WO2011010666A1 - PROTÉINE KINASE Cα UTILISÉE COMME BIOMARQUEUR DU DIAGNOSTIC DU CANCER - Google Patents

PROTÉINE KINASE Cα UTILISÉE COMME BIOMARQUEUR DU DIAGNOSTIC DU CANCER Download PDF

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WO2011010666A1
WO2011010666A1 PCT/JP2010/062252 JP2010062252W WO2011010666A1 WO 2011010666 A1 WO2011010666 A1 WO 2011010666A1 JP 2010062252 W JP2010062252 W JP 2010062252W WO 2011010666 A1 WO2011010666 A1 WO 2011010666A1
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cancer
pkcα
blood
protein kinase
substrate peptide
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PCT/JP2010/062252
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English (en)
Japanese (ja)
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佳樹 片山
貞勲 姜
潤 大石
健 森
陽次 朝見
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国立大学法人九州大学
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Priority to JP2011523676A priority Critical patent/JP5721628B2/ja
Publication of WO2011010666A1 publication Critical patent/WO2011010666A1/fr

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    • 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
    • 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
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/14—Post-translational modifications [PTMs] in chemical analysis of biological material phosphorylation

Definitions

  • the present invention relates to protein kinase C ⁇ (hereinafter sometimes referred to as PKC ⁇ ) as a cancer marker. Specifically, it relates to extracellular PKC ⁇ as a biomarker for cancer diagnosis of a plurality of cancer types.
  • PKC ⁇ protein kinase C ⁇
  • Non-Patent Documents 1 to 3 Over the past 30 years, the comparison between cancer tissues and normal tissues has mainly been involved in the discovery of biomarkers for cancer diagnosis (see Non-Patent Documents 1 to 3). In recent years, the search for biomarkers in blood, urine, or saliva has attracted great interest because it is easier to collect and handle samples compared to analysis of tissue samples (see Non-Patent Documents 4 to 7). For example, serum prostate specific antigen (PSA) can be assayed extensively to diagnose prostate cancer or to monitor recurrence of prostate cancer after surgical resection or radiation treatment (Non-Patent Document 3 and 8).
  • PSA serum prostate specific antigen
  • Phosphorylation by protein kinase activates target proteins in these intracellular signal transduction pathways and plays an important role in cell proliferation (see Non-Patent Documents 9 to 11).
  • Protein kinase C PLC
  • PLC Protein kinase C
  • serine / threonine kinases is one of the most important kinases and is classified into three subfamilies based on composition and activation characteristics.
  • PKC conventional or classical PKC
  • nPKC novel or non-classical PKC
  • ⁇ , ⁇ and ⁇ novel or non-classical PKC
  • ⁇ , ⁇ and ⁇ atypical PKC
  • PKC ⁇ is known to exhibit very little activity in normal tissues and is overexpressed or highly activated in most cancer cells (eg, liver cancer, breast cancer and melanoma) ( Non-patent documents 15 to 19).
  • the present invention has been made in view of the above situation, and includes the following cancer diagnostic markers, cancer diagnostic pharmaceutical compositions, cancer diagnostic kits, methods for evaluating cancer status, cancer preventive drugs and / or
  • the present invention provides a method for screening cancer drugs and the like.
  • a marker for cancer diagnosis comprising protein kinase C ⁇ .
  • Examples of the marker of the present invention include blood markers.
  • Examples of the marker of the present invention include those used for diagnosis of early cancer or recurrent cancer, and those used for diagnosis of at least two or more types of cancer, and further all types of cancer.
  • a pharmaceutical composition for cancer diagnosis comprising a substrate peptide of protein kinase C ⁇ or an antibody against protein kinase C ⁇ .
  • a kit for diagnosing cancer comprising the pharmaceutical composition of (2) above.
  • (4) A method in which a substrate peptide of protein kinase C ⁇ and a biological sample are reacted to detect the phosphorylated peptide, and the cancer state is evaluated using the obtained detection result as an index.
  • (5) A method of detecting protein kinase C ⁇ by reacting an antibody against protein kinase C ⁇ with a biological sample, and evaluating the cancer state using the obtained detection result as an index.
  • examples of the biological sample include blood.
  • a method for screening for preventive and / or therapeutic agents for cancer A step of administering a candidate substance to a cancer-bearing model non-human mammal, a step of detecting protein kinase C ⁇ in the blood of the non-human mammal, and a target cancer preventive drug and / or cancer treatment using the obtained detection result as an index
  • Said method comprising the step of selecting a drug.
  • the cancer diagnostic marker of the present invention is a marker in the blood and is easy to handle, can be used for diagnosis of multiple cancer types, and is particularly suitable for diagnosis of early cancer and recurrent cancer, Very useful.
  • each sample was analyzed by MALDI-TOF MS. It is a graph which shows the change of the level of the activated PKC (alpha) in the blood by the recurrence of the cancer after the surgical removal of the tumor. After phosphorylation of the substrate peptide and plasma collected at 0, 7, 14, 21, and 28 days after tumor resection, each sample was analyzed by MALDI-TOF MS.
  • PKC ⁇ Protein kinase C ⁇ plays a key role in differentiation, proliferation and apoptosis of cancer cells, and its activity is known to be higher in cancer cells than in normal cells.
