JP2001500847A - Novel method of detecting and treating cancer - Google Patents

Abstract

  (57) [Summary] The present invention provides new methods for diagnosing and treating cancer or BPH. Further, a therapeutic agent and a pharmaceutical composition for treating cancer and BPH are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION                       Novel method of detecting and treating cancer Related applications   This application is a continuation-in-part of USSN 08/691479, filed August 2, 1996. A part of USSN 08/714744 filed on September 16, 1996 It is a continuation application. The above application is incorporated herein by reference in its entirety. Field of the invention   The present invention relates, in part, to cancers, especially prostate cancer, and benign prostatic hyperplasia (BPH). Newly Established Assays for Diagnosing PLA, and PLA_TwoRegulate activity Drugs and PLA for treating cancer and BPH_TwoIdentify therapeutic agents that modulate activity How to do. Background of the Invention   Prostate cancer is the most common malignancy in adult men except skin cancer Ulcers, an increasingly common health problem in the United States. In 1994, It has been estimated that 38,000 deaths in the United States are due to the disease, This suggests that prostate cancer is the second most common cause of death in the same population as lung cancer. And Diagnosed and treated early when cancer is still confined to the prostate In such cases, the likelihood of healing is extremely high. Therefore, prostate cancer limited in tissue There is a great need for a sensitive method for the detection of   Extracellular phospholipase A_Two(PLA_Two) Enzymes reduce cell proliferation, chemotaxis and inflammation It is thought to regulate various responses, including: Extracellular PLA_TwoTwo major groups of enzymes Loop: spleen or group I and rheumatoid arthritis synovial fluid (RASF) or There is Group II. Group I enzymes are used in digestion and in growth and chemotaxis It also works in the regulation of Currently, RASF-PLA_TwoIs an arthritis, sepsis show It is primarily thought to play a role in inflammatory responses, including shock and lung injury. RASF-PLA_TwoLevels are all involved in the inflammatory response, MR by various factors, including IL-6, interleukin-1 and tumor necrosis factor. Regulated at NA level. Enhanced levels of PLA_TwoProstate cancer activity in rats Although reported in organizations (FH Faas et al.,The Journal of Urology, Vol 156, 243-248, 1996), RA in human prostate cancer or benign prostatic hyperplasia SF-PLA_TwoNo reports of changes in mRNA or polypeptide levels Not.   Maniatis (MOLECULAR CLONING Maniatis, et al., Cold Spring Harbor Labora tory, Cold Spring Harbor, New York). Isolated from cancer (PC), benign prostatic hyperplasia (BPH) and normal prostate (NP) Northern blot analysis was performed on equal amounts of mRNA. Probe is RASF- PLA_TwoWas synthesized from the full-length cDNA encoding The result is RASF-PLA_Twom The ratio of RNA was 10: 0.25: 1 for PC, BPH and NP, respectively. It was shown that. Loading differences were normalized using actin mRNA levels . As a result, RASF-PLA_TwoIs up-regulated in prostate cancer Has been shown to be potentially down-regulated in BPH Was.   Hereinafter, "PLA" used in this specification_Two"Means Group II or RASF-P LA_TwoMeans Summary of the Invention   For these and other purposes, cancer, especially prostate cancer, and benign prostate Methods for treating and treating various forms of abnormal cell proliferation, such as hypertrophy (BPH) Methods and new ways to monitor progression, remission or recurrence This is the subject of the present invention. In addition, cancer, especially prostate cancer, and BPH Screening for therapeutic agents and pharmaceutical compositions for treating abnormal cell proliferation Provide a way to In addition, the treatment of cancer, especially prostate cancer, and BPH The use of such an agent or composition is provided.   Therefore, according to one aspect of the present invention, PLA_TwoBinds to PLA_TwoBlock activation Methods are provided for screening for harmful compounds.   According to another aspect of the present invention, a PLA_TwoTo treat conditions associated with overexpression of Methods of using such inhibitory compounds are provided.   