WO2015083791A1 - Procédé et kit de détection du cancer du poumon - Google Patents

Procédé et kit de détection du cancer du poumon Download PDF

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WO2015083791A1
WO2015083791A1 PCT/JP2014/082122 JP2014082122W WO2015083791A1 WO 2015083791 A1 WO2015083791 A1 WO 2015083791A1 JP 2014082122 W JP2014082122 W JP 2014082122W WO 2015083791 A1 WO2015083791 A1 WO 2015083791A1
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lung cancer
antibody
amount
exosome
sample
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PCT/JP2014/082122
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Japanese (ja)
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幸嗣 植田
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国立研究開発法人理化学研究所
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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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention relates to a detection method and detection kit for lung cancer.
  • Non-patent Document 1 Non-patent Document 1
  • Non-Patent Documents 2 to 4 Delivery of therapeutic RNA via exosomes is already in the pioneering stage of cancer treatment.
  • Non-Patent Documents 7 to 9 Basically, a set of molecules expressed in the original solid tumor cells can be detected as an exosome component in the blood circulation.
  • exosome biomarkers are feasible, but it is difficult to separate exosomes from biological fluids, which significantly hinders efficient search for biomarker candidates.
  • ultracentrifugation-based methods are the most common method for separating exosomes from serum samples (Non-Patent Document 10), but their reproducibility, processing time and purity quantitatively analyze a large number of clinical samples. Not suitable for biomarker screening studies (Non-patent Document 11).
  • An object of the present invention is to provide a detection method and detection kit for lung cancer using a novel biomarker for lung cancer.
  • the detection method according to the present invention is a method for detecting lung cancer, and includes a step of measuring the amount of at least one of CD91 and CD317 in a sample collected from a living body.
  • the detection kit according to the present invention is a detection kit for detecting lung cancer, an antibody or peptide probe that recognizes CD91 in a sample collected from a living body, and an antibody that recognizes CD317 in a sample collected from a living body Or at least one of the peptide probes.
  • the present invention can provide a detection method and detection kit for lung cancer using a novel biomarker for lung cancer.
  • FIG. 1 is a schematic diagram of a workflow for searching an exosome biomarker in an example.
  • Exosomes were isolated from serum samples from 46 individuals by anti-CD9 antibody-conjugated monolithic chip (anti-CD9-MSIA chip) on a 12-channel automated pipetting platform.
  • the concentrated exosome fraction was analyzed by LC / MS / MS and subjected to label-free quantitative analysis by RefinerMS software on the Expressionist® proteome server system.
  • the quantified peptides were subjected to a two-step statistical analysis consisting of ANOVA and a feature exclusion method, and finally extracted biomarker candidate peptides were identified using a Sequest database search.
  • the identification threshold was set at a false positive rate (FDR) ⁇ 1%.
  • FDR false positive rate
  • FIG. 2 is a diagram illustrating a processing workflow used in biomarker screening analysis in the Expressionist® RefinerMS module in the embodiment.
  • FIG. 3 is a diagram showing the anti-CD9-MSIA chip in the example.
  • A is an enlarged view of an anti-CD9-MSIA chip in an example.
  • B shows the results of three measurements performed by LC / MS / MS after purifying the exosome fraction from a common serum sample using six independent anti-CD9-MSIA chips.
  • FIG. 4 shows a comprehensive overview of the proteome of 1601 identified exosomal proteins in the examples.
  • A is a pie chart showing the distribution of localization of proteins in cells.
  • B is the figure which used Fisher exact probability statistics (Fisher
  • FIG. 5 is a diagram showing a two-stage statistical selection of biomarker candidates in the examples.
  • a 3-group ANOVA was performed to compare NC, IP and ADC groups (a) or NC, IP and SCC groups (b). Peptides satisfying the reference value p ⁇ 0.001 were used for the second ranking selection.
  • the upport-vector-machine-recursive-feature-elimination (SVM-RFE) method based on cross confirmation was used for comparison of NC, IP and ADC groups, and the minimum biomarker set showing the minimum misclassification rate was calculated (c).
