EP0673438A1 - A method for the detection and treatment of prostate disease - Google Patents

A method for the detection and treatment of prostate disease

Info

Publication number
EP0673438A1
EP0673438A1 EP93920497A EP93920497A EP0673438A1 EP 0673438 A1 EP0673438 A1 EP 0673438A1 EP 93920497 A EP93920497 A EP 93920497A EP 93920497 A EP93920497 A EP 93920497A EP 0673438 A1 EP0673438 A1 EP 0673438A1
Authority
EP
European Patent Office
Prior art keywords
ngfr
sample
precancerous
tissue
antibody
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP93920497A
Other languages
German (de)
English (en)
French (fr)
Inventor
Daniel Djakiew
Michael E. Lewis
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cephalon LLC
Georgetown University
Original Assignee
Cephalon LLC
Georgetown University
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
Application filed by Cephalon LLC, Georgetown University filed Critical Cephalon LLC
Publication of EP0673438A1 publication Critical patent/EP0673438A1/en
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/48—Nerve growth factor [NGF]
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators

Definitions

  • the invention relates to diagnosis and treatment of prostate disease.
  • Prostate cancer is the most frequently diagnosed cancer, and the second leading cause of cancer deaths, among men in North America (Moon, T. 1992. J . Am . Geriat . Soc . 4j):662-627) . It is estimated that 30% of men over 50 years of age have latent cases that will remain benign throughout the life span of the individuals. The causes and determinants of progression of prostate cancer are unknown.
  • prostate cancer localized prostate neoplasias are frequently treated by surgery or radiation.
  • Metastatic prostate cancer is often treated with hormonal modulation, using, for example, GnRH analogs or anti-androgens. This provides an average progression-free survival time of 15 months, and an overall survival time of approximately 30 months.
  • the use of chemotherapeutics is limited by its lack of success in patients who are non-responsive to hormonal therapy.
  • Nerve growth factor receptors are present on the cell surface of many cell types, including sympathetic and CNS neurons, as well as peripheral nerve Schwann cells.
  • NGF interacts with target cells with biphasic equilibrium binding kinetics, reflecting two classes of receptors (Anures et al. 1977. Proc . Nat . Acad . Sci . USA 1:2785-2789), termed by Eveleth (1988. In Vitro Cell . Devel . Biol . 2 ⁇ : 1148-1153) as type I and type II NGF receptors.
  • the high-affinity NGF receptor has been recently identified as trk (Kaplan et al. 1991a. Science 252:554- 558; Kaplan et al. 1991b. Nature 3_5 :158-160; Klein et al. 1991. Cell 65:189-197; Ross, 1991. Cell Regulation 2 :685-690), a proto-oncogene that mediates tyrosine kinase activity.
  • the trk proto-oncogene was originally discovered as a transforming (i.e. cancer-causing) gene isolated from a colon carcinoma biopsy, and has since been well characterized (Coulier et al. 1989. Mol . Cell . Biol . : 15-23; Martin-Zanca et al.
  • the invention features a method of diagnosing a precancerous or cancerous condition in a human patient involving measuring the amount of nerve growth factor receptor (NGFR) present in a biological sample of the patient, a level of NGFR below a predetermined level indicating the presence of the precancerous or cancerous condition.
  • NGFR nerve growth factor receptor
  • NGFR Ne Growth Factor Receptor
  • predetermined level of NGFR is meant a level of NGFR that is standardized by repeated NGFR assays, or a level of NGFR established by concurrently run controls with biological samples known to be normal, i.e., free of any cancerous or precancerous condition.
  • the precancerous or cancerous condition can be present in any biological tissue of the human body, and is best exemplified, but not limited to, disease conditions of the prostate gland.
  • the biological sample can be in a living patient, can be isolated from the patient as a tissue sample, or can be a body fluid, e.g., urine, semen, or plasma.
  • the measuring can include administering a labeled anti-NGFR antibody, e.g., a humanized monoclonal antibody, to the patient, and determining the amount of the antibody that is proximal to the prostate gland.
  • the amount of antibody can be determined by imaging, e.g., by positron emission tomography (PET) or by single photon emission computed tomography (SPECT) .
  • PET positron emission tomography
  • SPECT single photon emission computed tomography