According to yet another aspect of the present invention, a PLA_TwoProvided by antagonist (inhibitor) Is done. PLA_TwoBinds to the binding molecule but only one PLA_Two-Evoked response or one or more PLA_Two-PLA to avoid evoked response_TwoThat mimics Is an antagonist. One PLA_TwoAction or one or more PLA_TwoBlock the action of PLA to harm_TwoOr PLA_TwoMolecule that interacts with PLA_Two Molecules that prevent the expression of are also preferred antagonists.   Other objects, features, advantages and aspects of the present invention will be apparent to those skilled in the art from the following description. It will be. However, the following description and specific examples will be helpful in understanding the present invention. While a better example is shown, it should be understood that it is provided by way of example only. Disclosure Various changes and modifications within the spirit and scope of the claimed invention are read in the following description. And other portions of the present disclosure will be readily apparent to those skilled in the art. U. Detailed description of the invention   The present invention is directed to cells, tissues and body fluids, including determination of normal and abnormal levels. PLA_TwoProtein or PLA_TwoQuantitative and quantitative methods for detecting mRNA levels And both qualitative and diagnostic assays. Thus, for example, a normal control fluid or Is PLA compared to tissue samples_TwoThe present invention for detecting protein overexpression Diagnostic assays may be used to detect the presence of cancer, including prostate cancer. Furthermore, before Presence of prostate specific antigen (PSA), digital tests, and transurethral ultrasound tests Is difficult to distinguish between prostate cancer and BPH,_Two The method of the present invention for quantifying protein levels distinguishes between BPH and prostate cancer It is particularly useful for For example, the PSA diagnostic test that is present ~ 80% of prostate cancer patients with PSA blood levels higher than about 99% of normal 62-81% is detected. In a host-derived sample, the PLA of the invention_TwoSuch as remains Assay techniques that can be used to determine levels of gene expression, are within the skill of the art. Is knowledge. Such assays include radioimmunoassay, reverse transcriptase PCR ( RT-PCR) assay, immunohistochemical assay, in situ high Hybridization assays, competition-binding assays, western blot analysis and And ELISA assays. Of these, ELISA is biological Often preferred for detecting gene-expressed proteins in body fluids. ELIS The A assay is available from PLA when not readily available from commercial sources._TwoAntibodies specific for Preferably, the method first includes preparing a monoclonal antibody. In addition, PLA_Two A reporter antibody that specifically binds to is generally prepared. Release reporter antibody Radioactive, fluorescent or enzymatic reagents, such as horseradish peroxidase Conjugate with a detectable reagent such as elemental or alkaline phosphatase.   To perform the ELISA, a solid support that binds the antibody, such as a polystyrene dish Above, PLA_TwoThe antibody specific for. Then, for example, bovine serum albumin Covers free protein binding sites on dishes by culturing with non-specific proteins I do. The sample to be analyzed is then cultured in a dish, during which time PLA_TwoIs Polysti Binds to the specific antibody bound to the dish. Wash off unbound sample with buffer. PL A_TwoReporter specifically bound to horseradish peroxidase Put the reporter antibody on the plate, and place the reporter antibody on PLA._TwoMonoclonal antibody bound to To be combined. Unbound reporter antibody is then washed away. Then, colorimetric temperament Add a reagent for peroxidase activity containing PLA_TwoFixed to Peroxidase produces a colored reaction product. Colors produced during a given period Depends on the amount of PLA present in the sample._TwoIt is proportional to the amount of protein. Typically, Quantitative results are obtained by comparison with a standard curve. Without limiting the invention, typically For quantitative diagnostic assays, a positive result indicating disease is when the blood level is Three standard deviations of the mean blood level of the population (99.86% of the population) blood levels higher than d deviations).   