WO2016013400A1 - Biomarqueur pour une utilisation dans le diagnostic du cancer du poumon - Google Patents

Biomarqueur pour une utilisation dans le diagnostic du cancer du poumon Download PDF

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Publication number
WO2016013400A1
WO2016013400A1 PCT/JP2015/069674 JP2015069674W WO2016013400A1 WO 2016013400 A1 WO2016013400 A1 WO 2016013400A1 JP 2015069674 W JP2015069674 W JP 2015069674W WO 2016013400 A1 WO2016013400 A1 WO 2016013400A1
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Prior art keywords
immune complex
lung cancer
gelsolin
complement component
cancer
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PCT/JP2015/069674
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English (en)
Japanese (ja)
Inventor
直敬 黒田
要 大山
中村　洋一
茂 河野
直哉 岸川
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国立大学法人 長崎大学
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Priority to JP2016535868A priority Critical patent/JPWO2016013400A1/ja
Publication of WO2016013400A1 publication Critical patent/WO2016013400A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G01N27/622—Ion mobility spectrometry
    • G01N27/623—Ion mobility spectrometry combined with mass spectrometry
    • 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
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • 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
    • 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

Definitions

  • the present invention relates to a lung cancer test method, drug efficacy evaluation method, and test kit targeting a novel biomarker for lung cancer.
  • An antibody is a type of protein produced from B lymphocytes. It recognizes non-self (bacteria, viruses, etc.) entering from outside the body and binds to them to activate leukocytes and macrophages for biological defense. Plays a big role. Thus, the antibody has a function of recognizing and eliminating non-self xenobiotics.
  • an immune complex may be formed when an antibody that recognizes its own normal cells or tissues is produced. As such diseases, autoimmune diseases are known, and it is also known that immune complexes are formed by viruses, bacterial infections, cancers and the like. Especially for cancer, since early detection greatly affects the prognosis and treatment policy, there is a great need for searching for a cancer-specific immune complex.
  • proteome analysis has begun to be used in many bioanalytical science research fields, and has been regarded as promising as a means for elucidating the cause and onset mechanism of diseases.
  • shotgun proteomics has been developed, in which all proteins are fragmented by enzymatic degradation and analyzed by high-performance liquid chromatography / tandem mass spectrometry (LC-MS / MS). And it has come to be able to perform with good reproducibility.
  • LC-MS / MS high-performance liquid chromatography / tandem mass spectrometry
  • LC-MS / MS high-performance liquid chromatography / tandem mass spectrometry
  • a method has also been reported in which immune complexes in serum are identified using an antibody microarray and antigen proteins that have formed colon cancer-specific immune complexes are comprehensively identified (Non-patent Document 1).
  • the present inventors have developed an “immuno complexome analysis method” that collects immune complexes and comprehensively identifies antigens in the immune complexes by proteomics, and uses this method only in patients with rheumatoid arthritis.
  • thrombospondin-1, platelet factor 4 two types of autoantigen proteins that are specifically detected.
  • thrombospondin-1, platelet factor 4 thrombospondin-1, platelet factor 4
  • Rho JH et al J Proteome Res. 2013; 12 (5): 2311-20.
  • An object of the present invention is to provide a test for diagnosis of lung cancer, a method for evaluating drug efficacy, a test kit, and the like.
  • Immune complexes are known to show high levels in malignant tumors other than autoimmune diseases.
  • autoantibody tests have been used for early diagnosis in lung cancer and colorectal cancer, and it is considered that there is a sufficient possibility that a cancer-specific immune complex is formed in serum. Therefore, the inventors conducted immunocomplexome analysis using serum derived from cancer patients (lung cancer, colon cancer, malignant lymphoma), and attempted to search for disease-specific immune complexes. As a result, a novel marker for lung cancer was found. As a result of further studies based on these findings, the present inventors have completed the present invention.