  • the inventor has identified a substrate peptide (Alphatomega) specific for PKC ⁇ from a library containing over 1,700 candidate peptides (Kang, J.-H. et al. (2008) Proteomics 8, 2006-2011. This peptide showed a higher phosphorylation rate in cancer cells and tissues than in normal tissues (Kang, J.-H. et al. (2008) Proteomics 8, 2006-2011. (Supra); Kang, J .- H. et al. (2008) J. Am. Chem. Soc., 130, 14906-14907. As such, alpha tomega can be used to detect PKC ⁇ activity.
  • the present inventor investigated whether or not activated PKC ⁇ was present in the blood, which was not conventionally known, using alpha-mega.
  • blood samples prepared from normal mice (control mice) and cancer xenograft mouse models (cancer-bearing mice) were analyzed by Western blot analysis and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). ).
  • MALDI-TOF MS matrix-assisted laser desorption / ionization time-of-flight mass spectrometry
  • the level of activated PKC ⁇ is higher in the blood of tumor-bearing mice (U87, A549, A431, HuH-7 and B16 melanoma) than the level observed in control mice, and the alpha tomega phosphorylation rate is , Increased in proportion to tumor size.
  • the addition of Ro-31-7549, a highly specific inhibitor of PKC ⁇ reduces the phosphorylation rate in a dose-dependent manner, while the addition of non-PKC ⁇ inhibitors rottlerin and H-89.
  • the phosphorylation rate was not significantly affected.
  • the level of activated PKC ⁇ decreased after cancer resection, but increased when the cancer recurred.
  • activated PKC ⁇ in blood can be a novel biomarker for cancer diagnosis, particularly a novel biomarker useful for diagnosis of multiple types of cancers, and 2) activated PKC ⁇ . It was found that the presence or absence of recurrent cancer (for example, recurrent cancer after surgical excision) or early cancer can be evaluated by monitoring the level of the cancer. Thus, the present inventor has been able to demonstrate for the first time that the existence of activated PKC ⁇ in blood and the discovery of a novel biomarker for cancer diagnosis based on this existence.
  • the marker for cancer diagnosis of the present invention contains PKC ⁇ as described above.
  • the amount of PKC ⁇ in a biological sample (especially blood, specifically plasma or serum) of a test animal is PKC ⁇ in a biological sample (especially blood, particularly plasma or serum) of a normal animal (control animal not showing cancer pathology). If the amount of the test animal is larger than the amount of the test animal, it can be determined that the subject animal has developed or is highly likely to develop cancer.
  • the detection target sample of PKC ⁇ that is substantially a cancer diagnostic marker is blood, among biological samples, the cancer diagnostic marker of the present invention is preferably a blood marker (particularly a plasma or serum marker). .
  • the cancer (cancer type) to be diagnosed or evaluated in the present invention is not particularly limited, and two or more types of cancer can be the target of diagnosis or evaluation, and in some cases, preferably all cancer types Can also be targeted for diagnosis and evaluation.
  • the cancer diagnostic marker of the present invention is capable of diagnosing (detecting cancer) when any cancer develops or is highly likely to develop regardless of the type of cancer. For example, it is useful in that it can be used for the first widespread diagnosis and detection of cancer at the stage before identifying the cancer type.
  • the preferred use of the marker for cancer diagnosis of the present invention includes diagnosis of recurrent cancer and early cancer.
  • an early stage cancer basically means the cancer of the stage to the stage I only or the stage II.
  • the pharmaceutical composition of the present invention is a pharmaceutical composition for diagnosing cancer characterized by containing a PKC ⁇ substrate peptide (hereinafter, PKC ⁇ substrate peptide) as described above.
  • the present invention includes a method for diagnosing cancer characterized by using a PKC ⁇ substrate peptide, and the use of the substrate peptide for producing a drug for cancer diagnosis.
  • PKC ⁇ substrate peptide may be any peptide that can be specifically phosphorylated by PKC ⁇ , and is not limited to, for example, 10 to 30 amino acid residues (preferably 10 to 20 amino acid residues, More preferred are substrate peptides consisting of 10 to 15 amino acid residues).
  • Specific examples of the peptide include a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (FKKQGSFAKKK), a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (FKKQGTFAKKK), and the like.
  • the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is referred to as “alpha tomega” in the present specification.
  • PKC ⁇ substrate peptides can be synthesized, for example, using an automated peptide synthesizer and following standard Fmoc chemistry procedures. After the synthesis, it can be purified by a known purification method using various chromatography or the like.
  • the blending ratio of the PKC ⁇ substrate peptide is not particularly limited, but is preferably 1 to 100% by weight, and more preferably 10 to 100% by weight.
  • Other components The pharmaceutical composition of the present invention may contain other components other than the PKC ⁇ substrate peptide as long as the effect of the present invention is not significantly impaired, and is not limited.
  • an antibody against a phosphorylated PKC ⁇ substrate peptide can be included.
  • the pharmaceutical composition of the present invention comprises a biological sample collected from a subject or a patient (test sample; particularly blood, in particular plasma, the PKC ⁇ substrate peptide in the composition). Or serum)), and then the phosphorylated PKC ⁇ substrate peptide is detected and quantified, and compared with the amount detected in healthy individuals, the possibility of onset and onset of cancer In addition, the degree of cancer pathology can be determined (diagnosed). In addition, by monitoring the amount of phosphorylated PKC ⁇ substrate peptide regularly in the same subject, it is also possible to determine the state of cancer (disease state) and the presence or absence of recurrence.
