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WO2015174539A1 - Méthode pour la détection de cellules - Google Patents

Méthode pour la détection de cellules Download PDF

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Publication number
WO2015174539A1
WO2015174539A1 PCT/JP2015/064124 JP2015064124W WO2015174539A1 WO 2015174539 A1 WO2015174539 A1 WO 2015174539A1 JP 2015064124 W JP2015064124 W JP 2015064124W WO 2015174539 A1 WO2015174539 A1 WO 2015174539A1
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cancer
cells
gene
ctc
cell
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Japanese (ja)
Inventor
晋作 十合
和久 高橋
泰生 浦田
正明 大内
友樹 志田
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Oncolys Biopharma Inc
Juntendo Educational Foundation
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Oncolys Biopharma Inc
Juntendo Educational Foundation
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Priority to JP2016519419A priority Critical patent/JP6840330B2/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a method for detecting cells, particularly a method for detecting circulating tumor cells.
  • Cancer acquires resistance to systemic treatment as a result of clonal evolution and selection. It is difficult and invasive to perform repeated biopsies to examine resistant oncogene mutations as a result of treatment.
  • blood tests using tumor markers are widespread, but non-neoplastic diseases such as chronic inflammatory diseases, smoking, diabetes, etc., the number may increase or early cancer may not be detected, etc. It cannot be said that the detection sensitivity and specificity of cancer are sufficient.
  • lung cancer occupies the top cause of cancer death and is the malignant disease with the worst prognosis among solid cancer types. Therefore, a technique for early detection is strongly demanded.
  • CTC circulating tumor cells
  • EMT epithelial-mesenchymal transition
  • Non-Patent Document 3 Science. 2013 Feb 1; 339 (6119): 580-4.).
  • CellSearch system which uses anti-EpCAM antibody as a marker, is the most popular CTC measurement method already approved by FDA, but it cannot detect CTC that caused EMT or detect dead CTC as false positive cells.
  • Non-small cell lung cancer NSCLC
  • CTC detection methods other than CellSearch system there has been no report that CTC could be detected with high sensitivity from patients with non-small cell carcinoma cancer.
  • non-small cell lung cancer particularly in early non-small cell lung cancer, It was not clear whether CTC existed.
  • Patent Document 1 Patent Document 1: WO2013 / 027427. It is not known what kind of cancer cells can be detected, or what kind of cancer can be detected depending on the degree of cancer progression, and it is optimal depending on the cancer cells to be detected or the type of cancer to be diagnosed. There were no studies on the conditions.
  • An object of the present invention is to provide a method for detecting circulating tumor cells with high sensitivity and high specificity, and thus with high accuracy.
  • the present inventor combined the adenovirus described in WO2013 / 027427 with a reagent for detecting epithelial cells and a reagent for detecting leukocytes, For example, it has been found that circulating tumor cells can be detected with high sensitivity and high specificity, and thus with high accuracy, and the present invention has been completed. That is, the present invention is as follows.
  • a step of detecting the growth of the recombinant virus utilizing the growth of a recombinant virus controlled by a telomerase reverse transcriptase promoter, a step of using a reagent for detecting epithelial-derived cells, and a step of using a reagent for detecting leukocytes A method for detecting a cell, comprising: (2) The method according to (1), wherein the step of detecting the growth of the recombinant virus uses a reporter gene. (3) The method according to (1) or (2), wherein the recombinant virus is a recombinant adenovirus.
  • the recombinant adenovirus contains an E1 gene, and the expression of the gene is controlled by a telomerase reverse transcriptase promoter.
  • the E1 gene comprises an E1A gene, an IRES sequence, and an E1B gene in this order.
  • the cell is a tumor cell floating in a body fluid.
  • the tumor cells floating in the body fluid are circulating tumor cells or cancer stem cells.
  • the method according to (6) or (7), wherein the body fluid is blood, lymph fluid, tissue fluid, intercellular fluid, spinal fluid or body cavity fluid.
  • the detection result of proliferation of the recombinant adenovirus is negative, the detection result using the detection reagent for the epithelial cells is positive, the detection result using the leukocyte detection reagent is negative, and the dead cell detection (9) to (10) when the detection result using the reagent for use is negative, the tumor cells floating in the body fluid are determined not to have undergone epithelial-mesenchymal transition and to survive.
  • a method for examining or diagnosing cancer comprising predicting or determining, or predicting prognosis or recurrence of cancer.
  • the determination result is Compared to the case where tumor cells floating in the body fluid were determined not to have undergone epithelial-mesenchymal transition, (i) the therapeutic effect of the subject's cancer was low, (ii) acquired treatment resistance (Iii)
  • a method for examining or diagnosing cancer which is an index for predicting that the prognosis is poor, (iv) the possibility of recurrence is high, or (v) the histopathological malignancy is high.
  • the determination result is Compared to when it is determined that tumor cells floating in the body fluid have undergone epithelial-mesenchymal transition, (i) the therapeutic effect of the subject's cancer is high, (ii) the prognosis is good (Iii) A method for examining or diagnosing cancer, which is used as an index to predict that recurrence is low or (iv) histopathological malignancy is low. (17) The method according to any one of (14) to (16), wherein the cancer is a solid cancer.
  • Solid cancer is brain tumor, cervical cancer, esophageal cancer, tongue cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, small intestine cancer, duodenal cancer, colon cancer, bladder cancer, kidney cancer, liver cancer, prostate cancer, uterine cancer, child
  • the method according to (17), wherein the solid cancer is lung cancer.
  • the method according to any one of (14) to (19), wherein the cancer is early cancer or oligo recurrence.
  • a cell detection kit comprising a recombinant virus controlled by a telomerase reverse transcriptase promoter, an epithelial cell detection reagent, and a leukocyte detection reagent.