  • the present invention [1] A method for examining lung cancer, comprising detecting gelsolin and / or complement component C9 in an immune complex recovered from a sample derived from a subject; [2] The inspection method according to [1], including the following steps: (1) a step of recovering an immune complex from a sample derived from a subject, (2) a step of decomposing the immune complex and separating a degradation product; (3) detecting gelsolin and / or complement component C9 contained in the immune complex by mass spectrometry of the separated degradation product; [3] The inspection method according to [1], including the following steps: (1) a step of recovering an immune complex from a sample derived from a subject, (2) detecting gelsolin and / or complement component C9 contained in the immune complex by an immunoassay; [4] The examination method according to any one of [1] to [3], wherein the lung cancer is lung adenocarcinoma; [5] The examination method according to any one of [1] to [4], wherein the sample is blood, serum
  • a method of determining whether or not the cancer is metastatic cancer including the following steps: (1) a step of recovering an immune complex from a sample derived from a subject having a history of developing lung cancer and having a cancer other than lung cancer; (2) a step of decomposing the immune complex and separating a degradation product; (3) detecting gelsolin and / or complement component C9 contained in the immune complex by mass spectrometry of the separated degradation product; [15] The determination method according to [13], including the following steps: (1) a step of recovering an immune complex from a sample derived from a subject having a history of developing lung cancer and having a cancer other than lung cancer; (2) detecting gelsolin and / or complement component C9 contained in the immune complex by an immunoassay; [16] The determination method according to any one of [13] to [15], wherein the lung cancer is lung adenocarcinoma; [17] The determination method according to any one of [13] to [16], wherein the sample is blood,
  • the present invention relates to a method for examining lung cancer, which comprises detecting gelsolin and / or complement component C9 in an immune complex recovered from a sample derived from a subject.
  • the immune complex refers to a complex comprising an antigen protein (self-antigen protein or non-self-antigen protein) and an antibody specific for the antigen.
  • the amino acid sequence of gelsolin to be detected in the present invention is registered in UniProt as accession numbers B7Z2X4, B7Z992, etc., respectively.
  • gelsolin may have the amino acid sequence shown in SEQ ID NO: 1 or 2, for example.
  • the complement component C9 to be detected in the present invention is registered in UniProt as an accession number P02748.
  • complement component C9 may have the amino acid sequence shown in SEQ ID NO: 3, for example.
  • the amino acid sequence constituting the protein may be the amino acid sequence specified above, or 1 to a plurality of amino acid sequences may be included in the amino acid sequence. Amino acids may be substituted, deleted, added or introduced.
  • gelsolin and complement component C9 may be full-length proteins or partial proteins.
  • gelsolin and “complement component C9” refer to the amino acid sequence specified by the above accession number, the amino acid sequence specified by the above sequence number, or 1 to It is used to mean a protein composed of an amino acid sequence in which a plurality of amino acids are substituted, deleted, added or introduced, or a partial protein thereof.
  • gelsolin detected or quantified in the test method of the present invention is a protein comprising an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2
  • complement component C9 is: It may be a protein comprising an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 3.
  • the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 is about 80% or more, preferably about 80% or more of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. Examples thereof include amino acid sequences having homology of 85% or more, more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 98% or more.
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 3 is about 80% or more, preferably about 85% or more, more preferably about 80% or more of the amino acid sequence represented by SEQ ID NO: 3. Examples include amino acid sequences having homology of 90% or more, particularly preferably about 95% or more, and most preferably about 98% or more.
  • homology refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably the algorithm uses a sequence of sequences for optimal alignment). The percentage of identical and similar amino acid residues relative to all overlapping amino acid residues in which one or both of the gaps can be considered).
  • Similar amino acids means amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn) ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is expected that substitution with such similar amino acids will not change the phenotype of the protein (ie, is a conservative amino acid substitution).
  • the gelsolin or complement component C9 transcript detected or quantified by the test method of the present invention includes DNA containing the same or substantially the same base sequence as that encoding the gelsolin or complement component C9.
  • DNA substantially identical to the base sequences encoding gelsolin and complement component C9 include, for example, about 50% or more, preferably about 60% or more, more preferably about 70% or more, and particularly preferably about base sequence. Examples thereof include DNA containing a base sequence having a homology of 80% or more, most preferably about 90% or more.
  • the lung cancer test method of the present invention may be, for example, a method including the following steps (test method I). (1) a step of recovering an immune complex from a sample derived from a subject, (2) a step of decomposing the immune complex and separating a degradation product; (3) A step of detecting gelsolin and / or complement component C9 contained in the immune complex by mass spectrometry of the separated degradation product
  • the subject to which the test method of the present invention can be applied is not particularly limited, and examples thereof include a subject who may be suspected of having or suffering from lung cancer.
  • lung cancer include small cell lung cancer and non-small cell lung cancer (lung adenocarcinoma, lung squamous cell carcinoma), and lung adenocarcinoma is more suitable for the examination method of the present invention.