  • Blood collected as a test sample may be diluted 1 to 100 times with a sample buffer or the like after being subjected to treatment such as plasma separation. Good.
  • phosphate buffered saline can be used as the sample buffer.
  • the test sample after the above treatment is brought into contact with the pharmaceutical composition of the present invention and used for the phosphorylation reaction with the PKC ⁇ substrate peptide.
  • the reaction medium is not particularly limited.
  • a buffer obtained by mixing Tris-HCl and magnesium chloride can be used.
  • adenosine triphosphate (ATP) can be used as a phosphate group-donating substrate. Etc.) can be used by mixing in the buffer.
  • the reaction between the test sample and the PKC ⁇ substrate peptide is carried out at 30 to 40 ° C. (preferably 37 ° C.) for 10 to 60 minutes (preferably 30 to 60 minutes).
  • Detection of the phosphorylated PKC ⁇ substrate peptide is not limited, but can be performed using a known immunoassay according to the conventional method.
  • Immunoassays include labeled immunoassay and immunoturbidimetry (TIA), but the former is preferred.
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • FSA fluorescent immunoassay
  • a labeled immunoassay method a Western blot method (in combination with electrophoresis method) or the like is preferably used as a method utilizing a combination with other separation methods. Among these, ELISA and / or Western blotting are preferable.
  • one or more types of monoclonal antibodies against phosphorylated PKC ⁇ substrate peptides may be used, and detection may be performed by combining two or more types of immunoassay methods, and further excellent in sensitivity and reliability. Detection can be performed.
  • a method for detecting a phosphorylated PKC ⁇ substrate peptide a method using a metal colloid such as a metal nanoparticle can also be employed. More specifically, the method involves phosphorylation of a substrate peptide by a protein kinase when the substrate peptide and protein kinase are reacted in the presence of a metal colloid, or when a metal colloid is added after the reaction of the substrate peptide and protein kinase. It is a method based on the principle that the level of oxidation can be evaluated based on the color change of the metal colloid.
  • This color change is caused by the presence of metal colloid in the phosphorylation reaction of the substrate peptide by protein kinase, causing the aggregation of the phosphorylated substrate peptide by the intervention of the metal colloid particles, and the color tone of the metal colloid is based on the degree of this aggregation. It changes. In addition, the color tone of the metal colloid changes depending on the degree of aggregation substantially proportional to the phosphorylation rate of the substrate peptide.
  • the reaction between the test sample and the PKC ⁇ substrate peptide is performed in the presence of the metal colloid, or after the reaction, the metal colloid is added to change the color tone of the sample.
  • the presence or absence of phosphorylation of the PKC ⁇ substrate peptide can be detected by comparing with a sample before the reaction or by comparing with a change in color tone in a control (reaction with a sample collected from a healthy person).
  • a buffer obtained by mixing HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) and magnesium chloride is used as the donor substrate.
  • metal colloids such as metal nanoparticles
  • metal colloids include gold colloid, silver colloid, platinum colloid, iron colloid, iron hydroxide colloid, aluminum hydroxide colloid, palladium colloid, and rhodium colloid.
  • gold colloid and silver colloid are preferable, and gold colloid is more preferable.
  • gold nanoparticles can be generally produced by a liquid phase reduction method in which a reducing solution is added to a solution containing gold ions to reduce the gold ions (see, for example, JP-A-2003-253310, JP (See Kaikai 2006-152438).
  • the particle size of the metal colloid is not particularly limited, but is preferably about 1 nm to 500 nm, more preferably 3 nm to 200 nm, still more preferably 5 nm to 100 nm, for example.
  • the metal colloid depends on the particle size, the gold colloid usually exhibits a red color, and the silver colloid exhibits a yellow color.
  • the color of the gold colloid changes from red to blue due to phosphorylation and aggregation of the PKC ⁇ substrate peptide. Based on such color change, the phosphorylated PKC ⁇ substrate peptide can be detected.
  • the color tone change of the metal colloid is preferably examined by measuring the absorbance in the range of about 600 nm to 800 nm, more preferably in the range of about 600 nm to 750 nm, and still more preferably about 700 nm.
  • Quantification of phosphorylated PKC ⁇ substrate peptide is not limited, for example, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used for the PKC ⁇ substrate peptide used in the reaction. This can be carried out by determining the phosphorylation rate (the ionic strength ratio between the phosphorylated substrate and the non-phosphorylated substrate).
  • the phosphorylated PKC ⁇ substrate peptide is detected by the above-described method using a metal colloid, it is based on the degree of color tone change of the metal colloid (specifically, the degree of color tone change with an appropriate control). Or by comparison with a calibration curve prepared in advance), the phosphorylated substrate peptide can be substantially quantified.
  • a commercially available kit can also be used for quantification or detection of the phosphorylated PKC ⁇ substrate peptide.
  • the cancer state can be evaluated using the detection result (detection amount) of the phosphorylated PKC ⁇ substrate peptide detected and quantified as described above as an index. . That is, the present invention detects a phosphorylated substrate peptide by reacting a PKC ⁇ substrate peptide with a biological sample (test sample; particularly blood, specifically plasma or serum), and uses the obtained detection result as an index to detect cancer. It includes a method for evaluating the condition.