PLA_TwoIs bound to a solid support and labeled PLA_TwoAnd host-derived The sample passes over the solid support and the amount of detectable label bound to the solid support is PLA_TwoCompetitive assays that can correlate with the amount of   Nucleic acid method as a marker for BPH and cancer, especially prostate cancer_TwomRNA It may be used to detect. Polymerase chain reaction (PCR) and other nucleic acid methods, 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. Wear. For example, reverse transcriptase PCR (RT-PCR) is a method for thousands of other mRNA species. It can be used to detect the presence of specific mRNAs in a complex mixture. This is a powerful technology. In RT-PCR, the mRNA species uses reverse transcriptase. First reverse transcribed into complementary DNA (cDNA); Amplify in a standard PCR reaction. Therefore, RT-PCR is simple for mRNA. A kind of presence is indicated by amplification. Therefore, mRNA is high in cells that produce it. RT-PCR to identify the presence of specific types of cells Can be used.   Clones arranged on a grid (ie, gridding) Detecting the expression of the gene; and It can be used both for quantifying the expression level of a gene. in this way , PLA_TwoThe cDNA encoding the gene is immobilized on a substrate. The substrate is glass, d Including but not limited to trocellulose, nylon or plastic Instead, it may be of an appropriate type. PLA_TwoDNA encoding the clone To the substrate, and then complement RNA or RNA isolated from the tissue of interest. Incubate with the analyte, which may be a sex DNA (cDNA) copy. Substrate binding claw Hybridization between the analyte and the analyte is performed by radioactive labeling of the analyte or Includes secondary molecules designed to detect fluorescent labels or hybrids It can be detected and quantified by several means, including but not limited to: gene Quantification of expression levels by examining signal strength from analytes from known standards This can be done by comparing with. In vitro transcription of target gene to standard, quantitative determination of yield , The material can then be used to generate a standard curve.   The above tests were performed on patients' body fluids such as blood, urine, saliva, tissue biopsy and necropsy material. And samples from tissue extracts (homogenates or solubilized tissues) it can. antibody   PLA_TwoPolypeptides, fragments or other derivatives thereof, or their Analogs, or cells that express them, are immune to producing antibodies against them. Can be used as a source. These antibodies are, for example, polyclonal and monoclonal. It can be a noclonal antibody. The present invention also includes chimeric, single chain, human Of Activated Antibodies and Fab Fragments or Fab Expression Libraries Things are included. Antibodies and fragments using various methods known in the art May be used for the production of   PLA_TwoAntibodies raised against direct injection of the polypeptide into animals, Can be obtained by administering the polypeptide, preferably to a non-human animal. it can. The antibody thus obtained then binds to the polypeptide itself . Thus, even if the sequence encodes only a polypeptide fragment, It can be used to generate antibodies that bind the native polypeptide of the body.   When preparing monoclonal antibodies, antibodies produced by continuous cell line cultures Any technique that provides a can be used. Examples include hybridoma technology (Kohler, G. and Milstein, C., Nature256: 495-497 (1975)), trioma technology , Human B-cell hybridoma post-surgery (Kozbor et al., Immunology Today,Four: 72 (1 983)) and EBV-hybridoma for producing human monoclonal antibodies Technology (Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss , Inc., pg. 77-96 (1985)).   The described technology for producing single-chain antibodies (US Pat. No. 4,946,778) Can be used to produce single-chain antibodies to the immunogenic polypeptide products of the invention. Can be. Also, transgenic mice or other organisms, such as other mammals Immunogenic PLA using animals_TwoMay be expressed.   