  • SVM-RFE upport-vector-machine-recursive-feature-elimination
  • FIG. 6 is a diagram showing 19 exosomal biomarker candidates identified in the examples. LC / MS / MS signal intensities for 19 biomarker candidates from 46 samples were represented in a box plot. UniProtKB entry protein names are shown at the top of the box plot.
  • N healthy control
  • IP interstitial pneumonia
  • ADC lung adenocarcinoma
  • SCC lung squamous cell carcinoma.
  • FIG. 7 is a diagram showing a verification test based on an exosome sandwich ELISA for CD91 in Examples.
  • A shows the principle of exosome sandwich ELISA.
  • SA-HRP Streptavidin-horseradish peroxidase. 212 independent serum samples were used to measure exosomal CD91 and CEA concentrations.
  • B shows the serum exosome concentration determined by CD9-CD9 sandwich ELISA.
  • C shows the concentration of CEA measured by a commercially available ELISA kit.
  • D shows the concentration of CD91 in exosomes determined by CD9-CD91 sandwich ELISA. The values are standardized using the exosome concentration calculated in (b).
  • the dashed line shows the cutoff value for CEA (c) at 5.0 ng / mL or exosomal CD91 (d) at 2.04 U / exosome.
  • Sensitivity (Sens) and specificity (Spec) for each small group of lung cancers are shown at the bottom of the box plot.
  • N Healthy control
  • IP Interstitial pneumonia
  • ADC_1_2 Stage I, II lung adenocarcinoma
  • ADC_3_4 Stage III, IV lung adenocarcinoma
  • SCC_1_2 Stage I, II lung squamous cell carcinoma
  • SCC_3_4 Stage III IV squamous cell carcinoma of the lung.
  • FIG. 8 is a diagram showing ROC curve analysis for exosomal CD91 and CEA in Examples.
  • the ROC curve for CEA (a), the ROC curve for CD91 of exosome (b), and the combined marker CEA + exosome CD91 (c) based on logistic regression are indicated by R, respectively.
  • Sensitivity (Sens), specificity (Spec), positive predictive value (PV +), negative predictive value (PV-) and area under the curve (AUC) are shown in each graph.
  • FIG. 9 is a diagram showing a verification test based on an exosome sandwich ELISA for CD317 in Examples. The OD450 value in CD9-CD317 sandwich ELISA is shown.
  • N Healthy control
  • ADC_1 Stage I lung adenocarcinoma
  • ADC_2 Stage II lung adenocarcinoma
  • ADC_3 Stage III lung adenocarcinoma
  • ADC_4 Stage IV lung adenocarcinoma
  • SCC_1 Stage I lung squamous epithelium Cancer
  • SCC_2 Stage II lung squamous cell carcinoma
  • SCC_3 Stage III lung squamous cell carcinoma
  • SCC_4 Stage IV lung squamous cell carcinoma.
  • the inventor of the present application has developed a chip that can purify exosomes in a short time with high reproducibility.
  • exosomes were purified using this chip, and an attempt was made to search for biomarkers in exosomes.
  • the present invention has been completed.
  • the lung cancer detection method according to the present invention includes a step of measuring the amount of at least one of CD91 and CD317 in a sample collected from a living body.
  • Other specific processes, and instruments and apparatuses to be used are not particularly limited.
  • sample used in the lung cancer detection method according to the present invention is a sample (biological sample) collected from a living body (subject).
  • samples collected from a living body include samples derived from body fluids such as blood, urine and saliva. Preferably, it is derived from blood.
  • samples derived from blood include whole blood, serum, and plasma.
  • sample is preferably whole blood, serum and plasma, more preferably serum and plasma.
  • One embodiment of the method for detecting lung cancer according to the present invention includes a step of collecting a sample from a living body (subject).
  • one embodiment of the lung cancer detection method according to the present invention includes a step of preprocessing a sample collected from a living body (subject). Examples of sample pretreatment include purification of collected whole blood to obtain serum, and purification of collected whole blood to obtain plasma.
  • Examples of the “living body (subject)” include mammals such as humans, mice, rats, rabbits, monkeys, and preferably humans.
  • Examples of the blood collection site include an axillary skin vein, a cephalic vein, and an ulnar skin vein.
  • lung cancer refers to lung adenocarcinoma, lung squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and a mixture thereof.