  • the sample used in the step (1) of the test method I of the present invention is a sample collected from a subject, and includes an antigen to be detected (ie, gelsolin and complementcompcomponent C9) and an antibody that binds to the antigen.
  • an antigen to be detected ie, gelsolin and complementcompcomponent C9
  • an antibody that binds to the antigen There is no particular limitation as long as it contains an immune complex.
  • samples include whole blood, plasma, serum, lymph, saliva, cerebrospinal fluid, body fluids such as joint fluid, or fractions thereof, mucous membranes, and tissue specimens (lung tissue) obtained by biopsy. Blood, serum or tissue is preferable, and serum is more preferable because of the advantage that there are few contaminants in the sample and the burden on the subject is small.
  • the immune complex in the sample can be collected using a method known per se.
  • methods for separating and purifying immunoglobulin e.g., salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, adsorption / desorption method using ion exchanger (e.g. DEAE), ultracentrifugation method, gel filtration method,
  • an active adsorbent such as protein A or protein G
  • the antibody constituting the immune complex may be any subclass, including IgG, IgM, IgA, IgD, or IgE, and IgG may include IgG1, IgG2, IgG3, and IgG4.
  • the immune complex recovered from the sample may be separated as it is, but may be reductively alkylated in advance to reduce the disulfide bond of the antibody.
  • the reducing agent include dithiothreitol (DTT).
  • DTT dithiothreitol
  • peptidase protease (protease) include trypsin and pepsin.
  • the separation of the degradation products can be performed according to a method known per se.
  • Such methods mainly utilize differences in molecular weight such as methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
  • Method using charge difference such as ion exchange chromatography, method utilizing specific affinity such as affinity chromatography, method utilizing hydrophobic difference such as liquid chromatography, isoelectric focusing
  • a method using the difference in isoelectric point, such as a method is used. These methods can be combined as appropriate.
  • it is preferable to use liquid chromatography because the degradation product is a peptide, and it is easy to perform the subsequent mass spectrometry.
  • the carrier for the liquid chromatography filler used in the separation is preferably silica gel or a spherical or crushed synthetic resin such as polyvinyl alcohol.
  • the average particle size is preferably about 0.5 ⁇ m to about 100 ⁇ m, more preferably about 1 ⁇ m to about 50 ⁇ m, and still more preferably about 3 ⁇ m to about 5 ⁇ m.
  • a person skilled in the art can select a suitable particle size in consideration of the fact that the smaller the average particle size, the larger the theoretical number of columns per unit length, but the greater the pressure loss of the column.
  • the carrier of the packing material for liquid chromatography used in the present invention is one in which a substituted or unsubstituted hydrocarbon group is bonded to the carrier, and in particular, one having a chain length of 8 or more carbon atoms.
  • a hydrocarbon group are not particularly limited, and include an octyl group, an octadecyl group, and a functional group obtained by modifying these ends, and an octadecyl group is preferably used.
  • the holding force depends on the chain length of the hydrocarbon group, those skilled in the art can adjust the holding force in the stationary phase by adjusting the number of carbon atoms in the chain length of the hydrocarbon group.
  • the polarity of a stationary phase can also be adjusted using a phenyl group, a diol group, a nitrile group, an amino group, etc. as a functional group.
  • Examples of the substituted or unsubstituted hydrocarbon group having a chain length of 8 or less carbon atoms include a trimethyl group, a butyl group, a phenyl group, a cyanopropyl group, and an aminopropyl group.
  • Suitable as the packing material for liquid chromatography of the present invention is, for example, a packing material for liquid chromatography in which a commercially available octyl group is chemically bonded to silica gel (Acclaim (R) PepMap100 C8), and octadecyl group on silica gel.
  • a packing material for liquid chromatography in which a commercially available octyl group is chemically bonded to silica gel (Acclaim (R) PepMap100 C8), and octadecyl group on silica gel.
  • Examples include chemically bonded packing materials for liquid chromatography [Acclaim (R) PepMap300 C18], [Acclaim (R) PepMap100 C18], and the like. These fillers are suitable in terms of stable quality and easy availability, but are not limited thereto.
  • Solvents to be contacted with the packing material for liquid chromatography include salts in a mixed solvent system such as water and organic solvents such as methanol, ethanol, propanol, isopropanol, acetonitrile, ethyl acetate, dioxane, tetrahydrofuran (THF) or DMSO.