  • the label used to make the labeled antibody can be, but is not necessarily, a radioisotope.
  • the method of diagnosing a cancerous or precancerous condition can further include reacting the sample with a labeled anti-NGFR antibody, and determining the amount of the antibody bound to the NGFR in the sample as an indication of the amount of NGFR present in the sample.
  • the amount of antibody can be determined when the biological sample is fixed and sectioned, for example, by immunohistochemistry. Alternatively, the amount of antibody can be determined when the biological sample is in solution, preferably after the sample has been homogenized or extracted. NGFR in solubilized tissue can be measured by an im unoassay.
  • the method of diagnosing a cancerous or precancerous condition can further include introducing a labeled nucleic acid probe to the sample, and determining the amount of the probe bound to NGFR-encoding RNA in the sample, as an indication of the amount of NGFR in the sample.
  • the sample can be, but is not of necessity, histologically fixed.
  • the amount of bound probe can be determined by in situ hybridization, or by solution hybridization.
  • the method of diagnosing a cancerous or precancerous condition can further include reacting the body fluid with a anti-NGFR antibody, and detecting the amount of the antibody bound to the NGFR in the body fluid as an indication of the amount of NGFR present in the body fluid.
  • the invention also features a method of treating a precancerous or cancerous condition in a human patient, involving administering a substantially pure preparation of NGFR to the patient.
  • the NGFR can preferably be human NGFR, but can be NGFR from any source.
  • the NGFR can be naturally occurring, or a recombinant form of the complete NGFR polypeptide, or can be a biologically active fragment of NGFR, a truncated fragment of NGFR, i.e., NGFRt, or the recombinant extracellular domain of NGFR, called RED.
  • a “substantially pure preparation of NGFR” is a preparation which is substantially free of the proteins with which NGFR naturally occurs in a cell.
  • “Homology” refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomeric subunit, e.g., if a position in each of two polypeptide molecules is occupied by leucine, then the molecules are homologous at that position.
  • the homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences. For example, 6 of 10, of the positions in two sequences are matched or homologous then the two sequences are 60% homologous.
  • NGFR amino acid sequences Leu-gly-val-ala-gly-pro and Leu-his-tyr-ala-gly-leu share 50% homology.
  • Applicants have discovered that the expression of NGFR is decreased in benign prostate hyperplasia tissue and prostatic adenocarcinoma tissue relative to normal prostate tissue, and is totally absent in four metastatic tumor cell lines of the human prostate. Accordingly, this invention permits a rapid means for diagnosing the neoplastic progression of the human prostate. NGFR can be an effective therapeutic when administered to a patient diagnosed with a cancerous or precancerous condition of the prostate gland.
  • Fig. 1 is a phase contrast image of a primary culture of normal prostate epithelial tissue showing vesicles (arrow) in the cytoplasm (A) , and of the TSU-prl human prostate, epithelial tumor cell line (E) .
  • Fig. 2 shows an immunoblot analysis of p75 NGFR in microsomal preparations of human prostate adenocarcinoma tissue (PA) , normal prostate (NP) , renal tissue (RT) ,
  • PA prostate adenocarcinoma tissue
  • NP normal prostate
  • RT renal tissue
  • A875 human melanoma cell line which overexpresses the p75 NGFR (A8) , and testis (T) .
  • Fig. 3 shows an immunoblot analysis of p75 NGFR in microsomal preparations of (A) five prostatic adenocarcinoma (P/i -PA ⁇ specimens, showing decreased or absent immunoreactivity of the adenocarcinoma specimens and benign prostatic hyperplasia (BP- L -BP g ) specimens in comparison with normal prostate (NP) tissue.
  • P/i -PA ⁇ prostatic adenocarcinoma
  • BP- L -BP g benign prostatic hyperplasia
  • Fig. 4 shows an immunoblot analysis of p75 NGFR in microsomal preparations of human prostate stroma (HS) , human prostate epithelial tumor cell lines, TSU-prl (TS) , DU-145 (DU) , PC-3 (PC) and LNCaP (LN) showing loss of expression of the p75 NGFR in the metastatic epithelial cell lines.
  • HS human prostate stroma
  • TS TSU-prl
  • DU-145 DU-145
  • PC-3 PC-3
  • LNCaP LNCaP
  • NGFR nerve growth factor receptor
  • TSU-prl human tumor epithelial cell line