  • “evaluating the state of cancer” means determining the presence / absence, progression, severity, treatment reactivity, prognosis, etc. of cancer (recurrent cancer, early cancer).
  • the evaluation may be performed by combining subjective amounts and the like in addition to the detected amount of phosphorylated PKC ⁇ substrate peptide. Although it can be evaluated by measuring the phosphorylated PKC ⁇ substrate peptide at a frequency of once or twice a year as in regular checkups, the change in the detected amount of the phosphorylated substrate peptide is periodically monitored. It is preferable to comprehensively evaluate the state of cancer by monitoring (including monitoring the presence or absence of cancer recurrence). As such an evaluation method, for example, if the detected amount of the phosphorylated PKC ⁇ substrate peptide exceeds a certain value, it is diagnosed that there is a high possibility that some cancer has developed.
  • the pharmaceutical composition of the present invention is a pharmaceutical composition for diagnosing cancer comprising an antibody against PKC ⁇ (hereinafter, anti-PKC ⁇ antibody).
  • anti-PKC ⁇ antibody an antibody against PKC ⁇
  • this invention includes the use of the anti-PKC (alpha) antibody for manufacturing the cancer diagnostic method characterized by using an anti- PKC (alpha) antibody and the chemical
  • An anti-PKC ⁇ antibody can be prepared as follows. (A) Preparation of polyclonal antibody (i) Preparation of antigen and its solution In preparing an anti-PKC ⁇ antibody, it is first necessary to prepare or obtain a protein to be used as an immunogen (antigen). As the antigen protein, purified PKC ⁇ can be used, but is not limited thereto.For example, the PKC ⁇ consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of PKC ⁇ , and PKC ⁇ . A protein having activity can also be used.
  • PKC ⁇ for example, PKC ⁇ derived from mammals, specifically, PKC ⁇ derived from human, PKC ⁇ derived from mouse, PKC ⁇ derived from rat, or the like can be used.
  • the base sequence and amino acid sequence information of PKC ⁇ derived from each animal can be obtained from the following accession numbers.
  • the method for preparing purified PKC ⁇ as an antigen is not limited.
  • PKC ⁇ can be produced using transformants of appropriate host cells (human-derived cultured fibroblasts, Chinese hamster ovary cells, yeast, etc.). And general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., alone or in appropriate combination. .
  • the obtained purified PKC ⁇ is dissolved in a buffer solution to prepare an antigen solution.
  • the adjuvant for example, commercially available Freund's complete adjuvant or Freund's incomplete adjuvant can be used. These adjuvants may be used alone or in combination of two or more, and are not limited.
  • Immunization and collection of antiserum Immunization is performed by administering a solution containing the purified PKC ⁇ to a mammal (eg, mouse, rat, rabbit, etc.). Administration is mainly performed by injecting intravenously, subcutaneously or intraperitoneally.
  • the dose per one dose of the antigen solution is not limited, and for example, it is preferably an amount that provides 2 to 500 ⁇ g of purified PKC ⁇ per animal, and more preferably 10 to 100 ⁇ g.
  • the administration interval is not limited, and is preferably, for example, an interval of several days to several weeks, more preferably an interval of 2 to 3 weeks.
  • the administration frequency is, for example, 2 to 10 times.
  • the collection of serum (antiserum) obtained by the above immunization is not limited, but for example, it is preferably performed 1 to 28 days after the last administration day, and more preferably 2 to 14 days later.
  • the antiserum can be collected from the blood of the immunized animal according to a conventional method.
  • the target antiserum ie, an antiserum containing an anti-PKC ⁇ antibody
  • the screening method is not limited, but, for example, collected antiserum and purified PKC ⁇ or recombinant PKC ⁇ as an antigen (produced by cultured human fibroblasts, Chinese hamster ovary cells, yeast, etc.) or those A known immunoassay can be performed using the mutated enzyme according to the conventional method.
  • Immunoassays include labeled immunoassay and immunoturbidimetry (TIA), but the former is preferred.
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • FIA fluorescent immunoassay
  • a labeled immunoassay method a Western blot method (in combination with electrophoresis method) or the like is preferably used as a method utilizing a combination with other separation methods. In the case of Western blotting, since the protein sample to be detected is denatured with heat and a surfactant and used, the antigen in the screening method becomes a mutant enzyme.
  • the anti-PKC ⁇ antibody contained in the target antiserum by the above screening is usually a polyclonal antibody.
  • known purification methods such as ammonium sulfate salting-out method, ion exchange chromatography, affinity chromatography, gel chromatography, etc., are used as appropriate, combining only one type or two or more types. Then you can do it.
  • the reaction site in PKC ⁇ to which the anti-PKC ⁇ antibody can specifically bind is not particularly limited.
  • antibody-producing cells include spleen cells, lymph node cells (particularly local lymph node cells), peripheral blood cells, etc. Among them, spleen cells and local lymph node cells (for example, sub-knee lymph node cells) are preferable. Is more preferable.
  • a fused cell can be obtained by performing cell fusion between the collected antibody-producing cells and myeloma cells.
  • myeloma cells for example, cell lines that are generally available in mammals such as mice can be used. Specifically, cell lines that have drug selectivity and cannot grow on HAT selection media (hypoxanthine, aminopterin and thymine-containing media) in an unfused state, but can grow in a state fused with antibody-producing cells. preferable.
