Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-11-03
2003-12-23
Fay, Zohreh (Department: 1614)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C436S086000, C436S105000
Reexamination Certificate
active
06667291
ABSTRACT:
FIELD OF THE INVENTION
This invention relates, in part, to newly developed assay for diagnosing cancers, particularly endometrial and mammary, endometriosis and endometrial fibroids along with methods for identifying therapeutic agents that modulate Endometrial steroid binding protein II activity for treatment of the above disorders.
BACKGROUND OF THE INVENTION
Endometrial cancer occurs at a rate of approximately 44,500 new cases per year with approximately 10,000 deaths per year. If diagnosed and treated early, when the cancer is still confined to the endometrium, cure can be achieved in approximately 95% of the cases by hysterectomy. Pap smears can show endometrial cancers but are effective in only 50% of the cases. For the remainder, abnormal vaginal bleeding is typically the first clinical sign of endometrial cancer. There is a great need for sensitive methods for the detection of organ-confined endometrial cancer.
Steroid binding proteins, including uteroglobin and CC10, are a class of proteins which bind steroids along with methylsulfonyl metabolites of polychlorinated biphenyls. The exact function of members of this class of protein is uncertain, but uteroglobin has been shown to inhibit PLA
2
mediated responses. The gene and gene product of the present invention display homology to uteroglobin and CC10, show elevated expression of mRNA in endometrial cancer samples and is expressed in mammary tissue. This gene encoded product will be referred to as Endometrial Steroid Binding Protein II (ESBPII), and their polypeptide and polynucleotide sequences are given in Table 1 and 2, respectively.
SUMMARY OF THE INVENTION
Toward these ends, and others, it is an object of the present invention to provide a new method for diagnosing, treating, and monitoring progression, remission or recurrence of various forms of abnormal cell growth, such as cancers, particulary endometrial and mammary cancer, and endometriosis and endometrial fibroids. Further provided are methods to screen for therapeutic agents and pharmaceutical compositions for treating abnormal cell growth, such as cancers, particular endometrial and mammary cancer, and endometriosis and endometrial fibroids. Further provided is the utilization of such agents or compositions for the treatement abnormal cell growth, particulary endometrial and mammary cancer and endometriosis and endometrial fibroids.
Thus, in accordance with one aspect of the present invention there are provided methods of screening for compounds which bind to and inhibit activation of the ESBPII.
In accordance with another aspect of the present invention there is provided a method of using such inhibiting compounds for treating conditions associated with over-expression of the ESBPII.
In accordance with yet another aspect of the present invention, there are provided ESBPII antagonists (inhibitors). Among the preferred antagonists are those which mimic ESBPII so as to bind to ESBPII binding molecules but not elicit a ESBPII -induced response or more than one ESBPII -induced response. Also among the preferred antagonists are molecules that bind to or interact with ESBPII so as to inhibit an effect of ESBPII or more than one effect of ESBPII or which prevent expression of ESBPII.
Other objects, features, advantages and aspects of the present invention will become apparent to those of skill in the art from the following description. It should be understood, however, that the following description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.
DESCRIPTION OF THE INVENTION
The present invention relates to diagnostic assays, both quantitative and qualitative for detecting levels of ESBPII protein or ESBPII mRNA in cells, tissues and bodily fluids, including determination of normal and abnormal levels. Thus, for instance, a diagnostic assay in accordance with the invention for detecting over-expression of ESBPII protein compared to normal control bodily fluids or tissue samples may be used to detect the presence of cancers, including endometrial and mammary cancer. Assay techniques that can be used to determine levels of gene expression, such as ESBPII of the present invention, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, reverse transcriptase PCR (RT-PCR) assays, gridding, immunohistochemistry assays, in situ hybridization assays, competitive-binding assays, Western Blot analyses and ELISA assays. Among these, ELISAs are frequently preferred to detect a gene's expressed protein in biological fluids. An ELISA assay initially comprises preparing an antibody, if not readily available from a commercial source, specific to ESBPII, preferably a monoclonal antibody. In addition a reporter antibody generally is prepared which binds specifically to ESBPII. The reporter antibody is attached to a detectable reagent such as radioactive, fluorescent or enzymatic reagent, for example horseradish peroxidase enzyme or alkaline phosphatase.
To carry out the ELISA, antibody specific to ESBPII is incubated on a solid support, e.g. a polystyrene dish, that binds the antibody. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the sample to be analyzed is incubated in the dish, during which time ESBPII binds to the specific antibody attached to the polystyrene dish. Unbound sample is washed out with buffer. A reporter antibody specifically directed to ESBPII and linked to horseradish peroxidase is placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to ESBPII. Unattached reporter antibody is then washed out. Reagents for peroxidase activity, including a colorimetric substrate are then added to the dish. Immobilized peroxidase, linked to ESBPII antibodies, produces a colored reaction product. The amount of color developed in a given time period is proportional to the amount of ESBPII protein present in the sample. Quantitative results typically are obtained by reference to a standard curve. Without limiting the instant invention, typically, for a quantitative diagnostic assay a positive result indicating the disease is one in which blood levels are higher than three standard deviations above the mean blood level for a normal healthy population of individuals (99.86% of the population).
A competition assay may be employed wherein antibodies specific to ESBPII attached to a solid support and labeled ESBPII and a sample derived from the host are passed over the solid support and the amount of label detected attached to the solid support can be correlated to a quantity of ESBPII in the sample.
Nucleic acid methods may be used to detect ESBPII mRNA as a marker for abnormal cell growth including endometrial cancer, mammary cancer, endometriosis and endometrial fibroids. Polymerase chain reaction (PCR) and other nucleic acid methods, such as ligase chain reaction (LCR) and nucleic acid sequence based amplification (NASABA), can be used to detect malignant cells for diagnosis and monitoring of various malignancies. For example, reverse-transcriptase PCR (RT-PCR) is a powerful technique which can be used to detect the presence of a specific mRNA population in a complex mixture of thousands of other mRNA species. In RT-PCR, an mRNA species is first reverse transcribed to complementary DNA (cDNA) with use of the enzyme reverse transcriptase; the cDNA is then amplified as in a standard PCR reaction. RT-PCR can thus reveal by amplification the presence of a single species of mRNA. Accordingly, if the mRNA is highly specific for the cell that produces it, RT-PCR can be used to identify the presence of a specific type of cell.
Hybridization to clones arrayed on a grid ca
Schmidt Carl J
Wang Xin-Min
Fay Zohreh
Licata & Tyrrell P.C.
SmithKline Beecham Corporation
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