  • Applicants then designed diagnostic procedures that make use of the observed inverse relationship between NGFR expression and neoplastic progression for early detection of cancerous or precancerous condition in a human patient. Once the condition is detected, the patient can be treated with a preparation of NGFR.
  • the situation in prostate tissue is provided as an example of how a decrease in NGFR levels can generally be used to indicate neoplastic transformation.
  • PC-3, DU-145 and LNCaP prostate epithelial tumor cell lines were obtained from the American Type Culture Collection (ATCC; Rockville, MD) .
  • the TSUprl cell line was provided by Dr. John Isaacs (Johns Hopkins University, Baltimore, MD) .
  • the human melanoma cell line (A875) was provided by the laboratory of Dr. Moses Chao (Cornell University, New York, NY) .
  • the PC-3, DU-145 and TSU-prl cell lines were maintained in RPMI 1640 medium supplemented with antibiotics/antimycotic (100 units/ml penicillin, 100 ⁇ g/ml streptomycin, 0.25 ⁇ g/ml fungizone) , 10% fetal bovine serum (FBS) , and 10 "7 M testosterone (T) .
  • the LNCaP cell line was maintained in Dulbecco's Modified Eagle's Medium (DMEM)/10% FBS and the A875 cell line was maintained in RPMI 1640 medium supplemented with 10% FBS.
  • DMEM Dulbecco's Modified Eagle's Medium
  • A875 cell line was maintained in RPMI 1640 medium supplemented with 10% FBS.
  • the BPH tissue was obtained from transurethral resections of the prostate; prostatic adenocarcinoma and normal prostate tissue from radical retropubic prostatectomy specimens; renal tissue from radical nephrectomy specimens and testis from orchiectomy specimens at Georgetown University Medical Center.
  • the human prostatic stromal cells and epithelial cells were isolated by enzymatic digestion of tissue samples in a collagenase solution as previously described (Djakiew et al. 1991. Cane . Res . 5JL: 3304-3310) .
  • the prostate stromal cells were cultured in 75 cm 2 tissue culture flasks (Costar, Cambridge, MA) maintained in RPMI 1640 medium containing 10% FBS/T.
  • the normal prostate epithelial cells were plated on glass coverslips and maintained in RPMI 1640 medium containing a serum free defined medium (SFDM) consisting of 2 ⁇ g/ml insulin, 10 ng/ml epidermal growth factor, 1 ⁇ g/ml transferrin, 10 ⁇ 7 M dexamethasone, 2 mM glutamine, 2.5 mg/ml bovine pituitary extract (UpState Biotechnology Inc., Lake Placid, N.Y.) , 10 ng/ml rat prolactin (NIH) and 10 ⁇ g/ml cholera toxin, all of which were also supplemented with 10% FBS/T and dihydrotestosterone (10 ⁇ 7 ) .
  • SFDM serum free defined medium
  • the cells were fixed in ice cold methanol for 2.5 minutes, blocked with 3% ovalbumin for one hour at 37°C to reduce non-specific binding, and incubated with murine anti-human p75 NGFR monoclonal antibody (1:80 dilution; Boehringer Mannheim, Indianapolis, IN) for three hours at 37°C.
  • the coverslips were rinsed with PBS (X6) and incubated in rhodamine conjugated sheep anti-murine IgG secondary antibody (1:400 dilution, Cappel, Durham, NC) for one hour.
  • Murine IgG partially purified from whole mouse serum (Cappel) by the method of Jemmerson and Margoliash (Jemmerson et al. 1981. Meth . Enzymol .
  • the subcellular fractions were resuspended in 50 mM Tris-HCI (pH 7.4) supplemented with 10 mM ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) and 10 ⁇ M phenylmethlysulfonylfluoride (PMSF) .
  • Tris-HCI pH 7.4
  • EGTA ethylenebis(oxyethylenenitrilo)tetraacetic acid
  • PMSF phenylmethlysulfonylfluoride
  • Nonreducing sample buffer was added to 50 ⁇ g of each microsomal and cytosolic protein sample. Subsequently, the samples were separated by sodium dodecyl sulfate- polyacrylamide gel (10%) electrophoresis (SDS-PAGE) . The protein from each sample was electrotransferred onto nitrocellulose and blocked for one hour in 5% nonfat dry milk in TBS (20 mM Tris-HCI, 500 mM NaCl, pH 7.5). The membranes were incubated overnight at room temperature with murine monoclonal anti-human p75 NGFR antibody (1:80 dilution) in TBS supplemented with Tween-20 (TTBS) and 1% gelatin (Bio-Rad, Richmond, CA) .
  • TBS murine monoclonal anti-human p75 NGFR antibody
  • the membranes were rinsed twice for 10 minutes with TTBS and reacted with horseradish peroxidase conjugated goat anti-mouse IgG (1:2000) in TTBS containing 1% gelatin for one hour and rinsed twice in TTBS and once in TBS. Immunoreactivity was visualized using 4-chloro-l-napthol hydrogen peroxide reaction substrate.
  • the prostate epithelial tumor cell line TSU-prl (Fig. IE) treated with 20 ⁇ g/ml hPS (Fig. IF) , or in the absence of hPS (not shown) , showed no immunofluorescence when stained for the p75 NGFR.