  • the lung cancer detection method according to the present invention is particularly suitable for the detection of lung adenocarcinoma and lung squamous cell carcinoma, and is more suitable for the detection of lung adenocarcinoma.
  • stage of lung cancer is not particularly limited, in the method for detecting lung cancer according to the present invention, when measuring the amount of CD91, it is also possible to detect lung cancer at an early stage, for example, stage I and stage II, which has been difficult to detect conventionally. Can do.
  • stage III and stage VI lung cancer when measuring the amount of CD317, it is possible to suitably detect stage III and stage VI lung cancer.
  • CD91 Protein-density lipoprotein-receptor-related-protein-1
  • CD317 Breast-stromal-antigen-1
  • the accession numbers of UniProtKB for protein and human CD317 protein are shown respectively.
  • CD91 includes not only the full-length CD91 protein but also fragments thereof, and also includes the precursor of CD91 protein. That is, for example, “measuring the amount of CD91” means measuring the amount of at least one of full-length CD91 polypeptide, a fragment of CD91 polypeptide, and a precursor of CD91 polypeptide.
  • the CD91 whose amount is measured can be, for example, CD91 579-605 .
  • CD317 includes not only the full-length CD317 protein but also fragments thereof, and also includes the precursor of CD317 protein.
  • “measuring the amount of CD317” means measuring the amount of at least one of a full length CD317 polypeptide, a fragment of a CD317 polypeptide, and a precursor of a CD317 polypeptide.
  • the CD317 whose amount is measured can be, for example, CD317 137-147 .
  • “measuring the amount of CD91” is intended to measure the abundance or concentration of CD91 in a biological sample or a sample obtained by purifying it.
  • the “amount of CD91” may be an absolute amount (absolute mass or absolute concentration) or a relative amount (relative mass or relative concentration). Further, for example, it may be a peak area in mass spectrometry, a luminescence intensity in luminescence measurement, or the like, or a value indicating how many times it is with respect to a predetermined standard.
  • “Amount of CD91” is preferably the amount of CD91 present on the exosome surface. Further, it may be preferable that the “amount of CD91” is CD91 per exosome. The same can be said for CD317.
  • lung cancer marker those whose amounts can be measured are collectively referred to as “lung cancer marker” below.
  • the amount of CD91 in a biological sample derived from a subject is measured, the amount of CD91 in a biological sample derived from a control subject not having lung cancer is measured, By comparing these, lung cancer can be detected. Specifically, when the amount of CD91 in the sample derived from the subject is compared with the amount of CD91 in the sample derived from the control subject, and the amount of CD91 in the sample derived from the subject is significantly increased. Can be determined that the subject has lung cancer or is likely to have lung cancer. The amount of CD91 in the sample derived from the control subject can be determined in advance.
  • the amount of CD91 in a biological sample derived from a control subject who does not have lung cancer is measured in advance, and a normal concentration range is determined based on this, and the amount of CD91 in the biological sample derived from the subject is normal. It can also be determined that the patient has lung cancer or is likely to have lung cancer when increasing outside the concentration range. That is, in one embodiment of the method for detecting lung cancer according to the present invention, when the measured amount of CD91 is increased compared to a control subject not having lung cancer, Or the process of determining with the high possibility of having lung cancer is further included.
  • the control subjects may include both control subjects and lung cancer patients who do not have lung cancer, or only lung cancer patients.
  • the significant difference is determined by a statistical method such as t-test, F-test, chi-square test, or Mann-Whitney's U test. I can judge.
  • another example of the method for detecting lung cancer according to the present invention is to measure the amount of CD317 instead of CD91, and the same can be said. Therefore, in another embodiment of the method for detecting lung cancer according to the present invention, whether the measured amount of CD317 is increased compared to a control subject not having lung cancer, has lung cancer? Or a step of determining that the patient is likely to have lung cancer.
  • the method for measuring the amount of the lung cancer marker is not particularly limited as long as the abundance of the lung cancer marker can be quantitatively or semi-quantitatively determined.
  • an antibody that recognizes the lung cancer marker to be measured was used.