  • a solvent in which formic acid is mixed in acetonitrile is brought into contact with the packing material for liquid chromatography, but is not limited thereto.
  • a concentration gradient is given to the solvent, and molecules with high polarity are eluted with a solvent with low acetonitrile concentration, for example, and the molecules with low polarity are eluted successively by increasing the acetonitrile concentration.
  • a concentration gradient is given to the solvent, and molecules with high polarity are eluted with a solvent with low acetonitrile concentration, for example, and the molecules with low polarity are eluted successively by increasing the acetonitrile concentration.
  • those skilled in the art can also set suitably about the elution time at the time of elution.
  • a salt such as ammonium sulfate, sodium citrate, potassium citrate, potassium phosphate, sodium phosphate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium chloride, potassium chloride, etc.
  • the pH of the solvent can be adjusted, but is not limited thereto.
  • the ionization of the solute can be controlled by adjusting the pH of the solvent.
  • the pH is 2 to 6 Is preferable, and 2 to 3 is more preferable.
  • the temperature in the present embodiment is preferably 0 to 40 ° C., more preferably 10 to 30 ° C.
  • mass spectrometry is a method of measuring an ionized analyte in a gas phase.
  • the analyzer is roughly divided into an ion source and a mass of an ionized analyte. It consists of a mass analyzer that measures the charge-to-charge ratio (m / z) and an ion detector that records the number of ions at each m / z value.
  • the most commonly used techniques for ionizing samples such as proteins and peptides include, for example, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and matrix-assisted laser desorption / ionization (MALDI). Is mentioned.
  • ESI is a method in which a solution-like sample is introduced into a capillary and sprayed and ionized by applying a high voltage, and can be analyzed in combination with a separation means such as chromatography or electrophoresis.
  • APCI is a method in which a sample solution is forcibly vaporized by heating at a high temperature of 400 to 500 ° C., and then ions are generated using the discharge of a corona needle.
  • MALDI is a method in which crystals obtained by mixing a sample in a matrix (such as an aromatic organic compound) are sublimated and ionized by laser pulses.
  • ESI or MALDI is preferably used for ionization of the sample of the present invention, but ESI is more preferable because it easily generates expensive ions.
  • the sample ionized by the ion source is applied and pulled into the mass spectrometer at a high voltage.
  • a voltage can be appropriately set by those skilled in the art as long as the sample can be analyzed, but if the voltage is too low, the ionized sample is not sufficiently transferred to the mass analyzer, and if the voltage is too high, Fragmentation occurs in the ionized sample.
  • a voltage of 1.0 kV to 2.5 kV, preferably 1.2 kV to 2.0 kV can be applied.
  • the mass analyzer separates ions within the selected mass-to-charge ratio (m / z).
  • the mass analyzer plays an important role in the sensitivity and resolution of analysis data, the accuracy of mass, and the abundance of information obtained from mass spectrum data.
  • Ion separation methods can be classified into six basic types: magnetic field type, electric field type, ion trap type, time-of-flight (TOF) type, quadrupole type, and Fourier transform cyclotron. It is a type. Each of these has advantages and disadvantages, and can be used alone or connected to each other.
  • tandem mass (MS / MS) analysis is used as a mass analysis method for fragmenting a sample in the middle of mass analysis and analyzing the fragmented ions.
  • Any of the mass spectrometry methods described above may be used to carry out the method of the present invention, and those skilled in the art can implement the method of the present invention by substantially the same or similar methods.
  • tandem mass spectrometry is preferably used.
  • ion trap tandem mass spectrometry is used in the examples described later.
  • Spectra obtained by mass spectrometry can be analyzed by known methods, but antigens can also be determined by comparison with public protein / peptide databases.
  • databases include, for example, the protein database of International Protein Protein Index provided by The Europe, Bioinformatics Institute, the protein database published in National Center Biotechnology Information (NCBI), and the protein database published in Swiss-Prot. Etc. Therefore, by comparing the spectral data obtained by mass spectrometry with the database, it can be determined whether the immune complex recovered from the sample contains gelsolin and / or complement component C9.
  • the lung cancer test method of the present invention may be a method including the following steps (test method II). (1) a step of recovering an immune complex from a sample derived from a subject, (2) A step of detecting gelsolin and / or complement component C9 contained in the immune complex by an immunological assay.
  • the sample used in the step (1) of the test method II of the present invention and the method for recovering the immune complex from the sample may follow the steps (1) of the test method I of the present invention.
