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WO2011047112A1 - Mesure de la pka pour dépister le cancer - Google Patents

Mesure de la pka pour dépister le cancer Download PDF

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Publication number
WO2011047112A1
WO2011047112A1 PCT/US2010/052597 US2010052597W WO2011047112A1 WO 2011047112 A1 WO2011047112 A1 WO 2011047112A1 US 2010052597 W US2010052597 W US 2010052597W WO 2011047112 A1 WO2011047112 A1 WO 2011047112A1
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WO
WIPO (PCT)
Prior art keywords
pka
cancer
activity
activated
antibody
Prior art date
Application number
PCT/US2010/052597
Other languages
English (en)
Inventor
Robert Puskas
Douglas Held
Original Assignee
Traxxsson, Llc
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 Traxxsson, Llc filed Critical Traxxsson, Llc
Publication of WO2011047112A1 publication Critical patent/WO2011047112A1/fr

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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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • the present invention is in the field of diagnostics and relates to methods for determining the presence, activity, and concentrations of certain protein biomarkers and their use in determining the presence of cancer.
  • the cAMP-dependent protein kinase (PKA) is activated by the binding of cAMP to the regulatory subunit ( ), of the molecule and results in the release of the active catalytic kinase subunit (C).
  • PKA cAMP-dependent protein kinase
  • C active catalytic kinase subunit
  • Most of the effects of cAMP in eukaryotic systems are the result of phosphorylation of proteins at serine or threonine residues by PKA.
  • PKA is localized subcellularly by binding to multidomain scaffolding proteins known as AKAPs which bind to the R subunits of the holoenzyme [1]. More than 50 AKAPs are known which localize PKA in various cell types.
  • the PKA-specific inhibitor (PKI) acts by binding with high affinity to the substrate binding site of the free active catalytic subunit [2].
  • NCI National Cancer Institute
  • Humphries et al demonstrated that oxidation of PKA inhibited its activity [6]. This inhibition could be overcome by the addition of the reducing agent dithiothreitol.
  • the current invention shows that activated (reduced) PKA activity is low in plasma or serum of cancer patients relative to that of controls.
  • the PSA (prostate-specific antigen) blood test for prostate cancer is of questionable value as a screening test. In fact the American Cancer Society no longer recommends that men routinely have PSA tests as part of their routine physical examinations [7]. There are several reasons for this lack of support for testing. When prostate cancer is present, the PSA test fails to detect 3 out of 4 cases [8]. In addition, when the PSA test comes back positive, 3 out 4 times it is a false positive - the patient does not have cancer [9] . Nonetheless, patients who have a positive PSA test typically will have a group of 12 or more biopsy samples taken from their prostate to verify if cancer is present.
  • Mammograms have a poorer record than PSA tests. While mammograms are purported to detect 85- 90% of breast cancers when they are present, the detected tumors on average are 1 1/2 inches in diameter when diagnosed. As for all cancers, earlier detection leads to better patient outcomes. What makes breast cancer screening costly is that an estimated 95% of the positive mammograms are false positives - the patient does not have cancer [12]. A positive mammogram frequently leads to a breast biopsy. A typical needle biopsy costs about $1,500; an invasive surgical biopsy (about 1/3 of all breast biopsies) costs about $5,000. This brings the national cost for the estimated 2 million unnecessary breast biopsies to more than $5.1 billion annually. Improved patient outcome could be accomplished by substituting a cancer confirmatory test for a biopsy, for a savings in health care costs of almost $5 billion annually
  • Cancer monitoring tests Beyond screening tests there are additional blood tests that are used to monitor cancer patients once cancer has been diagnosed. Many of these tests are not specific for cancer or specific for a particular type of cancer, rendering them useless as cancer screening tests. However, they can be an effective means for monitoring cancer treatment and testing for disease recurrence. These tests include CA-15.3 and CA27.29 for breast cancer, CA 125 for ovarian cancer, CEA for colon cancer and PSA for prostate cancer [13]. Other blood tests have been used to determine if a primary cancer has spread to other organs. These tests include assays for metastases to bone
  • liver (osteoprotegrin), and liver (E-selectin).
  • NCI National Cancer Institute
  • a method for determining the presence of cancer in a patient consisting of measuring the activity of activated PKA in a patient sample serum or plasma and determining that the activity of activated PKA is lower than that of a control sample or control population
  • a method is provided wherein, in addition to measuring the level of activated PKA in the sample, the level of anti-PKA antibody also is measured. Elevated levels of anti-PKA antibody relative to a control sample or control population plus low levels of activated PKA activity are used to determine the presence of cancer
  • a blood test that confirmed, monitored or screened for all types of cancer would be of benefit.
  • the test would serve as a universal confirmation test for presumptive positive PSA, Pap smear, and mammogram tests. Additionally it would be important if the test could determine the stage of diagnosed cancer and whether metastasis has occurred. Such a test could be used to monitor patient treatment, test for cancer recurrence, and it could be used by pharmaceutical companies to monitor the efficacy of cancer drugs under development.
  • Figure 1 is a bar graph of the levels of activity of activated PKA in prostate cancer patients relative to those of age and sex-matched normal controls.
  • Figure 2 is a bar graph of the levels of activity of activated PKA in prostate cancer patients relative to those of age and sex-matched normal controls.
  • Figure 3 is a bar graph of the levels of anti-PKA antibody relative to the levels of activated PKA activity.
  • Figure 4 is a receiver-operator curve (ROC) plot of the levels of activated PKA activity in cancer patients and age and sex-matched controls.
  • ROC receiver-operator curve
  • Serum samples from patients with early and late-stage cancer were obtained from ProMedDx, LLC.
  • blood samples from prostate cancer patients and normal controls presumably without cancer were assayed for activated PKA activity.
  • activated PKA in samples was mixed with a defined peptide used as a substrate.
  • Phosphorylation of the peptide was detected using biotinylated phosphoserine antibody, which was in turn was detected in an ELISA format using peroxidase- conjugated to streptavidin. Detection of the bound peroxidase was established using a color-producing peroxidase substrate included in the assay kit.
  • Bovine PKA catalytic unit was used at varying concentrations to develop a standard activity curve. The detail of the assay protocol is described below.
  • PKA diluent 25 mM KH 2 P0 4 , 5 mM EDTA, 150 mM NaCI, 50% (w/v) glycerol, 1 mg/ml BSA, 5 mM ⁇ -mercaptoethanol, pH 6.5
  • PKA diluent 25 mM KH 2 P0 4 , 5 mM EDTA, 150 mM NaCI, 50% (w/v) glycerol, 1 mg/ml BSA, 5 mM ⁇ -mercaptoethanol, pH 6.5
  • Coating buffer 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.4 e.
  • Coating wash buffer 20 mM HEPES, 150 mM sodium chloride, 30 mM sucrose, pH 7.0 with 0.1% BSA
  • Assay buffer 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.4 with 0.25% BSA and 0.1% Tween 20
  • Assay wash buffer 10 mM citrate, 150 mM sodium chloride, pH 5.1 with 0.1% Tween 20 i.
  • Detection antibody peroxidase-conjugated donkey anti-human IgG (H+L) (Jackson Prod.
  • Net anti-PKA Absorba nee (+ PKA)— Absorbance (- PKA)
  • Embodiment 1 indicate that the results of both assays together can be used to detect the presence of cancer.
  • the measurement of the levels of activated PKA activity can be used to determine the presence of cancer in individuals.
  • the measurement of levels of activated PKA activity and anti-PKA antibody can be used together to indicate the presence of cancer in individuals.
  • Extracellular protein kinase A as a cancer biomarker its expression by tumor cells and reversal by a myristate-lacking Calpha and Rllbeta subunit