  • a method using an immunological technique, a method using a peptide probe that recognizes a lung cancer marker to be measured, a liquid column chromatography method, a mass spectrometry method, and the like can be used.
  • Examples of the method using an antibody include an ELISA method, a quantitative western blotting method, a radioimmunoassay method, an immunochromatography method, and an immunoprecipitation method.
  • the type of ELISA method is not particularly limited, but is a so-called antigen measurement system (measurement of the amount of antigen contained in a biological sample), ELISA by direct adsorption method, ELISA by competition method, ELISA by sandwich method, and micro flow An ELISA specialized for the measurement of a very small amount of sample using a road system or microbeads can be mentioned.
  • the antibody that recognizes the lung cancer marker to be measured may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody. Based on information from public databases such as UniProt, those skilled in the art can easily determine an amino acid sequence suitable as an antigen for producing an antibody that recognizes CD91 and an antibody that recognizes CD317.
  • the antibody that recognizes CD91 is preferably an antibody specific to CD91.
  • the antibody that recognizes CD317 is preferably an antibody specific for CD317.
  • antibody is intended to mean a form including all classes and subclasses of immunoglobulins and functional fragments of antibodies.
  • the antibody is a concept including both natural antibodies of a polyclonal antibody and a monoclonal antibody, and includes an antibody produced using a gene recombination technique and a functional fragment of the antibody.
  • the “functional fragment of an antibody” refers to one having a partial region of the above-described antibody and having an antigen-binding ability (synonymous with a binding fragment).
  • Natural antibodies can be derived from any species including, but not limited to, humans, mice, rats, monkeys, goats, rabbits, camels, llamas, cows and chickens.
  • the antibody produced using gene recombination technology is not particularly limited, but chimeric antibodies such as humanized antibodies and primatized antibodies obtained by genetic modification of natural antibodies, synthetic antibodies, recombinant antibodies, Mutagenized antibodies and graft-bound antibodies (for example, antibodies to which other proteins and radioactive labels are conjugated or fused), and antibodies already produced using genetic recombination techniques are described above. These also include antibodies that have been modified in the same manner as when genetically modifying natural antibodies. Specific examples of functional fragments of antibodies include F (ab ′) 2 , Fab ′, Fab, Fv (variable fragment of antibody), sFv, dsFv (disulphide stabilized Fv) and dAb (single domain antibody). Etc.
  • the binding fragment includes an antibody fragment mutated in a range that maintains reactivity with the target lung cancer marker as a concept of the binding fragment.
  • the aforementioned mutation introduction is performed using a known technique such as a gene modification technique, which is appropriately selected by those skilled in the art.
  • the detection of the above-described antibody can be performed by forming a complex with a secondary antibody labeled with a reporter molecule capable of attracting a detectable signal.
  • the antibody described above may be pre-labeled with a reporter molecule.
  • the reporter molecule is, for example, an enzyme, a fluorophore-containing molecule, or a radionuclide-containing molecule.
  • Various reporter molecules are known and may be appropriately selected.
  • an enzyme for example, peroxidase, ⁇ -galactosidase, alkaline phosphatase, glucose oxidase, or acetylcholinesterase can be used as the enzyme.
  • a crosslinking agent such as a maleimide compound.
  • a known substance can be used according to the type of enzyme used. For example, when peroxidase is used as the enzyme, 3,3 ′, 5,5′-tetramethylbenzidine and the like can be used.
  • an alkaline phosphatase as an enzyme, paranitrol phenol etc. can be used.
  • radionuclide 125 I, 3 H, or the like can be used.
  • fluorescent dye fluoroless isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC) can be used.
  • a simple measurement system such as immunochromatography for detecting by aggregation of colloidal gold on which the secondary antibody is immobilized can also be used.
  • a peptide probe labeled with the above-mentioned reporter molecule can be used.
  • the peptide probe that recognizes CD91 is preferably a peptide probe specific for CD91.
  • the peptide probe that recognizes CD317 is preferably a peptide probe specific for CD317.
  • a luminescence measurement method including color development or fluorescence measurement, or a radiometric method can be used.
