[Brief description of drawings]

1 shows a HPLC analysis of CLN2p activity in normal (FIG. 1A) and tumor (FIG. 1B) tissues of patients with primary breast carcinoma. 0.2 mM pepstatin-A and 0.5 mM E-
Substrate GFFL-AFC (280 μM) in 50 mM ammonium formate buffer (pH 3.5) containing 64.
M) was incubated with 7 μg of protein in a final volume of 25 μl for 5 minutes at 37 ° C. The reaction was stopped by the addition of ice-cold acetone, followed by 12,000 xg.
It was centrifuged for 2 minutes. The supernatant was evaporated to dryness, the dried residue dissolved in acetonitrile and aliquots analyzed on a reverse phase C18 HPLC column. Substrate and product (L
-AFC) was detected at 340 nm with a variable wavelength detector set.

FIG. 2 shows a scattergram of CLN2p enzyme activity in breast tissue from normal subjects and tumors from patients with primary breast carcinoma. This data shows proteolytic enzyme activity (solid line shows mean) in normal tissues from tumor specimens of 220 patients and 8 non-cancerous subjects who underwent debulking surgery for megamammosis. .

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE, TR), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, G M, KE, LS, MW, MZ, SD, SL, SZ, TZ , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, B Z, CA, CH, CN, CO, CR, CU, CZ, DE , DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, I S, JP, KE, KG, KP, KR, KZ, LC, LK , LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, P T, RO, RU, SD, SE, SG, SI, SK, SL , TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW F-term (reference) 4B063 QA18 QA19 QQ02 QQ36 QR58                       QS28 QX01

Claims (34)