  • step (2) of the test method II of the present invention the detection of gelsolin and / or complement component C9 in the immune complex recovered from the subject-derived sample is carried out by using an antibody specifically recognizing gelsolin and complement component C9, respectively.
  • An antibody that specifically recognizes can be used for immunoassay (eg, ELISA, FIA, RIA, Western blot, etc.).
  • a sandwich ELISA method can be used as an immunoassay.
  • an antibody that specifically recognizes gelsolin and / or complement component C9 contained in the immune complex and gelsolin (or complement component C9) insolubilized on the carrier and gelsolin (or complement component C9) are specifically identified.
  • Quantification of gelsolin (or complement component C9) that formed immune complexes by measuring the activity of the labeling agent on the insolubilized carrier after reacting with the labeled antibody that recognizes the protein simultaneously or sequentially Can do.
  • the insolubilized antibody and the labeled antibody have an antigen recognition site that does not interfere with the binding of gelsolin (or complement component C9) to each other (for example, one antibody has gelsolin (or complement). the N-end of component C9) is recognized and the other antibody reacts with the C-end of gelsolin (or complement component C9)).
  • an antibody that specifically recognizes gelsolin (or complement component C9) and a labeled antibody that specifically recognizes gelsolin (or complement component C9) include gelsolin, complement component C9 polypeptide, Partial peptides having the antigenicity thereof, specifically, partial peptides having a portion corresponding to all or an epitope of the peptide sequence shown in Accession No. B7Z2X4 or B7Z992 in UniProt for gelsolin and Accession No. P02748 in UniProt for complement component C9 Can be produced by an existing general production method.
  • the antibody examples include an anti-gelsolin antibody that recognizes PRLKDKKMDAHP (SEQ ID NO: 4) as an epitope.
  • the antibody includes a polyclonal antibody, a natural antibody such as a monoclonal antibody (mAb), a chimeric antibody that can be produced using a gene recombination technique, a humanized antibody or a single chain antibody, and binding properties thereof. Fragments are included, but are not limited to these.
  • the antibody is a polyclonal antibody, a monoclonal antibody or a binding fragment thereof.
  • the binding fragment means a partial region of the aforementioned antibody having specific binding activity, and specifically includes, for example, F (ab ′) 2 , Fab ′, Fab, Fv, sFv, dsFv, sdAb and the like. (Exp. Opin. Ther. Patents, Vol.6, No.5, p.441-456, 1996).
  • the class of the antibody is not particularly limited, and includes antibodies having any isotype such as IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable, and IgG is more preferable in consideration of ease of purification.
  • a radioisotope for example, an enzyme, a fluorescent substance, a luminescent substance, or the like is used.
  • the radioisotope for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used.
  • the enzyme is preferably stable and has a large specific activity.
  • ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • the fluorescent substance for example, fluorescamine, fluorescein isothiocyanate and the like are used.
  • the luminescent substance for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
  • insolubilization of the antibody physical adsorption may be used, or a method using a chemical bond usually used to insolubilize or immobilize a protein or an enzyme may be used.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
  • gelsolin and / or complement component C9 contained in an immune complex is reacted with an insolubilized antibody (primary reaction), and a labeled antibody is further reacted (secondary reaction), and then on an insolubilized carrier.
  • the amount of gelsolin and / or complement component C9 in the immune complex can be quantified.
  • the primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times.
  • the labeling agent and the insolubilizing method can be based on those described above.
  • the antibody used for the solid phase antibody or the labeling antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving the measurement sensitivity. It may be used.
  • the method for examining lung cancer of the present invention is characterized in that gelsolin and / or complement component C9 contained in an immune complex contained in a sample derived from a subject is detected by an immunoassay. It is.
  • an antibody that specifically recognizes immune complex contained in a sample derived from a subject and gelsolin (or complement component C9) insolubilized on a carrier, and a labeled antibody that specifically recognizes human IgG And the gelsolin (or complement component C9) that formed the immune complex can be quantified by measuring the activity of the labeling agent on the insolubilized carrier.
  • the principle of this method is described in, for example, J Proteome Res. 2013 May 3; 12 (5): 2311-20, and those skilled in the art can appropriately set conditions and carry out the method of the present invention. it can.
  • the antibody production method, insolubilization method, labeling method, and the like may be as described above.
  • a measurement system for gelsolin and complement component C9 may be constructed by adding the usual technical considerations of those skilled in the art to the usual conditions and procedures in each method.