  • Mass spectrometry may be performed using a known mass spectrometer. Since mass spectrometry using a mass spectrometer is excellent in sensitivity and accuracy, accurate determination can be made. Furthermore, by using a multi-channel mass spectrometer capable of simultaneous multi-component analysis, the amount of two or more lung cancer markers (for example, CD91 and CD317) can be measured at a time. Furthermore, biomarkers relating to other diseases other than lung cancer can be simultaneously measured, and detection of various diseases can be attempted at once. In order to perform detection with higher accuracy, it is preferable to use a tandem mass spectrometer (MS / MS).
  • the mass spectrometer used in the detection method of the present invention is not particularly limited as long as it can be quantified, and conventionally known types of mass spectrometers such as a quadrupole type and a time-of-flight type can be used.
  • the ELISA method is preferable, and the sandwich ELISA is more preferable.
  • a sandwich ELISA for measuring the amount of CD91 present on the exosome surface will be described (see also FIG. 7 (a)).
  • an antibody capture antibody
  • a protein excluding CD91
  • Is bound Is bound, and then contacted with a sample containing exosomes to form a capture antibody-exosome complex. Further, it is brought into contact with a labeled anti-CD91 antibody (detection antibody) to form a capture antibody-exosome-detection antibody complex.
  • the capture antibody is preferably directed to a protein that has no significant difference in the expression level on the exosome surface between lung cancer patients and healthy individuals who do not have lung cancer.
  • proteins include CD9 (5H9 antigen, cell-growth-inhibiting gene-2 protein, leukocyte antigen-MIC3, Motility-related protein protein MRP-1, Tetraspanin-29; p24), CD63, CD81, and other molecules belonging to the tetraspanin family.
  • Anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody and the like reference document: International Publication WO2013 / 099925.
  • the amount existing on the exosome surface can be measured by the same method as described above.
  • the amount of CD91 and / or CD317 per exosome it is preferable to measure the amount of CD91 and / or CD317 per exosome.
  • the amount of exosome in the biological sample or a sample obtained by purifying the same is measured, and the result is used to normalize the separately measured amount of CD91 and / or CD317 to obtain CD91 and / or per exosome.
  • the amount of CD317 is calculated.
  • the “amount of exosome” may be an absolute amount (absolute mass or absolute concentration) or a relative amount (relative mass or relative concentration). Since the amount of exosome varies depending on the subject, lung cancer can be detected more accurately by standardization.
  • the method includes a step of measuring the amount of exosome in a sample collected from a living body.
  • the method includes a step of calculating the amount of CD91 per exosome using the measured amount of exosome and the measured amount of CD91.
  • a step of calculating the amount of CD317 per exosome using the measured amount of exosome and the measured amount of CD317 is included.
  • sandwich ELISA As a method for measuring the amount of exosome, sandwich ELISA can be mentioned. An example of a sandwich ELISA for measuring the amount of exosome will be described (see also FIG. 7 (a)).
  • a substrate to which a first antibody (capture antibody) against a protein existing on the exosome surface is prepared is prepared. And then contacted with a sample containing exosomes to form a capture antibody-exosome complex. Furthermore, it is contacted with a labeled second antibody (detection antibody) to form a capture antibody-exosome-detection antibody complex. Then, the amount of exosome is measured by measuring the color intensity.
  • the first antibody and the second antibody are preferably against a protein that has no significant difference in the expression level on the exosome surface between a lung cancer patient and a healthy person who does not have lung cancer.
  • Examples include CD9, CD63, CD81, and other molecules belonging to the tetraspanin family, and examples of the antibody include anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody.
  • the first antibody and the second antibody may be the same type of antibody, or may be different types of antibodies.
  • the first antibody and the second antibody may be of the same type or different types from those used in the sandwich ELISA for measuring the amount of CD91 and / or CD317 present on the exosome surface. May be.
  • exosomes may be purified from a sample collected from a living body. That is, one embodiment of the lung cancer detection method according to the present invention includes a step of purifying exosomes from a sample collected from a living body.
  • the method for purifying exosomes include a method using an antibody against a protein existing on the exosome surface, a method using an ultracentrifuge, and a method using a commercially available purification kit.
  • a mass spectrometry immunoassay pipette chip to which the antibody (for example, anti-CD9 antibody) is immobilized is used (see also Examples).