[Claims] 1. A step of: (a) contacting a breast tissue or vascular fluid sample to be tested with an anti-CLN2p antibody under conditions that allow the formation of a complex between the antibody and CLN2p in the sample; and (b) a breast cancer diagnostic method comprising the step of detecting said complex; diagnosing breast cancer by the increased amount of CLN2p in the sample being tested relative to the amount of CLN2p in a normal non-cancerous control sample. 2. CLN2p is a radioimmunoassay, Western blot assay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemistry assay, dot blot assay and slot. The method of claim 1, wherein the method is contacted with an antibody in an immunoassay selected from the group consisting of blot assays. 3. The method according to claim 1, wherein the antibody is a monoclonal antibody. 4. The method according to claim 1, wherein the antibody is a polyclonal antibody. 5. The method according to claim 2, wherein the immunoassay is an enzyme-linked immunosorbent assay or ELISA. 6. The method of claim 1, wherein the anti-CLN2p antibody is detectably labeled. 7. A labeled substance, wherein the complex is selected from the group consisting of immunofluorescent label, chemiluminescent label, phosphorescent label, enzyme label, radioactive label, avidin / biotin, colloidal gold particles, colored particles and magnetic particles. Or detected by an indicator.
The method described. 8. (a) Contacting a solid matrix having an antibody immunoreactive with CLN2p immobilized thereon with a breast tissue sample or vascular fluid sample of a patient to immobilize CLN2p in the sample Bound antibody; (b) removing unbound sample; (c) contacting the solid matrix with labeled antibody specific for bound CLN2p; and (d) in a patient sample. Of CLN2p in the patient's sample, including the step of quantifying the amount of CLN2p in the patient, and the step of comparing the amount obtained in step (d) with the amount of CLN2p in the normal non-cancerous control. Breast Cancer Diagnosis in Patients, Diagnosing Breast Cancer in. 9. The method of claim 8, wherein the sample is a breast carcinoma, tumor or cancer tissue. 10. The method of claim 8, wherein the sample is blood, serum or plasma. 11. The label is selected from the group consisting of an enzyme label, a fluorescent label, a chemiluminescent label, a phosphorescent label, a radioactive label, a colloidal gold, colored particles, magnetic particles, and biotin / avidin. The method described. 12. The method of claim 8, wherein the antibody is polyclonal or monoclonal. 13. The method of claim 8, wherein the CLN2p amount in the patient sample is at least 2-fold greater than the CLN2p amount in a normal control. 14. A step of measuring an enzyme activity level of CLN2p in a breast tissue or vascular fluid test sample; and a CLN2p activity level measured in the test sample,
A method of breast cancer diagnosis in an individual undergoing testing, which comprises comparing the CLN2p activity level in a normal sample control with an increase in CLN2p activity of the test sample relative to a control sample to diagnose breast cancer in the individual. 15. The method of claim 14, wherein the vascular fluid test sample is selected from the group consisting of blood, serum and plasma. 16. The method of claim 14, wherein the vascular fluid test sample is acidified prior to measuring CLN2p activity. 17. The test sample is acidified to have a pH between about 3 and about 5.5.
The method of claim 16. 18. The method of claim 14, wherein the CLN2p activity in the test sample is at least about 2 to 17 times greater than the CLN2p activity in the normal sample control. 19. The method of claim 14, wherein CLN2p activity is measured by the amount of enzyme substrate hydrolyzed by CLN2p in a test sample versus a normal control sample. 20. The substrate is leucine-aminotrifluoromethylcoumarin (L-
20. The method of claim 19, wherein the substrate and L-AFC product that have been hydrolyzed to AFC) product and that remain are detected by high performance liquid chromatography. 21. (a) Assaying an aliquot of a vascular fluid sample to determine CLN2p activity; and (b) comparing CLN2p activity in a test sample with CLN2p activity in a normal non-cancerous sample. A method for diagnosing breast cancer in a vascular fluid sample obtained from an individual undergoing a test, which comprises the steps of diagnosing breast cancer by increasing test sample CLN2p activity relative to normal sample CLN2p activity. 22. The method of claim 21, wherein the vascular fluid test sample is selected from the group consisting of blood, serum and plasma. 23. The method of claim 22, wherein the vascular fluid test sample is plasma. 24. The method of claim 21, wherein the CLN2p activity in the test sample is at least about 2 to 17 times greater than the CLN2p activity in the normal sample. 25. a) acidifying a vascular fluid sample to a pH between about 3.0 and about 5.5; b) assaying the acidified sample for CLN2p activity; and c) individual From an individual undergoing a test to diagnose breast cancer by increasing the CLN2p activity in a test sample relative to the CLN2p activity in a normal non-cancerous sample, including comparing the CLN2p activity in the test sample to the CLN2p activity in a normal non-cancerous sample. Breast cancer diagnostic method by determination of CLN2p enzyme level in the obtained vascular fluid sample. 26. The method of claim 25, wherein the vascular fluid test sample is blood, serum or plasma. 27. The method of claim 25, wherein the vascular fluid test sample is plasma. 28. Determining CLN2 proteolytic enzyme levels in a breast tissue or vascular fluid test sample obtained from a patient undergoing a test for cancer; and determining the CLN2 proteolytic enzyme level in the patient sample to be normal. Increasing the level of CLN2 proteolytic enzyme in a patient's test sample relative to the level of CLN2 proteolytic enzyme in a normal sample, including the step of comparing to the level of CLN2 proteolytic enzyme in the sample, can detect or diagnose breast cancer in the patient An assay for detecting or diagnosing breast cancer, such as allows. 29. The CLN2 proteolytic enzyme level in a patient test sample is compared to cathepsin D level; estrogen receptor level; and progesterone receptor level in the same patient sample; and further, normal non-cancerous. CLN2 in sample control
Elevated levels of CLN2 proteolytic enzyme, cathepsin D, estrogen receptor, and / or progesterone receptor in a test sample compared to proteolytic enzyme, cathepsin D, estrogen receptor, and / or progesterone receptor levels are 29. The assay method according to claim 28, which allows the diagnosis of breast cancer. 30. The assay method of claim 28, wherein the vascular fluid test sample is selected from the group consisting of blood, serum and plasma. 31. The assay method of claim 30, wherein the vascular fluid test sample is plasma. 32. The method of claim 28, wherein the level of CLN2 proteolytic enzyme is detected using high performance liquid chromatography. 33. The detected or diagnosed breast cancer is selected from the group consisting of primary breast cancer, non-primary breast cancer and metastatic breast cancer, 1, 8, 14, 21, 25 and
28. The method according to any one of 28. 34. a) an anti-CLN2p specific antibody in an amount suitable for at least one assay for detecting the presence or amount of CLN2p or a fragment thereof in a sample; b) a reagent concentration and / or a mixture. Instructions, including a description of at least one assay parameter selected from the group of reagent and sample relative amounts, retention time of the reagent and sample mixture, temperature and buffer conditions; and optionally c). CLN2p in a biological sample or vascular fluid with a detectable label or indicator for signaling complex formation of CLN2p, or a bindable fragment thereof, with an anti-CLN2p antibody in the sample Diagnostic system for detecting.