Therefore, among other things, PLA_TwoVarious antibodies, including prostate cancer, using antibodies against Types of cancer and BPH may be treated / inhibited. PLA_TwoBinding molecules and assays   PLA_TwoCan be used to isolate proteins that interact with it, Can be targets of interference. PLA_TwoAnd proteins between other factors PLA by inhibitors of quality-protein interactions_TwoLaunch drugs to regulate activity Can emit. The term "modulate" as used herein refers to PLA_TwoMachine Means to affect performance.   Therefore, the present invention also provides PLA_TwoThe present invention provides a method for identifying a binding molecule to PLA_TwoA gene encoding a protein of a binding molecule against, for example, Ligan Various methods known to those skilled in the art, such as dopanning and FACS sorting. Can be identified. Such methods are described, for example, in Coligan et al., Current Protocols in  Immunology 1 (Rivett, AJ Biochem.291: 1-10 (1993)): Chapter 5 (1991) And many other laboratory manuals.   For example, the yeast two-hybrid system re-establishes the activity of transcription activators. Using construction, in vivo, a first test protein and a second test protein A method is provided for detecting an interaction between the two. The method is disclosed in U.S. Pat. No. 5,283,173. Reagents are available from Clontech and Stratagene. Easily , PLA_TwoThe cDNA was fused to the Gal4 transcription factor DNA binding domain and Expression in the vesicle. The cDNA library members obtained from the cells of interest Fused to the transcriptional activation domain of 14. PLA_TwoEmit proteins that can interact with Depending on the cDNA clones that appear, Gal4 activity and Gal1-lacZ The transcriptional activity of reporter gene expression is reconstituted.   An alternative is to use recombinant PLA_TwoΛgt11, λZAP (Stratagene) or Screening of an equivalent cDNA expression library. Recombinant PLA_TwoTan The protein or its fragments are ligated to a small marker such as FLAG, HSV or GST. Fused to peptide tag. Peptide tags are typically used for cardiac creatine kinase Can have a kinase phosphorylation site or can be biotinylated Can be Recombinant PLA_TwoIs³²Can be phosphorylated with [P] or unlabeled It can be used and can be detected with streptavidin or an antibody against the tag. A λgt11 cDNA expression library was created from the cells of interest and recombinant PLA_Two And washed, PLA_TwoA cDNA clone that interacts with is isolated. T. Ma niatis et al. See (supra).   Another approach is to transfer the cDNA between a mammalian promoter and a polyadenylation site. Cloned into a vector and temporarily transfected into COS or 293 cells And then labeled PLA_Two, Preferably iodinated PLA_TwoAnd fixed and washed The bound protein is detected after 48 hours by culturing the PLA combined by radiography_TwoDetecting mammalian expression libraries Screening. Sims et al., Science241: 585-589 (1988) and McMahan  et al., EMBO J .;Ten: 2821-2832 (1991). Thus, the desired binding tank A pool of cDNAs containing cDNAs encoding proteins can be selected, and the desired cDN A was used for further subdivision of each pool, followed by transient transfection, binding and And autoradiography cycles. Separately, the cDNA of interest Transfect the entire cDNA library into mammalian cells and PLA coupled to_TwoCan be isolated by panning the cells on a dish containing After washing, cells that adhere after lysis are lysed and plasmid DNA is isolated and amplified in bacteria. Transfection and panning until a single cDNA clone is obtained Repeat the cycle. Seed et al., Proc. Natl. Acad. Sci. USA84: 3365 (1 987) and Aruffo et al., EMBO J .;6: 3313 (1987). Binding protein If secreted, assay supernatant from transiently transfected cells Once a binding or neutralization assay has been established for It can be obtained by the pooling method. Screen the supernatant General methods are described in Wong et al., Science228: 810-815 (1985).   Another alternative is PLA_TwoIsolation of proteins interacting directly with cells It is. PLA with GST or other small peptide tags_TwoA fusion protein And fix on beads. Biosynthetically labeled or labeled from the cells of interest A fresh protein extract is prepared, incubated with the beads, and washed with buffer. PLA_TwoProteins that interact with PEG are specifically eluted from the beads and analyzed by SDS-PA Analyze by GE. Binding partner primary amino acid sequence data Obtained by Thing. Optionally, cells can be tyrosine phosphorylated on cellular proteins And can be treated with agents that elicit a functional response. Examples of such agents include growth factors. Or cytokines such as interleukin-2.   