  • the method for detecting lung cancer according to the present invention preferably further includes a step of measuring the amount of CEA (carcinoembryonic antigen) in a sample collected from a living body.
  • CEA Carcinoembryonic antigen
  • sensitivity and specificity are improved, and lung cancer can be detected more accurately (see also Examples).
  • CEA has a known amino acid sequence, and the accession number of UniProtKB is P06731.
  • the method for detecting lung cancer according to the present invention may include a step of measuring the amount of CD91 and the amount of CD317 in a sample collected from a living body. Combining the amount of CD91 and the amount of CD317 may improve sensitivity and specificity and enable more accurate detection of lung cancer.
  • CEA includes not only the full length of the CEA protein but also a fragment thereof, and also includes a precursor of the CEA protein. That is, for example, “measuring the amount of CEA” means measuring the amount of at least one of a full-length CEA polypeptide, a fragment of CEA polypeptide, and a precursor of CEA polypeptide.
  • sensitivity refers to a ratio (true positive ratio) indicating a positive (abnormal subject value) when a test is performed on a population developing a specific disease.
  • specificity refers to a ratio (a ratio of true negative) indicating a negative (normal value) when a test is performed on a population not suffering from a specific disease.
  • “Positive predictive value” refers to the proportion of individuals who actually have a disease among subjects who show a positive result in a test. Further, the “negative predictive value” means the proportion of individuals who do not actually have a disease among subjects who show a negative test result.
  • the logistic regression score is calculated using the amount of CD91 and the amount of CEA.
  • a calculation method for example, the methods described in the literature: Pepe, M. S., Cai, T., and Longton, G. (2006), Biometrics 62, 221-229. If there is a significant difference between the two for this logistic regression score, it can be determined that the subject has lung cancer or is likely to have lung cancer.
  • the amount of CEA in the sample derived from the control subject can be determined in advance.
  • the amount of CEA and the amount of CD91 in a biological sample derived from a control subject are measured in advance, a logistic regression score is calculated, and based on this, a normal value range of the logistic regression score is determined. It can also be determined that the patient has lung cancer when the logistic regression score is out of the normal value range in the biological sample.
  • the same method as described above is used when combining the amount of CD317 and the amount of CEA, when combining the amount of CD91 and the amount of CD317, and when combining the amount of CD91, the amount of CD317 and the amount of CEA. be able to.
  • the significant difference is determined by a statistical method such as t-test, F-test, chi-square test, or Mann-Whitney's U test. I can judge.
  • the method for measuring the amount of CEA is not particularly limited as long as these abundances can be determined quantitatively or semi-quantitatively.
  • an immunological technique using an antibody recognizing CEA was used.
  • a method, a method using a peptide probe recognizing CEA, a liquid column chromatography method, a mass spectrometry method and the like can be used (the details are the same as described above).
  • the sample for measuring the amount of CEA may be the same sample as the sample for measuring the amount of CD91 and / or CD317, or may be a different sample.
  • the antibody that recognizes CEA is preferably an antibody specific for CEA.
  • the peptide probe that recognizes CEA is preferably a peptide probe specific for CEA.
  • the detection kit for lung cancer is a detection kit for detecting lung cancer, and recognizes an antibody or peptide probe that recognizes CD91 in a sample collected from a living body and CD317 in a sample collected from a living body. At least one of an antibody or a peptide probe.
  • the amount of CD91 and / or CD317 can be measured. Using the amount of CD91 and / or CD317, for example, [1. Lung cancer can be detected according to the method described in the section “Lung cancer detection method”.
  • the lung cancer detection kit according to the present invention preferably further comprises an antibody or peptide probe that recognizes CEA in a sample collected from a living body.
  • the peptide probe refers to a peptide probe that recognizes CD91, CD317 or CEA, preferably a peptide probe that specifically binds to CD91, CD317 or CEA. Specific examples include peptide sequences that specifically bind to CD91, CD317, or CEA.
  • the peptide probe included in the detection kit may include a non-natural amino acid in addition to a natural amino acid.
  • the lung cancer detection kit according to the present invention preferably further includes a substrate to which an antibody (capture antibody) for capturing an exosome in the sample is bound.