  • HAT selection media hyperxanthine, aminopterin and thymine-containing media
  • mouse myeloma cells for example, PAI, P3X63-Ag.8.U1 (P3U1), NS-I and the like can be used.
  • the cell fusion is performed, for example, by mixing antibody-producing cells and myeloma cells in a culture medium for animal cell culture such as RPMI-1640 medium without serum.
  • the mixing ratio of antibody-producing cells and myeloma cells is, for example, 5: 1.
  • the fusion reaction is preferably performed in the presence of a cell fusion promoter, and as the promoter, polyethylene glycol having an average molecular weight of 1000 to 6000 daltons or the like can be used.
  • antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).
  • the cells after the fusion reaction are cultured using, for example, a HAT selection medium. After the above culture, cells in which growth on the HAT selection medium is observed become fused cells (hybridomas).
  • the target hybridoma that is, a hybridoma producing an anti-PKC ⁇ antibody
  • the target hybridoma is screened from the hybridoma obtained by the above culture. Specifically, a culture supernatant containing a target anti-PKC ⁇ antibody is screened.
  • the screening method is not limited, for example, a part of the culture supernatant is collected and purified PKC ⁇ or recombinant PKC ⁇ (antigen-produced human-derived cultured fibroblasts, Chinese hamster ovary cells, yeast, etc.) ) Or a mutant enzyme thereof, and a known immunoassay can be performed according to the conventional method.
  • immunoassay examples include enzyme immunoassay (EIA) such as ELISA, radioimmunoassay (RIA), and fluorescent immunoassay (FIA).
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • FIA fluorescent immunoassay
  • Western blotting in combination with electrophoresis
  • the antigen in the screening method becomes a mutant enzyme.
  • the anti-PKC ⁇ antibody contained in the culture supernatant of the target hybridoma after the screening is an antibody obtained before cloning of the hybridoma, but may be an antibody consisting of a single molecule (monoclonal antibody), There is no limitation.
  • the reaction site in PKC ⁇ to which the anti-PKC ⁇ antibody can specifically bind (can be recognized) is not particularly limited.
  • Cloning of the target hybridoma by the above screening that is, establishment of a monoclonal antibody-producing cell line can be generally performed by selecting colonies derived from one cell by culturing using a limiting dilution method or the like.
  • the method of collecting the monoclonal antibody from the hybridoma obtained by the above cloning is not limited, but generally a cell culture method or ascites formation method can be employed.
  • the cell culture method the hybridoma obtained by cloning is cultured for 7 to 14 days under conditions of 37 ° C. and 5% CO 2 , for example, in an animal cell culture medium.
  • Monoclonal antibodies can be collected.
  • the ascites formation method for example, about 10 6 hybridomas obtained by cloning are administered into the abdominal cavity of a mammal of the same kind as the mammal from which the myeloma cells used for cell fusion are derived.
  • the desired monoclonal antibody can be collected from ascites or serum collected 7 to 14 days after the hybridoma is proliferated and administered.
  • any of the cell culture method and ascites formation method when purification of the antibody is required at the time of collecting or after collecting the monoclonal antibody, ammonium sulfate salting out method, ion exchange chromatography, affinity chromatography, gel chromatography, etc.
  • Known purification methods can be adopted and implemented as appropriate by combining only one type or a combination of two or more types.
  • the blending ratio of the anti-PKC ⁇ antibody is not particularly limited, but is preferably 1 to 100% by weight, and more preferably 10 to 100% by weight.
  • Other components The pharmaceutical composition of the present invention may contain other components other than the anti-PKC ⁇ antibody as long as the effect of the present invention is not significantly impaired, and is not limited. For example, it can contain a primary antibody detection reagent, a chromogenic substrate, and the like.
  • the pharmaceutical composition of the present invention is used for detecting and quantifying PKC ⁇ in a collected biological sample of a subject or patient (test sample; particularly blood, specifically plasma or serum),
  • test sample particularly blood, specifically plasma or serum
  • the presence or absence of cancer, the possibility of its onset, and the degree of the disease state can be determined (diagnosed) by comparison with the detected amount in healthy individuals.
  • the state (disease state) of cancer can also be judged by monitoring the amount of PKC ⁇ of the same subject regularly.
  • Detection of PKC ⁇ Blood or the like collected as a test sample is usually subjected to treatment such as serum separation, and is generally diluted 1 to 100 times with a sample buffer or the like.
  • sample buffer for example, phosphate buffered saline can be used.
  • the test sample after the treatment is brought into contact with the pharmaceutical composition of the present invention and used for a reaction with an anti-PKC ⁇ antibody (a monoclonal antibody is generally preferred).
  • the reaction between the test sample and the anti-PKC ⁇ antibody is carried out at 20 to 40 ° C. (preferably 30 to 37 ° C.) for 30 to 720 minutes (preferably 30 to 90 minutes).
  • PKC ⁇ can be detected by using a known immunoassay according to the conventional method.
  • Immunoassays include labeled immunoassay and immunoturbidimetry (TIA), but the former is preferred.
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • FSA fluorescent immunoassay
  • a labeled immunoassay method a Western blot method (in combination with electrophoresis method) or the like is preferably used as a method utilizing a combination with other separation methods.
  • ELISA and / or Western blotting are preferable.