Another alternative is immunoaffinity purification. Recombinant PLA_TwoLabeled or unlabeled cells Culture with extract, anti-PLA_TwoImmunoprecipitate with antibodies. Immunoprecipitate was converted to protein A -Collect on Sepharose and analyze by SDS-PAGE. Unlabeled protein Label by otinylation and detect on SDS gel with streptavidin. Join The partner protein is analyzed by microsequencing. In addition, Biochemical purification steps known to the supplier may be used prior to microsequencing. No.   Yet another alternative is to screen peptide libraries for binding partners. It is to do. Recombinantly tagged or labeled PLA_TwoUsing PLA_TwoAnd phase Select interacting peptides from a peptide or phosphopeptide library . Peptide sequencing may show up in interacting peptides A consensus peptide sequence is identified.   The P identified by any of these or other methods known to those skilled in the art LA_TwoBinding assays as well as putative binding partners as described above Can be used in the method. PLA_Two/ The existence of binding partner complex The assays were performed using yeast two-hive, for example, using antibodies specific for the complex. Performed by lid system, ELISA or immunoassay. PLA_Two/ Combined Complex in the presence of a test substance that interferes with or inhibits the formation of Is determined relative to a control lacking the test substance.   Free PLA_TwoOr assays for binding partners, e.g., specific ELISA or immunoassay using antibodies or radiolabeled PLA_Two With a cell or cell membrane, followed by a centrifugation or filter separation step. Do. PLA_Two/ Interfere with or inhibit the formation of binding partner interactions Free PLA in the presence of test substance_TwoOr increased amount of free binding partner Determined in comparison to a control without the test substance.   The polypeptides of the present invention can also be used in cells or cell-free preparations._Two PLA of binding molecule_TwoCan be used to assess binding capacity. Agonists and antagonists-assays and molecules   PLA_TwoPLA_TwoScreen for compounds that inhibit, promote or regulate It may be used in a leaning step. PLA_TwoOne standard about The assay uses [linoleoyl-1-¹⁴C] Labeled L-α-1-acyl-2 Using linoleoyl phosphatidylethanolamine,¹⁴Of C-labeled free fatty acids Track release. Using this assay or others, PLA_TwoAgonists Alternatively, any of the antagonists can be identified.   Potential PLA_TwoAn example of an antagonist is PLA_TwoBinds and interferes with enzyme activity Small molecules, such as organic molecules or peptides, antibodies, or, in some cases, oligos Nucleotides.   Potential antagonists include PLA, eg, fragments of binding molecules._Two Is closely related to the binding molecules of_TwoConjugation with polypeptide Includes small molecules or proteins that, when combined, inhibit its activity. (This specification The term "binding molecule" as used herein refers to the PLA of the present invention._TwoSpecifically binds to a polypeptide Refers to molecules that associate or interact with each other. The definition of the binding molecule is PLA_TwoActivity Other factors, co-factors, units or Or subunits. Such binding molecules are part of the present invention. Join The molecule is PLA_TwoNon-natural products such as antibodies and antibody-derived reagents that specifically bind to There may be. )   Potential antagonists also include antibodies prepared using antisense technology. The chisense construct. Using antisense technology, antisense DNA or Can control gene expression by triple-helix formation of RNA. Both of these Is based on the binding of a polynucleotide to DNA or RNA. For example, Mature PLA_TwoUsing the 5 'coding region of the polynucleotide sequence encoding Design an antisense RNA oligonucleotide about 10-40 base pairs in length. DNA oligonucleotides are designed complementary to the region of the gene involved in transcription (tri plehelix-Lee et al., Nucl. Acids Res.,6: 3073 (1979); Cooneyetal., Scien ce241: 456 (1988); Dervan et al., Science,2511360 (1991)) PLA_TwoPrevents transcription and production of the polypeptide. Antisense RNA oligo The nucleotide hybridizes to the mRNA in vivo and the PL of the mRNA molecule A_TwoBlocking translation into polypeptides (antisense-Okano, J. Neurochem.,Five 6 : 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expres sion, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above To deliver cells and express antisense RNA or DNA in vivo. , PLA_TwoPolypeptide production can also be inhibited. In this delivery , Gene therapy.   