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WO1998005349A1 - Nouveau procede de detection et de traitement du cancer - Google Patents

Nouveau procede de detection et de traitement du cancer Download PDF

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Publication number
WO1998005349A1
WO1998005349A1 PCT/US1997/013550 US9713550W WO9805349A1 WO 1998005349 A1 WO1998005349 A1 WO 1998005349A1 US 9713550 W US9713550 W US 9713550W WO 9805349 A1 WO9805349 A1 WO 9805349A1
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WO
WIPO (PCT)
Prior art keywords
pla2
cancer
prostate cancer
diagnostic
bph
Prior art date
Application number
PCT/US1997/013550
Other languages
English (en)
Inventor
Frank L. Tobin
Carl J. Schmidt
Francis E. Wilkinson
Original Assignee
Smithkline Beecham Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/714,744 external-priority patent/US5747264A/en
Application filed by Smithkline Beecham Corporation filed Critical Smithkline Beecham Corporation
Priority to CA002262624A priority Critical patent/CA2262624A1/fr
Priority to JP10508081A priority patent/JP2001500847A/ja
Priority to EP97934361A priority patent/EP0918532A4/fr
Priority to AU37439/97A priority patent/AU3743997A/en
Publication of WO1998005349A1 publication Critical patent/WO1998005349A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/918—Carboxylic ester hydrolases (3.1.1)
    • G01N2333/92—Triglyceride splitting, e.g. by means of lipase