  • one or two or more types of anti-PKC ⁇ monoclonal antibodies may be used, and detection may be performed by combining two or more types of immunoassays, and excellent detection with higher sensitivity and reliability can be performed. .
  • Quantification of PKC ⁇ Although quantification is not limited, it is preferable to quantify PKC ⁇ contained in the test sample from a calibration curve based on the relationship between the amount of antigen (antigen concentration: PKC ⁇ concentration) and the detected value.
  • the calibration curve is prepared in advance based on the detection value (detection data) obtained using purified PKC ⁇ as an antigen and using the above-described anti-PKC ⁇ antibody. Specifically, information on the detected value with respect to the antigen concentration is obtained by a method similar to the immunoassay method (ELISA, Western blotting method, etc.) described as the detection method, and prepared based on this. By comparing the calibration curve with the actual detection value (actual measurement value), the amount of PKC ⁇ in the test sample can be obtained.
  • a commercially available kit can also be used for quantification or detection of PKC ⁇ .
  • the state of cancer can be evaluated using the PKC ⁇ value determined or detected as described above as an index. That is, the present invention includes a method of detecting PKC ⁇ by reacting an anti-PKC ⁇ antibody with a biological sample (test sample), and evaluating the cancer state using the obtained detection result as an index.
  • evaluating the state of cancer means determining the presence / absence, progression, severity, treatment reactivity, prognosis, etc. of cancer (recurrent cancer, early cancer). Evaluation may be performed in combination with subjective symptoms in addition to the amount of PKC ⁇ .
  • the cancer status is comprehensively evaluated by periodically monitoring changes in the amount of PKC ⁇ . (Including monitoring for the recurrence of cancer).
  • an evaluation method for example, if the amount of PKC ⁇ exceeds a certain value, it is diagnosed that there is a high possibility that some cancer has developed. If the amount of PKC ⁇ is reduced in a cancer case that is already being treated, the treatment is successful. It is also possible to diagnose that the case where the amount of PKC ⁇ exceeds a certain value and changes at a high value cannot be treated and recovered.
  • Cancer Diagnostic Kit A cancer diagnostic kit can be used for diagnosing cancer, and includes, for example, the pharmaceutical composition for cancer diagnosis described in the above sections 3-1 and 3-2. Kits can be mentioned.
  • the substrate peptide when the PKC ⁇ substrate peptide is included, the substrate peptide is in a powder state or a solution state (pure water, buffer, physiological saline, etc.) in consideration of stability (storage stability) and ease of use. In a state of being dissolved in a refrigerated or frozen state (-30 ° C to -80 ° C).
  • the kit contains an anti-PKC ⁇ antibody, the antibody is preferably provided in a dissolved state in consideration of stability (storage stability) and ease of use.
  • the kit can contain other components in addition to the above PKC ⁇ substrate peptide or anti-PKC ⁇ antibody.
  • other components include a primary antibody detection reagent and a chromogenic substrate that can be used for ELISA and Western blotting.
  • the kit only needs to have at least the above PKC ⁇ substrate peptide or anti-PKC ⁇ antibody as a component. Therefore, all the components essential for the diagnosis of cancer may be provided together with the PKC ⁇ substrate peptide or the anti-PKC ⁇ antibody or not, and there is no limitation.
  • the method of screening for a cancer preventive and / or therapeutic agent of the present invention is a method comprising the following steps (a) to (c).
  • the screening method of the present invention may include other steps as necessary.
  • (b) A step of detecting PKC ⁇ in the blood of the above cancer-bearing model non-human mammal detection step
  • a step of selecting a target cancer preventive and / or cancer therapeutic agent using the obtained detection result as an index evaluation step
  • the tumor-bearing model non-human mammal to be the test animal and the control animal are not particularly limited, but usually the same kind of non-human mammal is used in that an accurate comparative experiment can be performed.
  • non-human mammals of the same litter are used, more preferably non-human mammals of the same sex and the same age are used.
  • the test animals and the control animals have the same breeding conditions other than the amount of feed intake.
  • a cancer-bearing model non-human mammal in which tumor is developed by transplanting a tumor tissue or tumor cell to a normal non-human mammal by a known method can be used.
  • a non-human mammal obtained by surgically excising a tumor tissue or tumor cell from the above cancer-bearing model non-human mammal can also be used.
  • a screening method for a preventive drug for recurrent cancer can be performed.
  • the non-human mammal after the excision is also included in the cancer-bearing model non-human mammal.
  • the control animal is not limited as long as it is suitable for use as a control for comparison with a test animal.
  • a normal non-human mammal not transplanted with a tumor tissue or the like is used, and a candidate substance is used.
  • Non-administered cancer-bearing model non-human mammals or test animals prior to administration of candidate substances can also be used as control animals.
  • Candidate substances to be administered to a test animal are not limited, but various naturally or artificially synthesized peptides, proteins (including enzymes and antibodies), nucleic acids (polynucleotide (DNA, RNA), oligonucleotides (siRNA, etc.) ), Peptide nucleic acids (PNA), etc.), low molecular or high molecular organic compounds, and the like.
  • the candidate substance can be administered orally or parenterally, and is not limited. In any case, known administration methods, administration conditions, and the like can be adopted. The dose can be appropriately set in consideration of the type and condition of the test animal, the type of candidate substance, and the like.