  • a measurement system for gelsolin and complement component C9 may be constructed by adding the usual technical considerations of those skilled in the art to the usual conditions and procedures in each method.
  • Hiroshi Irie “Radioimmunoassay” Kelsha, published in 1974
  • Hiroshi Irie “Continue Radioimmunoassay” published in Kodansha, 1974
  • Enzyme Immunoassay edited by Eiji Ishikawa et al. 53)
  • the test method of the present invention tests whether or not a subject suffers from lung cancer by detecting gelsolin and / or complement component C9 in an immune complex recovered from a subject-derived sample by the above-described method. be able to. For example, when gelsolin and / or complement component C9 is detected in an immune complex collected from a sample derived from a subject, it can be determined that the subject is likely to have lung cancer. In particular, when both gelsolin and complement component C9 can be detected in the immune complex, it can be determined that the possibility of suffering from lung cancer is higher than when only one of them is detected.
  • immune complexes are collected from samples derived from individuals that have been confirmed not to have lung cancer (negative control) and individuals that have been clinically determined to have lung cancer (positive control).
  • the detection level of gelsolin and / or complement component C9 in the immune complex recovered from the sample from the subject is compared to that of the positive and negative controls.
  • the test method of the present invention may further include a step of determining that the subject is suffering from lung cancer when gelsolin and / or complement component C9 is quantified at a higher level than the detection or control group in the subject. .
  • the therapeutic effect can be evaluated. It can be carried out. That is, gelsolin and / or complement component C9 in the immune complex collected from samples collected over time from patients treated or treated with lung cancer were measured and compared, respectively. It can be determined that a therapeutic effect is observed if the level of immune complexes including / complementcompcomponent C9 tends to be suppressed or cannot be detected.
  • the present invention also provides for detecting and comparing gelsolin and / or complement component C9 in immune complexes collected from samples collected over time from patients undergoing treatment or undergoing treatment for lung cancer and compared over time.
  • the present invention relates to a method for evaluating therapeutic effects on lung cancer patients.
  • the evaluation method of the present invention may be, for example, a method including the following steps (evaluation method I). (1) recovering the immune complex from a sample collected over time from a patient with or during treatment of lung cancer, (2) a step of decomposing the immune complex and separating a degradation product; (3) A step of detecting gelsolin and / or complement component C9 contained in the immune complex by mass spectrometry of the separated degradation products and comparing them over time.
  • the evaluation method of the present invention may be a method including the following steps (evaluation method II), for example. (1) recovering the immune complex from a sample collected over time from a patient with or during treatment of lung cancer, (2) A step of detecting gelsolin and / or complement component C9 contained in the immune complex by immunoassay and comparing them over time.
  • the evaluation method of the present invention may further include a step of determining that a therapeutic effect is observed when the level of immune complex containing gelsolin and / or complement component C9 after treatment is reduced as compared to before treatment. Good.
  • the lung cancer testing method to a patient having a history of lung cancer and having a cancer other than lung cancer, it is possible to determine whether the cancer is a metastatic cancer of the lung cancer. . That is, when gelsolin and / or complement component C9 is detected in an immune complex collected from a sample collected from the cancer patient, it can be determined that the cancer is highly likely to be a metastatic cancer of lung cancer.
  • Lung cancer has developed in the past and may have already been treated at the time of determination, or may be under treatment at the time of determination.
  • the sample is preferably derived from the cancer tissue to be determined.
  • the determination criteria may be in accordance with the determination criteria described in the above lung cancer inspection method. With this determination method, an appropriate treatment policy can be selected.
  • the present invention also includes detecting gelsolin and / or complement component C9 in an immune complex recovered from a sample derived from a subject having a history of developing lung cancer and having a cancer other than lung cancer.
  • the present invention relates to a method for determining whether cancer is a metastatic cancer of lung cancer.
  • the determination method of the present invention may be, for example, a method including the following steps (determination method I).
  • the determination method of the present invention may be, for example, a method including the following steps (determination method II). (1) a step of recovering an immune complex from a sample derived from a subject having a history of developing lung cancer and having a cancer other than lung cancer; (2) A step of detecting gelsolin and / or complement component C9 contained in the immune complex by an immunoassay. Further, in the determination method of the present invention, when gelsolin and / or complement component C9 is detected in an immune complex collected from a sample collected from the cancer patient, the cancer is likely to be a metastatic cancer of lung cancer. May be further included.