Another potential antagonist is PLA_TwoAnd bind this to a binding molecule (eg, , Substrates), which are inaccessible and interfere with normal biological activity Is a molecule. Examples of small molecules include, but are not limited to, small peptides or peptides. Peptide-like molecules and organic compounds are included.   PLA_TwoIs ubiquitous in animal hosts and has many biological functions, including many etiologies. Involved. Therefore, PLA_TwoFor compounds and drugs that can inhibit the function of It is desired.   In addition, the present invention is directed to excess forms of cancer, including various forms of cancer, including BPH and prostate cancer. PLA_TwoA method of treating an abnormal condition related to activity, comprising the method of inhibiting as described above. Compound (antagonist) together with a pharmaceutically acceptable carrier_TwoActivity directly Or binding molecule PLA_TwoBy blocking binding to the polypeptide Provided is a method comprising administering to a subject in an amount effective to inhibit. Compositions and kits   PLA_TwoMay be used in combination with a suitable pharmaceutical carrier. . Such compositions comprise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable amount. It contains an acceptable carrier or excipient. Such carriers include, but are not limited to, foods. Saline, buffered saline, dextrose, water, glycerol, ethanol and its Include combinations. The formulation must suit the mode of administration.   The present invention further comprises one or more components of the composition of the present invention described above. Pharmaceutical packs and kits containing one or more filled containers. Administration   The polypeptides and other compounds of the present invention can be used alone or as a therapeutic compound. It may be used in combination with any other compound.   Pharmaceutical compositions include, for example, topical, oral, anal, vaginal, intravenous, intraperitoneal, among others. Any effective, including administration by intra, intramuscular, subcutaneous, nasal or intradermal routes It may be administered in a conventional manner.   Generally, a pharmaceutical composition is effective in treating or preventing a particular indication or multiple indications It is administered in an amount. Generally, the composition will comprise at least about 10 μg / kg body weight Is administered in an amount of In most cases, these are about Will be administered in no more than 8 mg. Preferably, in most cases, The amount is from about 10 μg to about 1 mg / kg body weight per day. The optimal dosage is Considering the indication, its severity, route of administration, complications, etc., each treatment modality And indications are determined by standard methods. vaccine   Another aspect of the invention is a method of eliciting an immunological response in an animal, particularly a mammal. PLA suitable for producing antibodies_TwoOr its fragment or Inoculates an animal with a variant and treats the animal with various forms of cancer, including BPH or prostate cancer. From defending against fire. Yet another aspect of the invention relates to a method of A method of eliciting an immunological response, comprising eliciting an immunological response and producing antibodies. PLA in vivo to protect the animal from disease_TwoOr its fragment PLA by gene therapy to express_TwoOr its A method comprising delivering a gene encoding a fragment or variant. Related.   A further aspect of the present invention is a method wherein, when introduced into an animal, particularly a mammalian host, its dynamics. PLA in things_TwoImmunity to genes or proteins encoded from them An immunological composition for eliciting a biological response, comprising:_TwoGene or Recombinant PLA containing DNA expressing and encoding the antigen of the protein to be loaded_Two It relates to a composition comprising a gene or a protein encoded therefrom.   