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Zoology (AREA)
  • Hospice & Palliative Care (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Cette invention concerne un procédé permettant de dépister la présence d'un cancer par mesure du taux d'activité enzymatique et des auto-anticorps dans le sang d'un individu. En particulier, cette invention concerne des procédés pour mesurer l'activité de la protéine kinase A (PKA) activée dépendante de l'AMPc et les anticorps anti-PKA, un kit pour mesurer l'activité de la PKA activée, et l'utilisation des taux mesurés de ces analytes pour déterminer la présence d'un cancer.
PCT/US2010/052597 2009-10-15 2010-10-14 Mesure de la pka pour dépister le cancer WO2011047112A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US25176909P 2009-10-15 2009-10-15
US61/251,769 2009-10-15

Publications (1)

Publication Number Publication Date
WO2011047112A1 true WO2011047112A1 (fr) 2011-04-21

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WO (1) WO2011047112A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2614375A2 (fr) * 2010-09-09 2013-07-17 Traxxsson, LLC Procédés combinés de diagnostic d'un cancer chez un patient

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080213918A1 (en) * 2001-04-11 2008-09-04 Bristol-Myers Squibb Company Polynucleotides encoding two novel human G-protein coupled receptors, HGPRBMY28 and HGPRBMY29, and splice variants thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080213918A1 (en) * 2001-04-11 2008-09-04 Bristol-Myers Squibb Company Polynucleotides encoding two novel human G-protein coupled receptors, HGPRBMY28 and HGPRBMY29, and splice variants thereof

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"MBL International corporation Reagents for Research", MESACUP PROTEIN ASSAY KIT, 10 August 2001 (2001-08-10), Retrieved from the Internet <URL:http://web.archive.org/web/20010815170411/www.mblintl.com/home/alphken.htm> *
"MESACUP Protein Kinase Assay Kit", DATASHEET, 2008, 4 H CONSTITUTION WAY, WOBUM, MA 01801 *
CHO ET AL.: "Extracellular protein kinase A as a cancer biomarker: Its expression by tumor cells and reversal by a myristate-lacking C-alpha and RII-beta subunit overexpression", PROC. NAT. ACADEMY SCI., vol. 97, no. 2, 2000, pages 835 - 840 *
JAY ET AL.: "Rapid increase in PKA activity during long-term potentiation in the hippocampal afferent fibre system to the prefrontal cortex in vivo", EUROPEAN JOURNAL OF NEUROSCIENCE, vol. 10, 1998, pages 3302 - 3306 *
SANDRINI ET AL.: "Regulatory Subunot Type I-alpha of Protein Kinase A (PRKARIA): A Tumor- Supressor Gene for Sporadic Thyroid Cancer", GENES, CHROMOSOMES & CANCER, vol. 35, 2002, pages 182 - 192 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2614375A2 (fr) * 2010-09-09 2013-07-17 Traxxsson, LLC Procédés combinés de diagnostic d'un cancer chez un patient
EP2614375A4 (fr) * 2010-09-09 2014-02-26 Traxxsson Llc Procédés combinés de diagnostic d'un cancer chez un patient
AU2011299088B2 (en) * 2010-09-09 2015-12-24 Traxxsson, Llc Combination methods of diagnosing cancer in a patient
EP3211421A1 (fr) * 2010-09-09 2017-08-30 Traxxsson, LLC Combinaison de procédés de diagnostic du cancer chez un patient

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