  • antibodies for capturing exosomes include antibodies to proteins (excluding CD91 and CD317) present on the exosome surface.
  • the antibody is preferably directed to a protein having no significant difference in the expression level on the exosome surface between a lung cancer patient and a healthy person who does not have lung cancer. Examples of such a protein include CD9, CD63, CD81. And other molecules belonging to the tetraspanin family, and examples of the antibody include anti-CD9 antibody, anti-CD63 antibody, and anti-CD81 antibody.
  • the capture antibody and the substrate may be separately included.
  • either one of the capture antibody and the substrate may be included.
  • the base material examples include materials such as protein highly adsorbed polypropylene.
  • the substrate is preferably a microwell plate (eg, 96 well). This is because a large number of samples can be processed in parallel.
  • the lung cancer detection kit according to the present invention further includes various reagents (secondary antibodies, reporter molecules, buffers, etc.) and instruments (plates and pipettes) for immunologically detecting antibodies or peptide probes as necessary. Etc.), at least one of a use instruction of the detection kit, a control sample used at the time of measurement, control data used when analyzing the measurement result, and the like.
  • reagents secondary antibodies, reporter molecules, buffers, etc.
  • instruments plates and pipettes
  • the detection kit for lung cancer according to the present invention is preferably, for example, a detection kit based on the ELISA method, and more preferably a detection kit based on the sandwich ELISA method.
  • the result obtained by carrying out the detection method described in the section “Lung cancer detection method” can be used as one of diagnostic materials for diagnosis by a doctor.
  • treatment can be performed.
  • examples of treatment include chemotherapy, radiation treatment, and surgery performed by a doctor, and in some cases, a specialist other than the doctor.
  • the detection method according to the present invention can detect early lung cancer, early diagnosis and early treatment can be realized.
  • the detection method according to the present invention it can be detected that the cancer is stage I or II, or stage III or IV lung cancer, so that the result obtained by the doctor performing the method according to the present invention is used. Based on this, diagnosis or treatment suitable for each stage can be adopted.
  • a doctor determines that “possibility of having lung cancer” based on the result obtained by performing the method according to the present invention
  • other examination methods X-rays
  • CT examination CT examination
  • endoscopy etc.
  • smoking history subject age and family history, etc.
  • a definitive diagnosis of the presence or absence of lung cancer is made based on the result of a biopsy, and a treatment policy is established.
  • a doctor determines that "there is a possibility of having lung cancer"
  • a predetermined reference value is set in advance, and if the measured value of the subject exceeds the reference value, It may be determined that there is a possibility that
  • the detection method according to the present invention is a method for detecting lung cancer, and includes a step of measuring the amount of at least one of CD91 and CD317 in a sample collected from a living body.
  • the detection method according to the present invention has lung cancer when the amount of at least one of the measured CD91 and CD317 is increased compared to a control subject not having lung cancer, Alternatively, it is preferable to further include a step of determining that there is a high possibility of having lung cancer.
  • the amount of CD91 is the amount of CD91 present on the exosome surface
  • the amount of CD317 is preferably the amount of CD317 present on the exosome surface.
  • the detection method according to the present invention preferably further includes a step of measuring the amount of CEA in a sample collected from a living body.
  • the lung cancer may be stage I or II.
  • the lung cancer may be stage III or IV.
  • the detection method according to the present invention it is preferable to measure the amount of at least one of CD91 and CD317 by ELISA.
  • the sample is preferably whole blood, serum or plasma.
  • the detection kit according to the present invention is a detection kit for detecting lung cancer, an antibody or peptide probe that recognizes CD91 in a sample collected from a living body, and an antibody that recognizes CD317 in a sample collected from a living body Or at least one of the peptide probes.
  • the detection kit according to the present invention preferably further includes a base material to which an antibody for capturing the exosome in the sample is bound.
  • the antibody for capturing the exosome is preferably an anti-CD9 antibody.
  • the detection kit according to the present invention preferably further comprises an antibody or peptide probe that recognizes CEA in a sample collected from a living body.
  • biomarkers were searched and verified.
  • the outline of biomarker search is as shown in FIG.
  • the eluted peptide was ionized with a spray voltage of 2000 V, and MS data was acquired by the data-dependent fragment method.