  • the DU-145, PC-3 and LNCaP cell lines also did not show p75 NGFR immunostaining.
  • Fig. 2 shows the results of an immunoblot with the p75 NGFR antibody of microsomal fractions of normal prostate and adenocarcinoma tissue specimens.
  • a 75 kD immunoreactive protein was evident in the normal prostate tissue.
  • the prostate adenocarcinoma tissue exhibited reduced expression of the NGFR protein. Renal tissue was used as the negative control, whereas testis tissue and the A875 melanoma cell line were used as the positive controls.
  • Fig. 3A prostatic adenocarcinoma specimens were examined.
  • NGF can bind to both the low- affinity NGFR as well as to the higher affinity trk.
  • the low-affinity NGFR is present on cells in very large excess (Vale et al. 1985. Meth . in Enzymology 109:21-39) .
  • the interaction of NGF with trk would increase, in turn activating a tyrosine kinase activity in the affected cells.
  • All of the diagnostic methods of the invention are designed to detect and quantitate the amount of NGFR in a tissue.
  • the diagnostic methods of the invention can be used on a living patient, on tissue that has been removed from the patient, or on bodily fluids.
  • the amount of NGFR measured in che tissue, or in the patient is compared to the amount of NGFR present in normal tissue.
  • the amount of NGFR present in normal tissue can be measured side-by-side with experimental tissue, or, if possible, a standard level of NGFR can be established, where it is shown by repetitive measurements that the level of NGFR routinely falls within an expected concentration range.
  • the diagnostic methods of the invention are carried out as follows: Example 1
  • an anti-NGFR antibody preferably a humanized monoclonal antibody
  • a suitable radioisotope for imaging is administered to the patient.
  • the amount of radioactive uptake is then determined in the region of the tissue being examined, e.g., in the region of the prostate gland.
  • Imaging is carried out by positron emission tomography (PET) , single photon emission computed tomography (SPECT) , or other imaging techniques that can accurately measure radio- labeled antibody.
  • PET positron emission tomography
  • SPECT single photon emission computed tomography
  • Anti-NGFR antibodies useful for the diagnostic methods of the invention can include any polyclonal or monoclonal antibody that binds specifically to NGFR, or to a fragment of NGFR, e.g., NGFRt.
  • One example of such an antibody is the monoclonal antibody 8211 (Eveleth, D.D. 1988. In Vitro Cell Develop . Biol . 14 . : 1148; Herlyn, M. et al. 1983. Cancer Invest 1:215) which is commercially available from Boehringer Mannheim
  • Antibody 8211 was used above to measure a reduction in NGFR in precancerous and cancerous prostate cells.
  • Other suitable antibodies include those disclosed by Ross et al. (U.S. 4,786,593, hereby incorporated by reference), Johnson (U.S. 4,855,241, hereby incorporated by reference) , and DiStefano and Clagett-Da e (WO 92/09631, hereby incorporated by reference) . These references also disclose methods that can be useful for the generation of other suitable antibodies.
  • the antibody is labeled with a suitable detectable element, e.g., a radioisotope.
  • a suitable detectable element e.g., a radioisotope.
  • the antibody can be chemically radiolabelled with Iodine-123 or Technecium 99m, either of which is useful for SPECT imaging.
  • the antibody can also be labelled with non- radioactive labels, e.g., a paramagnetic ion.
  • the antibody After it is labeled, the antibody can be detected in the human body by PET, SPECT, or Nuclear Magnetic Resonance (NMR) imaging, or another imaging method most suitable for the particular label on the antibody, using techniques which are known to those skilled in the art.
  • a reduction in uptake of the labeled antibody is diagnostic of the presence of a precancerous or cancerous condition in the tissue being examined.
  • the tissue sample is suitably fixed and sectioned, and the tissue section reacted with anti-NGFR antibody to immunohistochemically determine the amount of NGFR.
  • the tissue sample is removed from the patient, fixed, stained, and counterstained as described by Ross et al. (U.S.
  • the tissue is suspended in solution.
  • the tissue sample is suitably homogenized to extract NGFR by any of a number of methods known to those skilled in the art that permit the maintenance of the NGFR antigen.
  • the level of NGFR is determined by immunoassay. Quantitation of the antigen is carried out by using an NGFR antibody disclosed above.