  • the present invention also provides a test kit for testing lung cancer that can be preferably used in the test method of the present invention.
  • the test kit comprises an antibody specifically recognizing gelsolin and / or an antibody specifically recognizing complement component C9.
  • each antibody can specifically recognize different epitopes of gelsolin and complement ⁇ ⁇ ⁇ component C9.
  • Examples of the antibody specifically recognizing gelsolin and the antibody specifically recognizing complement component C9 include the antibodies described above in the test method of the present invention.
  • Reaction for detecting gelsolin and / or complement component C9 in addition to an antibody that specifically recognizes gelsolin and / or an antibody that can specifically detect complement component C9 as a reagent constituting the test kit of the present invention It may further contain other substances that are necessary in the above, and that do not adversely affect the reaction when stored in the coexisting state.
  • the reagent may be provided with a separate reagent containing gelsolin and / or other substances required in the reaction to detect complementcompcomponent C9.
  • the reaction for detecting gelsolin and / or complement component C9 is an antigen-antibody reaction
  • the other substance includes, for example, a reaction buffer, a competitor antibody, a labeled secondary antibody (for example, a primary antibody).
  • test kit may contain protein A or protein G as a reagent for collecting immune complexes, and these may be immobilized on a carrier (sepharose column, magnetic beads, etc.). Good.
  • Example 1 Collection of detection sample of immune complex specific to lung adenocarcinoma patients 28 lung adenocarcinoma patients (Stage III to IV; age, 34-81 years; 17 men, 11 women), colon cancer patients 11 (Stage I-IV; age, 51-93 years; 2 men, 9 women; 6 preoperative CEA positive, 1 preoperative CA19-9 positive, 4 preoperative p53 positive), malignant lymphoma patients Serum was collected from 9 (age, 61-81 years; 5 men, 4 women) and 11 healthy individuals (age, 30-65 years; 9 men, 2 women).
  • IC immunocomplex
  • PBS phosphate buffered saline
  • the beads were resuspended in 100 ⁇ l of 10 mM dithiothreitol and incubated for 45 minutes at 56 ° C., after which 100 ⁇ l of 55 mM iodoacetamide was added and incubated in the dark at room temperature for 30 minutes. Subsequently, trypsin solution (25 mM ammonium bicarbonate) was added at a final concentration of 0.5 mg / ml and the mixture was incubated at 37 ° C. overnight. TFA (10%, v / v) was added to stop the digestion, and then the supernatant containing the antigen and antibody-derived peptide digest was collected using a magnet. Finally, the mixture was concentrated to about 80 ⁇ l under reduced pressure.
  • the mobile phase B content in the mobile phase was increased from 5 to 24% in 10 minutes (0.1% formic acid in 90% acetonitrile [Kanto Kagaku, Tokyo , Japan]) (mobile phase A: 0.1% formic acid); mobile phase B content increased from 24 to 34% in 70 minutes; mobile phase B content increased from 34 to 50% in 16 minutes; mobile phase B content 0.1 Increase from 50 to 100% in minutes; maintain mobile phase B content of 100% for 9 minutes (total separation time: 105 minutes).
  • Process the mass spectrometer to three tandem MS scans of the three most intense precursor masses per unit time (determined in real time by Xcaliber® software [Thermo Fisher Scientific, Waltham, MA, USA]) Set.
  • the collision energy was 35%. All spectra were measured with a total mass / charge ratio range of 400-1500.
  • the transfer capillary temperature was set to 200 ° C.
  • MS / MS data was extracted using Proteome Discoverer V3.3 (Thermo Fisher Scientific, Waltham, MA, USA).
  • the filter reference values (single, double, and triple charge peptides with related factors [XCorr] and protein probabilities [P]) were adjusted so that the false-discovery rate (FDR) was 5%.
  • the FDR was calculated by dividing the number of unique peptides in the reverse database by the number of key unique peptides in the forward database. Proteins were identified for peptides longer than 6 amino acids. Keratin and trypsin were excluded as potential matches.
  • Antigen proteins identified by proteomic analysis of the serum samples were found in 418 serum samples from lung adenocarcinoma patients, 160 in serum samples from colon cancer patients, and 127 in serum samples from malignant lymphoma patients. It was. In addition to the healthy individuals analyzed this time, the results of comparative analysis with autoimmune diseases (ANCA-related vasculitis, Takayasu arteritis, mixed connective tissue disease, dermatomyositis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus) Antigen proteins specifically detected in cancer patients were 10 types in serum samples from lung adenocarcinoma patients and 2 types in serum samples from colorectal cancer patients, and were not found in malignant lymphoma.