PLA_TwoOr its fragments do not themselves produce antibodies, but Fusion proteins that will have protein stabilization and immunogenic and protective properties It may be fused with a co-protein capable of producing quality. This Such a fusion recombinant protein is preferably further provided with glutathione-S-trans Antigenic complement proteins such as spherase (GST) or beta-galactosidase Relatively large to solubilize proteins and proteins and facilitate their production and purification. Includes a major coprotein. In addition, coproteins provide systemic stimulation of the immune system In addition, it may act as an adjuvant. Coprotein as first protein May be attached to either the amino or carboxy terminus.   The present invention also relates to a vaccine comprising the immunogenic recombinant protein together with a suitable carrier. Also included. Since proteins can be broken down in the stomach, parenteral administration (eg, skin Lower, intramuscular, intravenous, intradermal, etc.). Suitable for parenteral administration The prescription includes antioxidants, buffers, antimicrobial agents, and the prescription with the recipient's blood. Aqueous and non-aqueous sterile injectable solutions, which may contain isotonic solutes; suspending agents; Or sterile aqueous and non-aqueous suspensions which may contain a thickening agent. The prescription is Unit or multi-dose containers, for example, sealed ampules and vials Lyophilization, which may be present in the medium and requires only the addition of a sterile liquid carrier immediately before use It may be stored in a state. Vaccine prescriptions also include immunogenicity of prescriptions such as oil-in-water To With adjuvant systems to enhance and other systems known in the art Good. The dose depends on the specific activity of the vaccine and is easily determined by routine experimentation it can.   PLA_TwoHas been described, but this description Additions or deletions that do not substantially affect the immunogenicity of fragments and recombinant proteins A similar protein with a loss or substitution (eg, 50% or more (With sequence homology).   The present invention also provides PLA_TwoAltered PLA for the production of animals with induced disease_Two Methods for producing transgenic animals having levels are provided. Non-human tiger The transgenic animal may be a fertilized egg or embryo of a suitable host, LA_TwoMay be obtained by transfecting with a nucleic acid encoding For example U.S. Patent Nos. 4,736,866; 5,175,385; 5,175,384 and 5,175. See 386. PLA modified transgenic animals obtained_TwoLebe May be used as a model for studying Particularly useful transgenics Animals are PLA_TwoDisplay detectable phenotype associated with altered expression of polypeptide Is what you do. The ability to then restore or worsen the phenotype associated with the drug You may screen for strength. Example   The present invention is further described by the following examples. The embodiments merely refer to specific examples. It is provided to explain the invention by reference. These examples are specific to the present invention. However, the present invention does not limit the scope of the disclosed invention.   All examples are standard and well known to those skilled in the art unless otherwise described. Performed using technology. Conventional molecular biology techniques of the following examples are described in Sambrook et al. , MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed .; Cold Spring Harbor La boratory Press, Cold Spring Harbor, N.Y. (1989) (herein, "Sam brook "). Can be.   Unless otherwise specified, all parts and amounts described in the following examples are by weight. Depends on quantity.   Unless otherwise noted, the size separation of the fragments in the following examples was performed by Sambro ok or for example Goeddel et al., Nucleic Acids Res.8: Others such as 4057 (1980) Agarose and polyacrylamide gel electrophoresis described in many references (PAGE) using standard techniques. Example 1   mRNA is prepared from normal and cancerous prostate tissue and sized on agarose gel. The fractions are then transferred to a nylon membrane (Northern blot). The blot is then radiated Activity [³²PLA labeled with [P] -dCTP_TwoHybridize to specific cDNA probe Let it go. After incubation, the blots are washed under stringent conditions (Sambrook ) And then exposed to film. MRNA levels as a function of disease Changes in the strength of the signal seen in diseased versus normal tissue Seen as change. Example 2   The following is the PLA in serum observed by the ELISA assay._TwoLevel These are the results. A. Healthy men without prostate disease (17 individuals): 4.85 ng / ml ± 3.2 9 B. Healthy women (20 individuals): 5.31 ng / ml ± 3.44 C. Mixed healthy men and women (37 individuals): 5.11 ng / ml ± 3.33 D. Patients with advanced prostate cancer (10 individuals): 32.65 ng / ml ± 18. 