  • the particular format of immunoassay can be any of several types of enzyme immunoassay, as described (Tijssen, P. , Practice and Theory of Enzyme Immunoassays, Elsevier 1985) or radioimmunoassay (Chard, T.
  • Example 4 For measurement of a decrease in NGFR expression in a tissue sample from a patient, the tissue sample is suitably fixed and sectioned, and reacted with a labeled nucleic acid probe to determine, by in situ hybridization, the amount of NGFR mRNA. For this test, the tissue sample is frozen, sectioned, and reacted with an NGFR nucleic a ⁇ id probe, as described by Springer et al. (1990.
  • the tissue sample can be suitably homogenized and extracted, and the level of NGFR mRNA determined by a suitable hybridization assay, e.g. a dot blot, Northern blot, polymerase chain reaction (PCR) , or solution hybridization assay.
  • a suitable hybridization assay e.g. a dot blot, Northern blot, polymerase chain reaction (PCR) , or solution hybridization assay.
  • the tissue sample is suitably homogenized and the RNA extracted by methods that are known to those skilled in the art. Suitable nonradioactive hybridization assays have been disclosed (Guesdon, J.L., 1992, J . Immunol . Methods 150: 33-49; Kricka, L.J., ed.
  • a decrease in NGFRt in a body fluid sample from a patient the sample is first obtained from the patient.
  • Preferred body fluids include urine, seminal fluid, blood, plasma, mucous, or any other fluid that can be collected from a human patient. The level of
  • NGFRt is determined by immunoassay or NGF ligand binding assay.
  • the body fluid is assayed for the presence of NGFRt using an immunoassay as disclosed by Johnson (U.S. 4,855,241, supra) or DiStefano and Clagett- Dame (WO 92/09631, supra) or another suitable immunoassay as referenced above.
  • the body fluid sample is assayed for the presence of NGFRt using a ligand binding assay with either radio-iodinated NGF or biotinylated NGF, followed by suitable precipitation and detection methods, as are known to those skilled in the art.
  • a reduction in ligand binding to NGFRt in the body fluid, relative to the normal level in the fluid is diagnostic of the presence of precancerous or cancerous cells. Therapy
  • NGFR NGFR
  • a suitable therapeutic treatment for a patient diagnosed with a reduction in NGFR or NGFRt is systemic administration of a recombinant form of a human NGFRt, which has the effect of substituting for the loss in prostatic NGFR as a binding compartment for endogenous NGF.
  • This polypeptide also referred to as the recombinant extracellular domain (RED) of the NGFR, has been cloned and expressed (Vissavajjhala, P. et al. 1990. J . Biol . Chem . 265:4746-4752) .
  • substantially pure NGFR polypeptide can be produced in quantity using standard techniques known to one skilled in the art (see, e.g., Scopes, R. Protein Purification: Principles and Practice, 1982 Springer Verlag, NY) .
  • the NGFR protein or fragment can be purified using conventional methods of protein isolation known to one schooled in the art, e.g., methods including but not limited to precipitation, chromatography, immunoadsorption, or affinity techniques.
  • the polypeptide can be purified from starting material using the cloned NGFR gene, or using a recombinant form of the NGFR DNA or cDNAs genetically engineered into an overproducing cell line.
  • the NGFR preparation can be administered to a human patient in a pharmaceutically acceptable buffer (e.g., physiological saline, which may also contain stabilizing agents as excipients) .
  • a pharmaceutically acceptable buffer e.g., physiological saline, which may also contain stabilizing agents as excipients
  • the therapeutic NGFR preparation is administered in accordance with the location and condition of the neoplasia to be treated.
  • the therapeutic preparation can be administered systemically, orally, parenterally, transdermally, or trans ucosally.
  • Administration can be in a sustained release formulation using a biodegradable biocompatible polymer, or by on-site delivery using micelles, gels or liposomes, or by transgenic modes.
  • the NGFR preparation is administered systemically, e.g., by an intravenous, subcutaneous, or intramuscular injection.
  • An appropriate therapeutic dose is an amount of NGFR which effects a reduction in, or postpones progression of, a neoplasia.
  • the dosage can be, but is not necessarily, in the range of 0.001 - 100.0 g/kg body weight, or a range that is clinically determined as appropriate by those skilled in the art.