  • Detection step In this step, PKC ⁇ is detected from the blood (blood sample) of the test animal to which the candidate substance has been administered.
  • a well-known method can be applied as a blood sample collection method.
  • the number of detections, the detection period, and the like of PKC ⁇ are not particularly limited, and can be appropriately set in consideration of the efficacy of the target cancer preventive drug or cancer therapeutic drug.
  • the method for detecting and quantifying PKC ⁇ in blood is not limited, but in Sections 3-1. And 3-2 (especially 3-1. (4) and 3-2. (4)). The described method can be adopted as appropriate.
  • the candidate substance can be selected as a target cancer therapeutic agent.
  • the control animal can be the test animal itself before administration of the candidate substance.
  • a cancer-bearing model non-human mammal obtained by surgically excising tumor tissue or the like, or a cancer-bearing model non-human mammal once cancer treatment has been completed by a method other than surgical excision (medical method, etc.)
  • the candidate substance Can be selected as a cancer preventive drug (specifically, a preventive drug for recurrent cancer).
  • the control animal can be the test animal itself immediately before administration of the candidate substance (before recurrence).
  • cancer screening based on the amount of PKC ⁇ in the blood can be screened for a plurality of cancer types (preferably all cancer types), a cancer-bearing model non-human mammal
  • a cancer-bearing model non-human mammal As described above, when a cancer-bearing model non-human mammal of a specific cancer type is prepared and used, a preventive and / or therapeutic drug for the specific cancer type can be screened.
  • the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
  • MALDI-TOF MS analysis ⁇ -cyano-4-hydroxycinnamic acid (CHCA) matrix (10 mg / ml) was prepared in 50% water / acetonitrile and 0.1% TFA. The matrix and sample were mixed in a 20: 1 ratio. A total volume of 1 ⁇ l of the sample / matrix mixture was applied to the sample plate and dried to allow crystallization.
  • the Voyager DE RP biospectrometry workstation (Applied Biosystems) was used using a positive or negative ion reflectron mode. The acceleration voltage was 20 kV and the extraction delay time was 100 ns. Typically, 100 laser shots were averaged to improve the signal / noise ratio. All spectra were analyzed using Data Explore software (Applied Biosystems).
  • the phosphorylation rate was defined as the ionic strength ratio of phosphorylated to non-phosphorylated and calculated according to existing explanations (Kang, J.-H. et al. (2007) J. Am. Soc. Mass Spectrom. 18, 106-112 .; Kang J.-H. et al. (2007) J. Am. Soc. Mass Spectrom. 18, 1925-1931.).
  • B16 melanoma 5-week-old BALB / c male mice were used, and for human cancers (A549, U87, HuH-7 and A431), BALB / c nude mice (weight approximately 20 g) were used.
  • B16 melanoma cells 100 ⁇ l Hank's balanced salt solution (Invitrogen), for human cancer (A549, U87, HuH-7 and A431), 1 ⁇ 10 7 cells in 100 ⁇ l matrigel (BD Biosciences) were injected subcutaneously in the back. Mice were inoculated. The cancer was grown until the average diameter was about 1.2 cm.
  • the phosphorylation of the substrate peptide was performed in 30 ⁇ l buffer (20 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 100 ⁇ M ATP and 2 mg / ml plasma) containing 30 ⁇ M synthetic peptide. After incubation at 37 ° C for 60 minutes, the samples were analyzed by MALDI-TOF MS.
  • each sample was analyzed by MALDI-TOF MS.
  • MALDI-TOF MS MALDI-TOF MS.
  • a higher phosphate rate of the substrate peptide was confirmed than that of normal mouse blood (FIG. 2).
  • mice with 0.6 cm, 0.9 cm and 1.2 cm B16 melanoma or U87 tumors was prepared.
  • the phosphorylation rate increased with increasing tumor size.
  • mice with tumor size ⁇ 0.9 cm showed a significant increase in phosphorylation rate (P ⁇ 0.05) (FIG. 6).
  • P ⁇ 0.05 phosphorylation rate
  • Phosphorylated peptides have been detected by a variety of techniques including MS-based analysis, radioactive assays using [ 32 P] ATP chip technology, and homogeneous assays. Of these assays, the use of MS-based analysis has increased significantly over the past decade. The most widely used MS technique is MALDI-TOF MS, which is highly sensitive and fast in identification. Accordingly, MALDI-TOF MS was also used in the phosphorylated peptide assay in this example. Dilution methods were incorporated and samples were diluted in CHCA to avoid hindering crystal formation and ionization by contaminants or biomolecules. As a result, phosphorylated peptides can be sufficiently detected (FIG. 1).
  • PKC ⁇ is widely expressed in many tissues and plays a key role in cancer cell differentiation, proliferation and apoptosis.
  • PKC ⁇ activity is higher in tumor cells than in normal cells.
  • DAG diacylglycerol
  • PS phosphatidylserine
  • Ca 2+ cofactors
  • PKA protein kinase A
  • MAPKAP-2 mitogen-activated protein kinase activated protein kinase 2
  • PRAK p38 regulated / activated kinase
  • GSK3 glycogen synthase kinase 3
  • MAPKAP-K1b mitogen and stress activated protein kinase 1 (MSK1), protein kinase B ⁇ (PKB ⁇ ), plasma and glucocorticoid-inducible kinase (SGK), p70 ribosomal protein S6 by H-89 (10 ⁇ M) Inhibition of kinase (S6K1), Rho-related protein kinase-II (ROCK-II), checkpoint kinase 1 (CHK1), and AMP-activated protein kinase (AMPK) was> 80%, but for PKC ⁇ ⁇ 30%.