7   Eight (80%) of 10 patients with advanced prostate cancer are healthy men and women PLA at least three standard deviations above the mixed mean_TwoBlood level. E. FIG. Patients with prostate cancer in remission (10 individuals): 7.77 ng / ml ± 8. 67   Only 1 out of 9 patients (11.1%) with prostate cancer in remission are healthy men Above the standard deviation of at least 3 times the female and mixed mean_TwoAt blood level Was. F. Patients with stabilized prostate cancer (5 individuals): 8.74 ng / ml ± 8.12   Only one (20%) of five patients with stabilized prostate cancer are healthy men and women Above the standard deviation at least 3 times the sexual mixed mean_TwoBlood level. G. FIG. Patients with benign prostatic hyperplasia (13 individuals): 4.59 ng / ml ± 3.2 9   PLA above at least 3-fold standard deviation of the healthy male and female mixed mean_Twoblood No patient had a level of benign prostatic hyperplasia. H. Patients with prostatitis (7 individuals): 8.47 ng / ml ± 9.73   Only 1 (14.3%) of 7 patients with prostatitis are healthy men and women PLA at least three standard deviations above the mixed mean_TwoBlood level.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 35/00 A61P 43/00 111 43/00 111 A61K 31/7105 // A61K 31/7105 31/711 31 / 711 C12N 9/00 C12N 9/00 9/20 9/20 G01N 33/15 Z G01N 33/15 33/573 A 33/573 33/574 A 33/574 33/68 33/68 A61K 37/54 ( 81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY) , KG, KZ, MD, RU, J, TM), AL, AM, AU, BB, BG, BR, CA, CN, CZ, EE, GE, GH, HU, IL, IS, JP, KG, KP, KR, LK, LR, LT, LV, MD, MG, MK, MN, MX, NO, NZ, PL, RO, SG, SI, SK, TR, TT, UA, US, UZ, YU (72) Inventor Wilkinson, Francis E United States 19355 Pennsylvania 307, Charleston Green, Malvern

Claims (1)

[Claims] 1. A method for treating cancer comprising administering to a patient a therapeutically effective amount of a PLA ₂ antagonist. 2. The method according to claim 1, wherein the cancer is prostate cancer. 3. Cells of an individual with prostate cancer, comprising analyzing for abnormally high levels of PLA ₂ polypeptide in tissues and body fluids, diagnostic methods for cancer. 4. The diagnostic method according to claim 3, wherein the cancer is prostate cancer. 5. Diagnostic method for identifying an individual cell, tissue and analyzing for the abnormally high levels of PLA ₂ polypeptide in a body fluid, prostate cancer and BPH or normal prostate tissue with prostate cancer. 6. 4. The method of claim 3, wherein the diagnostic process comprises an ELISA. 7. 4. The method according to claim 3, wherein the diagnostic process is immunohistochemistry. 8. Method of diagnosing cancer or BPH comprising analyzing for the abnormally high or low transcription levels PLA _2. 9. 9. The method according to claim 8, wherein the method is Northern blot analysis. 10. The method according to claim 8, which is RT-PCR. 11. 9. The method according to claim 8, wherein the method is in situ hybridization. 12. 9. The method of claim 8, wherein the method is gridding. 13. 9. The diagnostic method according to claim 8, wherein the cancer is prostate cancer. 14． The diagnostic method according to claim 8 for BPH. 15. PLA ₂ to use a specific enzymatic assay methods bind to PLA _2, to identify the inhibition or activation compounds. 16. Method of inducing immunological response in produce antibodies, animals sufficient to protect against cancer, animal that includes a PLA ₂ or a fragment or variant thereof is inoculated into animals. 17． Inoculated with PLA ₂ to the animal, cancer, in particular that to produce antibodies for use in diagnostic tests for prostate cancer, and BPH, a method of inducing immunological response in the animal. 18. PLA ₂ levels altered production method of a transgenic animal for the animal production with PLA ₂ induced diseases.