  • the methods of the invention can be carried out by administering or detecting any protein that is substantially homologous to a NGFR protein.
  • allelic variations include allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridizes under high or low stringency conditions (e.g., washing at 2xSSC at 40 °C with a probe length of at least 40 nucleotides) to a naturally occurring NGFR nucleic acid (for other definitions of high and low stringency see Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989, 6.3.1 - 6.3.6, hereby incorporated by reference) ; and polypeptides or proteins specifically bound by antisera to a NGFR protein, especially by antisera to the active site or binding domain of a NGFR protein.
  • the term also includes chimeric polypeptides that include biologically active fragments of the NGFR protein.
  • the invention also includes any biologically active fragment or analog of a NGFR protein.
  • biologically active is meant possessing in vivo or in vitro receptor binding activity which is characteristic of the NGFR molecule described in Zupan et al. (1989. J. Biol . Chem . 264 : 11714-11720) . Since a NGFR protein receptor exhibits a range of physiological properties and since such properties may be attributable to different portions of the NGFR molecule, a useful NGFR fragment or NGFR analog is one that exhibits a biological activity in any biological assay for NGFR activity, as described above. This includes natural or induced proteins with significant affinity for NGF, i.e., those with a Kd of less than 10 ⁇ M.
  • ⁇ -macroglobulin Koo et al. 1989. Journal of Neuroscience Research 2 . :247-261
  • an example of an induced protein is a monoclonal antibody to NGF.
  • a NGFR protein fragment or analog possesses 10%, preferably 40%, or at least 90% of the activity of a member of the NGFR protein family, in any in vivo or in vitro NGFR activity assay.
  • Preferred analogs include NGFR (or biologically active fragments thereof) whose sequences differ from the wild-type sequence only by conservative amino acid substitutions, for example, substitution of one amino acid for another with similar characteristics (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not abolish the polypeptide's biological activity.
  • Other useful modifications include those which increase peptide stability.
  • Such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) or D-amino acids in the peptide sequence.
  • Analogs can differ from a naturally occurring member of the NGFR protein family in amino acid sequence or in ways that do not involve sequence, or in both.
  • Analogs of the invention will generally exhibit at least 70%, more preferably 80%, more preferably 90%, and most preferably 95% or even 99%, homology with a segment of 20 amino acid residues, preferably more than 40 amino acid residues, or more preferably the entire sequence of a naturally occurring NGFR polypeptide sequence.
  • Alterations in primary sequence include genetic variants, both natural and induced. Also included are analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non- naturally occurring or synthetic amino acids, e.g., ⁇ or ⁇ amino acids. Alternatively, increased stability may be conferred by --..yclizing the peptide molecule, or by exposing the polypeptide to phosphorylation-altering enzymes, e.g., kinases or phosphatases.
  • phosphorylation-altering enzymes e.g., kinases or phosphatases.
  • glycosylation can be modified, e.g., by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps, e.g., by exposing the polypeptide to glycosylation affecting enzymes derived from cells that normally provide such processing, e.g., mammalian glycosylation enzymes; phosphorylation can be modified by exposing the polypeptide to phosphorylation-altering enzymes, e.g., kinases or phosphatases.
  • phosphorylation-altering enzymes e.g., kinases or phosphatases.
  • the invention also includes biologically active fragments of the NGFR polypeptides.
  • fragment as applied to a polypeptide, will ordinarily be at least about 20 residues, more typically at least about 40 residues, or preferably at least about 60 residues in length. Fragments of a NGFR polypeptide can be generated by methods known to those skilled in the art. The ability of a candidate fragment to exhibit a biological activity of a NGFR protein can be assessed by methods known to those skilled in the art as described herein. Also included are NGFR polypeptides containing residues that are not required for biological activity of the peptide, or that result from alternative mRNA splicing or alternative protein processing events. What is claimed is:

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Oncology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Hospice & Palliative Care (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
EP93920497A 1992-09-11 1993-09-09 A method for the detection and treatment of prostate disease Withdrawn EP0673438A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US94351292A 1992-09-11 1992-09-11
US943512 1992-09-11
PCT/US1993/008446 WO1994006935A1 (en) 1992-09-11 1993-09-09 A method for the detection and treatment of prostate disease

Publications (1)

Publication Number Publication Date
EP0673438A1 true EP0673438A1 (en) 1995-09-27

Family

ID=25479795

Family Applications (1)

Application Number Title Priority Date Filing Date
EP93920497A Withdrawn EP0673438A1 (en) 1992-09-11 1993-09-09 A method for the detection and treatment of prostate disease

Country Status (5)

Country Link
EP (1) EP0673438A1 (ja)
JP (1) JPH08506654A (ja)
AU (1) AU5102493A (ja)
CA (1) CA2143945A1 (ja)
WO (1) WO1994006935A1 (ja)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU689145B2 (en) * 1993-10-18 1998-03-26 Walter And Eliza Hall Institute Of Medical Research, The A method for enhancing neurone survival and agents useful for same
US5837694A (en) * 1993-10-18 1998-11-17 The Walter And Eliza Hall Institute Of Medical Research Method for enhancing neurone survival and agents useful for same
US6548062B2 (en) * 2000-02-29 2003-04-15 Cephalon, Inc. Method of treating cancer with anti-neurotrophin agents
FR2846426B1 (fr) 2002-10-28 2004-12-10 Bio Merieux Procede de dosage du ngf pour le diagnostic in vitro du cancer du sein et utilisation en therapie

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4782015A (en) * 1984-03-30 1988-11-01 Syntex (U.S.A.) Inc. Method for determining the presence of malignant cells

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9406935A1 *

Also Published As

Publication number Publication date
AU5102493A (en) 1994-04-12
JPH08506654A (ja) 1996-07-16
CA2143945A1 (en) 1994-03-31
WO1994006935A1 (en) 1994-03-31

Similar Documents

Publication Publication Date Title
KR100762995B1 (ko) 암의 진단과 치료를 위한 ａｉｍｐ2ｄｘ2의 용도
JP4375932B2 (ja) 悪性新生物の診断および処置
US6630326B2 (en) Cancer diagnosis and therapy based on mutations in TGF-β receptors
JP2010046086A (ja) 悪性新生物の診断および処置
JPH09509570A (ja) Ｍｔｓ−１遺伝子により転移性癌の診断
US8188051B2 (en) Metadherin polypeptides, encoding nucleic acids and methods of use
US20030180295A1 (en) Angiocidin: a Cys-Ser-Val-Thr-Cys-Gly specific tumor cell adhesion receptor
EP1064005B1 (en) Urokinase plasminogen activator receptor as a target for diagnosis of micrometastases
KR20060057992A (ko) ｐ38-ＤＸ2 및 이의 용도
WO1994006935A1 (en) A method for the detection and treatment of prostate disease
WO1994006935A9 (en) A method for the detection and treatment of prostate disease
JP2004537981A (ja) マイトジェンオキシゲナーゼ調節因子
US6509316B2 (en) Pharmaceutical compositions, methods, and kits for treatment and diagnosis of lung cancer
US20050214219A1 (en) 24p3 receptors and uses thereof
Younes et al. Fodrin as a differentiation marker. Redistributions in colonic neoplasia.
US5942402A (en) Method of detecting and treating cancer
US20080020461A1 (en) Kinesin-like proteins and methods of use
Tokiwa et al. Mucosal cell proliferation activity of the gallbladder in children with anomalous arrangement of the pancreaticobiliary duct
YAMAGATA et al. Levels of vitamin K, immunoreactive prothrombin, des‐γ‐carboxy prothrombin and γ‐glutamyl carboxylase activity in hepatocellular carcinoma tissue
US20050123981A1 (en) Method of diagnosing, monitoring, staging, imaging and treating gastrointestinal cancer
Lemaire et al. Bombesin-like peptides in alveolar macrophage: increased release in pulmonary inflammation and fibrosis
WO2004015390A2 (en) Lung cancer target proteins and use thereof
JP2009523009A (ja) 結腸直腸癌を検出するための化合物及び方法
CA2340938A1 (en) Antibodies specific for bladder nuclear matrix proteins blca-1 to blca-6
EP3968025A1 (en) Methods for the determination of invasive ability of brain tumour cells and for the diagnosis and prognosis of brain tumour

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19950331

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU NL PT SE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Withdrawal date: 19960909