Definitions

  • This invention relates, in part, to newly developed assay for diagnosing cancers, particularly prostate cancer, and benign prostate hyperplasia (BPH), and methods for identifying agents which modulate PLA2 activity and therapeutic agents that modulate PLA2 activity for treating cancers and BPH
  • Extracellular Phospho pase A2 (PLA2) enzymes appear to mediate a variety of responses including cellular proliferation, chemotaxis and inflammation
  • PHA2 enzymes pancreatic or group I and rheumatoid arthritis synovial fluid (RASF) or group II.
  • RASF-PLA2 functions in digestion and also in modulating proliferation and chemotaxis
  • RASF-PLA2 is predominantly thought to play a role in inflammatory responses including arthritis, septic shock and lung injury
  • the level of RASF- PLA2 is regulated at the mRNA level by a variety agents including ⁇ nterleuk ⁇ n-6, ⁇ nterleuk ⁇ n-1 and tumor necrosis factor, all of which are involved in inflammatory responses
  • elevated leveis of PLA2 enzyme activity have been reported in a prostate cancer tissue in rats (F H. Faas et al , The Journal of Urology. Vol 156, 243- 248, 1996), there do not appear to be any reports of alterations of RASF-PLA2 mRNA or polypeptide level in prostate cancer or benign prostate hyperplasia in humans
  • PLA2 antagonists include those which mimic PLA2 so as to bind to PLA2 binding molecules but not elicit a PLA2- mduced response or more than one PLA2- ⁇ nduced response
  • preferred antagonists include molecules that bind to or interact with PLA2 so as to inhibit an effect of PLA2 or more than one effect of PLA2 or which prevent expression of LA2
  • the present invention relates to diagnostic assays, both quantitative and qualitative for detecting levels of PLA2 protein or PLA2 mRNA in cells, tissues and bodily fluids, including determination of normal and abnormal levels
  • a diagnostic assay in accordance with the invention for detecting over- expression of PLA2 protein compared to normal control bodily fluids or tissue samples may be used to detect the presence of cancers, including prostate cancer
  • the present method of quantifying protein PLA2 protein level is particularly useful for discriminating between BPH and prostate cancer, since the existing methods such as prostatic specific antigen (PSA), digital examination, and transurethral ultrasound tests have difficulty discnminating between prostate cancer and BPH
  • PSA prostatic specific antigen
  • the existing PSA diagnostic tests detect 20-28% of BPH patients and 62- 81% of prostate cancer patients with PSA blood levels above approximately 99% of the normal population Assay techniques that can be used to determine levels of gene expression, such as PLA2 of the present invention, in a sample derived from a host are well-known to those of skill in the art
  • An ELISA assay initially comprises preparing an antibody, if not readily available from a commercial source, specific to PLA2, preferably a monoclonal antibody.
  • a reporter antibody generally is prepared which binds specifically to PLA2.
  • the reporter antibody is attached to a detectable reagent such as radioactive, fluorescent or enzymatic reagent, for example horseradish peroxidase enzyme or alkaline phosphatase.
  • PLA2 antibody specific to PLA2 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 se ⁇ jm albumin.
  • a non-specific protein such as bovine se ⁇ jm albumin.
  • PLA2 binds to the specific antibody attached to the polystyrene dish. Unbound sample is washed out with buffer.
  • a reporter antibody specifically directed to PLA2 and linked to horseradish peroxidase is placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to PLA2- Unattached reporter antibody is then washed out.
  • Reagents for peroxidase activity including a colorimetric substrate are then added to the dish.
  • the amount of color developed in a given time period is proportional to the " amount of PLA2 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 PLA2 attached to a solid support and labeled PLA2 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 PLA2 in the sample.
  • Nucleic acid methods may be used to detect PLA2 mRNA as a marker for BPH and cancer, particularly prostate cancer.
  • 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 monitonng of various malignancies.
  • LCR ligase chain reaction
  • NASABA nucleic acid sequence based amplification
  • RT-PCR reverse-transc ⁇ ptase PCR
  • cDNA complementary DNA
  • 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 can be used to both detect the expression of and quantitate the level of expression of that gene
  • a cDNA encoding the PLA2 gene is fixed to a substrate.
  • the substrate may be of any suitable type including but not limited to glass, nitrocellulose, nylon or plastic.
  • DNA encoding the PLA2 clone is attached to the substrate and then incubated with the analyte, which may be RNA or a complementray DNA (cDNA) copy of the RNA, isolated from the tissue of interest.
  • Hybridization between the substrate bound clone and the analyte can be detected and quantitated by several means including but not limited to radioactive labeling or fluorescence labeling of the analyte or a secondary molecule designed to detect the hybrid Quantitation of the level of gene expression can be done by comparison of the intensity of the signal from the analyte compared with that determined from known standards
  • the standards can be obtained by in vitro transcription of the target gene, quantitatmg the yield, and then using that material to generate a standard curve.
  • the above tests can be carried out on samples derived from patients' bodily fluids and tissue extracts (homogenates or solubi zed tissue) such as from blood. urine, saliva, tissue biopsy and autopsy material.
  • tissue extracts homogenates or solubi zed tissue
  • the PLA2 polypetide its fragments or other derivatives, or analogs thereof, or celis expressing them can be used as an immunogen to produce antibodies thereto
  • These antibodies can be, for example, polyclonal or monoclonal antibodies
  • the present invention also includes chime ⁇ c, single chain, and humanized antibodies, as well as Fab fragments, or the product of a Fab expression library Various procedures known in the art may be used for the production of such antibodies and fragments
  • Antibodies generated against PLA2 can be obtained by direct injection of the polypeptide into an animal or by administering the polypeptide to an animal, preferably a nonhuman The antibody so obtained will then bind the polypeptide itself
  • any technique which provides antibodies produced by continuous cell line cultures can be used Examples include the hyb ⁇ doma technique (Kohler, G and Milstein, C , Nature 255495-497 (1975)), the t ⁇ oma technique, the human B-cell hyb ⁇ doma technique (Kozbor et al , Immunology Todav 4 72 (1983)) and the EBV-hyb ⁇ doma technique to produce human monoclonal antibodies (Cole et al , pg 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R Liss, Inc (1985)) Techniques described for the production of single chain antibodies (U S Patent