  • Ro-31-7549 is a highly specific inhibitor of PKC ⁇ (> 90% at 5 ⁇ M) and Ro-31-7549 resulted in a dose-dependent decrease in phosphorylation rate, while rottrelin Alternatively, no significant decrease was observed with H-89 (FIG. 5).
  • N-myristoylation is an acylation process that attaches myristic acid to the N-terminal glycine of proteins. Protein N-myristoylation results in fragile and reversible protein-membrane interactions.
  • active PKC ⁇ present in the extracellular space can be derived from the secretion of PKC ⁇ docked to the membrane.
  • angiogenesis involves angiogenesis-promoting factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumor necrosis factor (TNF) and interleukin 8 (IL-8), and interferon (IFN).
  • VEGF vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • TNF tumor necrosis factor
  • IL-8 interleukin 8
  • IFN interferon
  • Increased blood vessel density increases the supply of oxygen and nutrients to cancer cells, leading to cancer growth.
  • biomarkers for the diagnosis of specific tumors such as PSA for prostate cancer and human epidermal growth factor receptor 2 (HER2) for breast cancer have been discovered and used clinically.
  • few biomarkers have been discovered that can be used for early stage tumor diagnosis and extensive diagnostic screening of one or more cancers.
  • the level of activated PKC ⁇ was higher in the blood of five types of cancer mice than in the blood of normal mice (FIG. 2).
  • Plasma activated PKC ⁇ could be useful as a diagnostic marker for multiple cancers.
  • Blood biomarkers are also used to monitor cancer recurrence after surgical resection or to monitor radiotherapy or anticancer drug therapy.
  • the substrate peptide was phosphorylated for 1 hour at 37 ° C. Thereafter, the phosphorylation rate was determined by MALDI-TOF-MS.
  • the cancer diagnostic marker of the present invention is a marker in the blood and is easy to handle, can be used for diagnosis of multiple cancer types, and is particularly suitable for diagnosis of early cancer and recurrent cancer, It is extremely useful.
  • SEQ ID NO: 1 Synthetic peptide
  • SEQ ID NO: 2 Synthetic peptide

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Abstract

Cette invention concerne un nouveau biomarqueur du diagnostic du cancer, qui est simple et extrêmement fiable. Le marqueur du diagnostic du cancer se caractérise en ce qu’il renferme la protéine kinase Cα.
PCT/JP2010/062252 2009-07-22 2010-07-21 PROTÉINE KINASE Cα UTILISÉE COMME BIOMARQUEUR DU DIAGNOSTIC DU CANCER WO2011010666A1 (fr)

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KR101784414B1 (ko) 2015-10-26 2017-10-11 연세대학교 산학협력단 말디 질량 분석 이미징 방법을 활용한 소분자 화합물의 정성 및 분포 결정방법
WO2019221245A1 (fr) * 2018-05-17 2019-11-21 学校法人慈恵大学 Nouveau marqueur du cancer du foie
WO2021112225A1 (fr) * 2019-12-05 2021-06-10 国立大学法人九州大学 Procédé de diagnostic du cancer, composition de diagnostic du cancer, kit de diagnostic du cancer, procédé d'évaluation de l'état du cancer, et procédé de dépistage d'agents pour la prévention et/ou le traitement du cancer

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101784414B1 (ko) 2015-10-26 2017-10-11 연세대학교 산학협력단 말디 질량 분석 이미징 방법을 활용한 소분자 화합물의 정성 및 분포 결정방법
WO2019221245A1 (fr) * 2018-05-17 2019-11-21 学校法人慈恵大学 Nouveau marqueur du cancer du foie
JPWO2019221245A1 (ja) * 2018-05-17 2021-08-19 学校法人慈恵大学 新規肝癌マーカー
JP7267527B2 (ja) 2018-05-17 2023-05-02 学校法人慈恵大学 新規肝癌マーカー
WO2021112225A1 (fr) * 2019-12-05 2021-06-10 国立大学法人九州大学 Procédé de diagnostic du cancer, composition de diagnostic du cancer, kit de diagnostic du cancer, procédé d'évaluation de l'état du cancer, et procédé de dépistage d'agents pour la prévention et/ou le traitement du cancer
JP2021089220A (ja) * 2019-12-05 2021-06-10 国立大学法人九州大学 がんを診断するための方法、がん診断用組成物、がん診断用キット、がんの状態を評価する方法、並びにがん予防薬及び／又は治療薬をスクリーニングする方法
EP4060335A4 (fr) * 2019-12-05 2024-02-28 Univ Kyushu Nat Univ Corp Procédé de diagnostic du cancer, composition de diagnostic du cancer, kit de diagnostic du cancer, procédé d'évaluation de l'état du cancer, et procédé de dépistage d'agents pour la prévention et/ou le traitement du cancer
JP7454756B2 (ja) 2019-12-05 2024-03-25 国立大学法人九州大学 がんを診断するための方法、がん診断用組成物、がん診断用キット、がんの状態を評価する方法、並びにがん予防薬及び／又は治療薬をスクリーニングする方法

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