  • the measurement scan was performed at m / z 400-1600, resolution 60,000, AGC target value 1.0 ⁇ 10 6 ion count.
  • the top 20 intensities of precursor ions in each measurement scan were subjected to low resolution MS / MS acquisition using a normal CID scan mode with an AGC target value of 5000 ion counts in a linear ion trap.
  • Exosome sandwich ELISA assay 1 An exosome capture antibody solution (5 ⁇ g / mL anti-CD9 antibody in PBS, 50 ⁇ L / well) was loaded onto a Nunc MaxiSorp flat-bottom 96 well plate (Thermo Fischer Scientific) and incubated at 4 ° C. overnight. Blocking solution (5% BSA in PBS, 150 ⁇ L / well) was added and incubated on a plate shaker for 60 minutes at ambient temperature. After three washes with PBS, (5 ⁇ L serum + 95 ⁇ L PBS) was loaded into the upper 48 wells and (30 ⁇ L serum + 70 ⁇ L PBS) were loaded into the lower 48 wells, respectively.
  • Exosome sandwich ELISA assay 2 Exosome capture antibody solution (5 ⁇ g / mL anti-CD9 antibody in PBS, 50 ⁇ L / well) was loaded onto a Nunc MaxiSorp flat-bottom 96 well plate (Thermo Fischer Scientific) and incubated for 60 minutes at ambient temperature. Blocking solution (5% BSA in PBS, 150 ⁇ L / well) was added and incubated on a plate shaker for 30 minutes at ambient temperature. After washing 3 times with PBS, (10 ⁇ L serum + 90 ⁇ L PBS) was loaded into the wells. After 3 hours incubation, the plates were washed 3 times with PBS.
  • HRP-labeled anti-CD317 antibody 250 ng / mL was loaded into the wells (100 ⁇ L / well). After incubation for 60 minutes, the plate was washed 3 times with PBS, followed by loading One-step Ultra TMB-ELISA Substrate Solution (Thermo Fischer Scientific) into the well (100 ⁇ L / well). After 15 min incubation, the reaction was stopped with 2N HCl (100 ⁇ L / well). The OD at 450 nm was measured immediately.
  • the true prediction rate in the ADC patient group is 181 peptides and the true prediction rate in the SCC patient group is 90.9% 32 peptides with 100% were determined as final candidate biomarkers (FIG. 5 (c) and FIG. 5 (d), respectively).
  • CD91, ITA2B (Integrin alpha-IIb) and CD317 are expressed on the surface of exosomes and can be easily measured by exosome sandwich ELISA. Therefore, preferred exosome biomarker candidates in subsequent large-scale verification tests Met.
  • the power of detection was particularly high in ADC patients. Furthermore, it was found that the amount of blood exosome CD317 increases depending on the stage of lung adenocarcinoma.
  • the present invention can be used for detection of lung cancer. Therefore, the present invention can be widely used in the diagnostic medical field and the health medical field.

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Abstract

L'invention concerne un procédé de détection du cancer du poumon, le procédé comprenant une étape consistant à mesurer la/les quantité(s) de CD91 et/ou de CD317 dans un échantillon prélevé sur un organisme vivant. L'invention concerne également un kit de détection du cancer du poumon, ledit kit de détection contenant un anticorps ou un échantillon de peptide capable de reconnaître le CD91 dans un échantillon prélevé sur un organisme vivant et/ou un anticorps ou un échantillon de peptide capable de reconnaître le CD317 dans un échantillon prélevé sur un organisme vivant.
PCT/JP2014/082122 2013-12-04 2014-12-04 Procédé et kit de détection du cancer du poumon WO2015083791A1 (fr)

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* Cited by examiner, † Cited by third party
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CN110655580A (zh) * 2019-10-31 2020-01-07 中国科学院深圳先进技术研究院 一种杂交瘤细胞株及其用途
CN110655580B (zh) * 2019-10-31 2021-04-09 中国科学院深圳先进技术研究院 一种杂交瘤细胞株及其用途
WO2023176963A1 (fr) * 2022-03-17 2023-09-21 株式会社島津製作所 Procédé d'analyse de biomolécules présentes dans des vésicules

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