  • PLA2 could be used to isolate proteins which interact with it and this interaction could be a target for interference Inhibitors of protein-protein interactions between PLA2 and other factors could lead to the development of pharmaceutical agents for the modulation of PLA2 activity
  • Inhibitors of protein-protein interactions between PLA2 could lead to the development of pharmaceutical agents for the modulation of PLA2 activity
  • this invention also provides a method for identification of binding molecules to PLA2
  • Genes encoding proteins for binding molecules to PLA2 can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting Such methods are described in many laboratory manuals such as, for instance, Coligan et al., Current Protocols in Immunology 1 (Rivett. A.J. Biochem. J. 29/; 1-10 (1993)): Chapter 5 ( 1991 ).
  • the yeast two-hybrid system provides methods for detecting the interaction between a first test protein and a second test protein, in vivo, using reconstitution of the activity of a transcriptional activator.
  • the method is disclosed in U.S. Patent No. 5,283,173; reagents are available from Clontech and Stratagene. Briefly, PLA2 cDNA is fused to a Gal4 transcription factor DNA binding domain and expressed in yeast cells. cDNA library members obtained from cells of interest are fused to a transactivation domain of Gal4. cDNA clones which express proteins which can interact with PLA2 will lead to reconstitution of Gal4 activity and transactivation of expression of a reporter gene such as GaU-lacZ.
  • An alternative method is screening of ⁇ gtl 1, ⁇ ZAP (Stratagene) or equivalent cDNA expression libraries with recombinant PLA2.
  • Recombinant PLA2 protein or fragments thereof are fused to small peptide tags such as FLAG, HSV or GST.
  • the peptide tags can possess convenient phosphorylation sites for a kinase such as heart muscle creatine kinase or they can be biotinylated.
  • Recombinant PLA2 can be phosphorylated with 32 [p] or used unlabeled and detected with streptavidin or antibodies against the tags, ⁇ gtl IcDNA expression libraries are made from cells of interest and are incubated with the recombinant PLA2, washed and cDNA clones isolated which interact with PLA2. See, e.g., T. Maniatis et al, supra.
  • Another method is the screening of a mammalian expression library in which the cDNAs are cloned into a vector between a mammalian promoter and polyadenylation site and transiently transfected in COS or 293 cells followed by detection of the binding protein 48 hours later by incubation of fixed and washed cells with a labelled PLA2, preferably iodinated, and detection of bound PLA2 by autoradiography.
  • a labelled PLA2 preferably iodinated
  • pools of cDNAs containing the cDNA encoding the binding protein of interest can be selected and the cDNA of interest can be isolated by further subdivision of each pool followed by cycles of transient transfection, binding and autoradiography.
  • the cDNA of interest can be isolated by transfecting the entire cDNA library into mammalian cells and panning the cells on a dish containing PLA2 bound to the plate Cells which attach after washing are lysed and the plasmid DNA isolated, amplified in bacteria, and the cycle of transfection and panning repeated until a single cDNA clone is obtained See Seed et al, Proc Natl Acad Sci USA 84 3365 ( 1987) and Aruffo et al , EMBO J 6 3313 ( 1987) If the binding protein is secreted, its cDNA can be obtained by a similar pooling strategy once a binding or neutralizing assay has been established for assaying supematants from transiently transfected cells General methods for screening supe
  • Biosynthetically labeled or unlabeled protein extracts from the cells of interest are prepared, incubated with the beads and washed with buffer. Proteins interacting with PLA2 are eluted specifically from the beads and analyzed by SDS-PAGE Binding partner primary ammo acid sequence data are obtained by microsequencing
  • the cells can be treated with agents that induce a functional response such as tyrosine phosphorylation of cellular proteins
  • agents that induce a functional response such as tyrosine phosphorylation of cellular proteins
  • An example of such an agent would be a growth factor or cytok e such as ⁇ nterleuk ⁇ n-2.
  • Another alternative method is immunoaffinity purification Recombinant
  • PLA2 is incubated with labeled or unlabeled cell extracts and immunoprecipitated with anti- PLA2 antibodies
  • the immunoprecipitate is recovered with protein A- Sepharose and analyzed by SDS-PAGE.
  • Unlabelled proteins are labeled by biotmylation and detected on SDS gels with streptavidin Binding partner proteins are analyzed by microsequencing Further, standard biochemical purification steps known to those skilled in the art may be used prior to microsequencing
  • PLA2 Recombinant tagged or labeled PLA2 is used to select peptides from a peptide or phosphopeptide library which interact with PLA2 Sequencing of the peptides ieads to identification of consensus peptide sequences which might be found in interacting proteins PLAo binding partners identified by any of these methods or other methods which would be known to those of ordinary skill in the art as well as those putative binding partners discussed above can be used in the assay method of the invention. Assaying for the presence of PLA2 binding partner complex are accomplished by, for example, the yeast two-hybrid system, ELISA or immunoassays using antibodies specific for the complex. In the presence of test substances which interrupt or inhibit formation of PLA2 binding partner interaction, a decreased amount of complex will be determined relative to a control lacking the test substance.
  • Assays for free PLA2 or binding partner are accomplished by, for example, ELISA or immunoassay using specific antibodies or by incubation of radiolabeled PLA2 with cells or cell membranes followed by centrifugation or filter separation steps. In the presence of test substances which interrupt or inhibit formation of PLA2/binding partner interaction, an increased amount of free PLA2 or free binding partner will be determined relative to a control lacking the test substance.
  • Polypeptides of the invention also can be used to assess PLA2 binding capacity of PLA2 binding molecules in cells or in cell-free preparations.
  • the PLA2 may be employed in a process for screening for compounds which either inhibit, promote or modulate the enzymatic activity of PLA2.
  • One standard assay for PLA2 uses [linoleoyi-1- ⁇ C] labeled L- ⁇ -l-acyl-2- linoleoylphosphatidylethanolamine as a substrate and follows the release of ⁇ C labeled free fatty acid. This assay or others could be used to identify either agonists or antagonists of PLA2.
  • Examples of potential PLA2 antagonists are small molecules such as organic molecules or peptides, antibodies, or in some cases an oligonucleotide, which binds to PLA2 and prevents enzymatic activity.
  • Potential antagonists also include small molecules or proteins which are closely related to the binding molecules of the PLA2, e.g. a fragment of the binding molecules, which have lost biological function, and when bind to the PLA2 polypeptide inhibit its activity.