  • autoimmune diseases ANCA-related vasculitis, Takayasu arteritis, mixed connective tissue disease, dermatomyositis, scleroderma, Sjogren's syndrome, systemic lupus
  • Lung adenocarcinoma-specific proteins identified by proteomic analysis on lung adenocarcinoma-derived serum samples are abbreviated in Table 1.
  • Table 2 abbreviates colon cancer-specific proteins identified by proteome analysis on serum samples derived from colon cancer.
  • Example 3 Verification of Abnormal Expression of Gelsolin Protein Lung Cancer (Lung Adenocarcinoma, 28; Squamous Cell Cancer, 3; Small Cell Cancer, 2) 33 Patients, Colon Cancer 20 Patients, Malignant Lymphoma 9 Patients, Urine Gelsolin concentration in each serum of 20 patients with tract cancer and 20 patients with non-cancerous pulmonary disease (interstitial pneumonia / pulmonary tuberculosis) was measured using a commercially available ELISA kit. In this example, the concentration of Gelsolin as a free antigen was quantified, not the concentration of immune complex consisting of Gelsolin and an antibody specific for Gelsolin.
  • the Gelsolin concentration in non-lung cancer patients is 0.95 times in colorectal cancer patients, 0.84 times in malignant lymphoma patients, 0.87 times in urinary tract cancer patients, and non-cancerous lung It was 1.64 times in disease patients, and no abnormal high or low expression of Gelsolin was observed in lung cancer patients. Therefore, specific detection of immune complexes consisting of Gelsolin and antibodies specific to Gelsolin in lung cancer patients is not due to abnormal expression of Gelsolin, but antibodies specific to Gelsolin and Gelsolin specifically immunize in lung cancer patients. It was presumed to have been detected by forming a complex.
  • Example 4 Verification of Abnormal Expression Specific to Organs of Gelsolin
  • immunocomplexome analysis was performed using serum samples of 20 patients with non-cancerous lung disease (interstitial pneumonia / pulmonary tuberculosis).
  • non-cancerous lung disease interstitial pneumonia / pulmonary tuberculosis.
  • no specific antibodies to Gelsolin and Gelsolin were detected in the patient, suggesting the specific formation of an immune complex composed of antibodies specific to Gelsolin and Gelsolin in lung cancer patients.
  • Example 5 Immunocomplexome analysis on serum of urinary tract cancer patients Each serum of 20 patients with urinary tract cancer (Tanaka et al, Cancer Research, 55, 3228-3232, 1995) whose relevance to Gelsolin has been pointed out Immunocomplexome analysis was performed using the sample. As a result, Gelsolin was not detected at all in the patient, strongly suggesting the specific formation of an immune complex consisting of Gelsolin and an antibody specific to Gelsolin in lung cancer patients.

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Abstract

La présente invention concerne : une méthode de test du cancer du poumon, ladite méthode comprenant les étapes consistant à détecter la gelsoline et/ou le composant C9 du complément dans un complexe immun collecté à partir d'un échantillon provenant d'un sujet ; et une trousse pour tester le cancer du poumon, ladite trousse contenant un anticorps susceptible de reconnaître plus précisément la gelsoline et/ou un anticorps susceptible de reconnaître plus précisément le composant C9 du complément.
PCT/JP2015/069674 2014-07-24 2015-07-08 Biomarqueur pour une utilisation dans le diagnostic du cancer du poumon WO2016013400A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009516178A (ja) * 2005-11-10 2009-04-16 ユニバーシティー オブ ケンタッキー 肺癌診断アッセイ
JP2010532480A (ja) * 2007-06-29 2010-10-07 アボット・ラボラトリーズ 肺癌に対する素因をスクリーニングするための方法およびマーカーの組み合わせ

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009516178A (ja) * 2005-11-10 2009-04-16 ユニバーシティー オブ ケンタッキー 肺癌診断アッセイ
JP2010532480A (ja) * 2007-06-29 2010-10-07 アボット・ラボラトリーズ 肺癌に対する素因をスクリーニングするための方法およびマーカーの組み合わせ

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YANG J ET AL.: "Prognostic significance of MCM2, Ki-67 and gelsolin in non-small cell lung cancer", BMC CANCER, vol. 6, pages 203 *
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