  • Binding molecules refer to molecules that specifcally bind to or interact with PLA2 polypeptide of the present invention.
  • binding molecules are other factors, co-factors, units or subunits which enhance PLA2 activity or diminish it Such binding molecules are a part of the present invention Binding molecules also may be non-naturally occurring, such as antibodies and antibody-derived reagents that bind specifically to PLA2 )
  • a potential antagonist also includes an antisense construct prepared through the use of antisense technology
  • Antisense technology can be used to control gene expression through t ⁇ ple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA
  • the 5' coding portion of the polynucleotide sequence, which encodes for the mature PLA2 is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transc ⁇ ption (t ⁇ ple helix -see Lee et al , Nucl Acids
  • Another potential antagonist is a small molecuie which binds to the PLA2 making it inaccessible to binding molecules (e g substrates) such that normal biological activity is prevented
  • small molecules include, but are not limited to, small peptides or peptide-hke molecules and organic compounds
  • PLA2 are ubiquitous in the animal host and are responsible for many biological functions, including many pathologies Accordingly, it is desirous to find compounds and drugs which can inhibit the function of a PLA2
  • This invention additionally provides a method of treating an abnormal condition related to an excess of PLA2 activity, such as BPH and va ⁇ ous forms of cancer, including prostate cancer, which comprises administering to a subject the inhibitor compounds (antagonists) as hereinabove described along with a pharmaceutically acceptable carrier in an amount effective to inhibit PLA2 activity directly or by blocking binding of binding molecules to PLA2 polypeptide
  • compositions and Kits The compounds which inhibit such PLA2. may be employed in combination with a suitable pharmaceutical earner
  • a suitable pharmaceutical earner Such compositions comprise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient
  • a earner includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof
  • the formulation should suit the mode of administration
  • the invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention
  • Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds
  • compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, mtrape ⁇ toneal, intramuscular, subcutaneous, intranasal or lntradermal routes, among others
  • compositions generally are administered in an amount effective for treatment or prophylaxis of a specific indication or indications
  • the compositions are adrmnistered m an amount of at least about 10 ⁇ g/kg body weight In most cases they will be administered in an amount not in excess of about 8 mg/kg body weight per day
  • dose is from about 10 ⁇ g kg to about 1 mg kg body weight, daily It will be appreciated that optimum dosage will be determined by standard methods for each treatment modality and indication, taking into account the indication, its seventy, route of administration, complicating conditions and the like
  • Vaccine Another aspect of the invention relates to a method for inducing an immunological response in an animal, particularly in a mammal, which comprises inoculating the animal with PLA2, or a fragment or variant thereof, adequate to produce antibody to protect said animal from BPH or various forms of cancer, including prostate cancer.
  • Yet another aspect of the invention relates to a method of inducing immunological response in an animal which comprises, through gene therapy, delivering gene encoding PLA2. or a fragment or a variant thereof, for expressing PLA2, or a fragment or a variant thereof in vivo in order to induce an immunological response to produce antibody to protect said animal from disease.
  • composition which, when introduced into an animal, particularly mammalian host, induces an immunological response in that animal to a given PLA2 gene or protein coded therefrom, wherein the composition comprises a recombinant PLA2 gene or protein coded therefrom comprising DNA which codes for and expresses an antigen of said PLA2 gene or protein coded therefrom.
  • the PLA2 or a fragment thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties.
  • fused recombinant protein preferably further comprises an antigenic co- protein, such as Glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-proteins which solubilize the protein and facilitate production and purification thereof.
  • GST Glutathione-S-transferase
  • beta-galactosidase relatively large co-proteins which solubilize the protein and facilitate production and purification thereof.
  • the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system.
  • the co-protein may be attached to either the amino or carboxy terminus of the first protein.
  • the present invention also includes a vaccine formulation which comprises the immunogenic recombinant protein together with a suitable carrier. Since the protein may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection).
  • parenterally including subcutaneous, intramuscular, intravenous, intradermal etc. injection.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient: and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the formulations may be presented in unit- dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • PLA2 Whilst the invention has been described with reference to PLA2, it is to be understood that this covers fragments of the naturally occurring protein and similar proteins (for example, having sequence homologies of 50% or greater) with additions, deletions or substitutions which do not substantially affect the immunogenic properties of the recombinant protein.
  • the present invention also provides a method for the production of transgenic animals with altered PLA2 levels for the productions of animals bearing PLA2 induced diseases.
  • Transgenic, non-human, animals may be obtained by transfecting appropriate fertilized eggs or embryos of a host with nucleic acids encoding the PLA2 disclosed herein, see for example U.S. Patents 4,736,866; 5,175,385; 5,175,384 and 5,175,386.
  • the resultant transgenic animal may be used as a model for the study of altered PLA2 levels.
  • useful transgenic animals are those which display a detectable phenotype associated with the altered expression of the PLA2 polypeptide. Drugs may then be screened for their ability to reverse or exacerbate the relevant phenotype.
  • mRNA is prepared from normal and cancerous prostate tissue, size fractionated on an agarose gel and then transferred to a nylon membrane (northern blot) This blot is then hybridized to a PLA2 specific cDNA probe that has been labeled with radioactive [32p]-dCTP. Following incubation, the blot is then washed under stnngent conditions (desc ⁇ bed in Sambrook) and then exposed to film. Changes in mRNA levels as a function of disease will be seen as a change in intensity of the signal seen in diseased versus normal tissue.

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Abstract

L'invention concerne un nouveau procédé de diagnostic et de traitement de cancers ou de l'adénome prostatique ainsi que des agents et des compositions pharmaceutiques pour le traitement de cancers et de l'adénome prostatique.
PCT/US1997/013550 1996-08-02 1997-07-31 Nouveau procede de detection et de traitement du cancer WO1998005349A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002262624A CA2262624A1 (fr) 1996-08-02 1997-07-31 Nouveau procede de detection et de traitement du cancer
JP10508081A JP2001500847A (ja) 1996-08-02 1997-07-31 癌の検出および処置の新規方法
EP97934361A EP0918532A4 (fr) 1996-08-02 1997-07-31 Nouveau procede de detection et de traitement du cancer
AU37439/97A AU3743997A (en) 1996-08-02 1997-07-31 A novel method of detecting and treating cancer

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US69147996A 1996-08-02 1996-08-02
US08/691,479 1996-08-02
US08/714,744 US5747264A (en) 1996-08-02 1996-09-16 Method of diagnosing and monitoring prostate cancer
US08/714,744 1996-09-16

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999042830A1 (fr) * 1998-02-23 1999-08-26 Diadexus Llc Procedes mettant en application pla2 en tant que marqueur de metastases et afin de diagnostiquer des cancers selectionnes
JP2003514770A (ja) * 1999-09-14 2003-04-22 ディ − ファーム リミテッド 抗増殖性医薬の燐脂質プロドラッグ
WO2004064822A1 (fr) * 2002-06-07 2004-08-05 Kieran Francis Scott Procede d'inhibition de la proliferation de cellules cancereuses

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WO1994000766A1 (fr) * 1992-06-23 1994-01-06 Auckland Uniservices Limited Gravidine: dosages, genes ou fragments d'adn, et leur application

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BR. J. CANCER, 1993, Vol. 68, MURATA et al., "Expression of Group-II Phospholipase A2 in Malignant and Non-Malignant Human Gastric Mucosa", pages 103-111. *
BR. J. CANCER, 1997, Vol. 75, No. 11, DE SOUZA et al., "Enhancement of Paclitaxel Activity Against Hormone-Refractory Prostate Cancer Cells in Vitro and in Vivo by Quinacrine", pages 1593-1600. *
See also references of EP0918532A4 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999042830A1 (fr) * 1998-02-23 1999-08-26 Diadexus Llc Procedes mettant en application pla2 en tant que marqueur de metastases et afin de diagnostiquer des cancers selectionnes
JP2003514770A (ja) * 1999-09-14 2003-04-22 ディ − ファーム リミテッド 抗増殖性医薬の燐脂質プロドラッグ
WO2004064822A1 (fr) * 2002-06-07 2004-08-05 Kieran Francis Scott Procede d'inhibition de la proliferation de cellules cancereuses
AU2003229143B2 (en) * 2002-06-07 2009-07-23 Kieran Francis Scott Method of inhibiting prostate cancer cell proliferation
EP1531803A4 (fr) * 2002-06-07 2009-08-05 Kieran Francis Scott Procede d'inhibition de la proliferation de cellules cancereuses
US7919458B2 (en) 2002-06-07 2011-04-05 Scott, Kieran, Francis Method of inhibiting prostate cancer cell proliferation

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JP2001500847A (ja) 2001-01-23
EP0918532A1 (fr) 1999-06-02
EP0918532A4 (fr) 2001-01-03
CA2262624A1 (fr) 1998-02-12
AU3743997A (en) 1998-02-25

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