  • the tumor cells floating in the body fluid are circulating tumor cells or cancer stem cells.
  • the kit according to (26) or (27), wherein the body fluid is blood, lymph, tissue fluid, intercellular fluid, spinal fluid or body cavity fluid.
  • Solid cancer is brain tumor, cervical cancer, esophageal cancer, tongue cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, small intestine cancer, duodenal cancer, colon cancer, bladder cancer, kidney cancer, liver cancer, prostate cancer, uterine cancer, child
  • the kit according to (32) which is at least one selected from the group consisting of cervical cancer, ovarian cancer, thyroid cancer, gallbladder cancer, pharyngeal cancer, sarcoma, and melanoma.
  • the kit according to (32), wherein the solid cancer is lung cancer.
  • the present invention is a method for detecting tumor cells floating in a body fluid, wherein a biological sample collected from a subject is contacted with the following recombinant adenovirus (a):
  • a biological sample collected from a subject is contacted with the following recombinant adenovirus (a):
  • a biological sample collected from a subject is contacted with the following recombinant adenovirus (a):
  • a recombinant adenovirus
  • a human telomerase reverse transcriptase promoter a polynucleotide comprising an E1A gene, an IRES sequence and an E1B gene in this order, and miR-142, miR-15, miR-16, miR-21, miR-126, miR-181,
  • a polynucleotide comprising a target sequence for at least one microRNA selected from the group consisting of miR-223, miR-296, miR-125, miR-143, miR-145, miR-199 and let-7 is an adenovirus genome
  • a replication cassette incorporated into the E1 region of A labeling cassette in which a reporter gene and a promoter capable of controlling the expression of the gene are incorporated in the E3 region of the adenovirus genome;
  • Recombinant adenovirus comprising a gene encoding a fiber protein that binds to CD46
  • c Reagent for detecting epithelial-derived cells
  • the present invention also detect
  • the tumor cells floating in the body fluid include circulating tumor cells.
  • blood, lymph, tissue fluid, spinal fluid or body cavity fluid can be used as the biological sample.
  • dead cells can be detected using (d) a reagent for detecting dead cells.
  • the tumor cells floating in the body fluid are those that have undergone epithelial-mesenchymal transition or those that have not undergone epithelial-mesenchymal transition.
  • the detection result of reporter gene expression by the recombinant adenovirus of (a) is positive, the detection result using the reagent of (b) is negative, and the detection using the reagent of (c)
  • the detection result is negative, it can be determined that the tumor cells floating in the body fluid have undergone epithelial-mesenchymal transition.
  • the detection result of reporter gene expression by the recombinant adenovirus of (a) is negative
  • the detection result using the reagent of (b) is positive
  • the detection result using the reagent of (c) is negative
  • it can be determined that the tumor cells floating in the body fluid have not undergone epithelial-mesenchymal transition.
  • the detection result of reporter gene expression by the recombinant adenovirus of (a) is negative
  • the detection result using the reagent of (b) is positive
  • the detection result using the reagent of (c) is negative
  • the detection result using the reagent (d) is negative
  • it can be determined that the tumor cells floating in the body fluid have not undergone epithelial-mesenchymal transition and are alive.
  • the present invention uses the detection result detected by the above method as an index to determine the presence or absence of cancer in a subject, to predict or determine the success and / or resistance of cancer treatment, or to determine the prognosis or recurrence of cancer.
  • a method for testing or diagnosing cancer characterized by predicting.
  • the determination results indicate that the tumor cells floating in the body fluid are epithelial-mesenchymal.
  • the therapeutic effect of the subject's cancer is low, (ii) acquired resistance to treatment, (iii) poor prognosis, (iv) recurrence Or (v) a method for examining or diagnosing cancer, which is an index for predicting that the degree of histopathological malignancy is high.
  • the determination results indicate that the tumor cells floating in the body fluid are epithelial-mesenchymal.
  • the subject's cancer treatment effect is high, (ii) the prognosis is good, (iii) the possibility of recurrence is low, or (Iv) An index for predicting that histopathological malignancy is low.
  • examples of the cancer include solid cancer.
  • the solid cancer examples include brain tumor, cervical cancer, esophageal cancer, tongue cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, small intestine cancer, duodenal cancer, colon cancer, bladder cancer, It is at least one selected from the group consisting of kidney cancer, liver cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, thyroid cancer, gallbladder cancer, pharyngeal cancer, sarcoma, and melanoma.
  • the solid cancer is preferably lung cancer.
  • the cancer is early cancer or oligo recurrence.
  • the tumor cell which floats in a bodily fluid is a cancer stem cell.
  • the labeling cassette further comprises miR-142, miR-15, miR-16, miR-21, miR-126, miR-181, miR-223, miR-296, miR-125, miR-
  • it comprises a target sequence for at least one microRNA selected from the group consisting of 143, miR-145, miR-199 and let-7.
  • the reporter gene is a gene encoding a protein that emits fluorescence or a gene encoding an enzyme protein that generates a luminescent substance or a coloring substance by an enzymatic reaction.
  • the promoter capable of controlling the expression of the reporter gene is preferably a human telomerase reverse transcriptase promoter or a cytomegalovirus promoter.
  • examples of the fiber protein that binds to CD46 include those containing at least the fiber knob region of the 34 or 35 type adenovirus fiber protein.
  • examples of the reagent for detecting epithelial-derived cells include those containing an antibody against an epithelial cell adhesion molecule, and examples of the reagent for detecting leukocytes include those containing an antibody against CD45. According to the present invention, tumor cells floating in a body fluid can be detected with high sensitivity, high specificity, and hence high accuracy.
  • tumor cells floating in the body fluid having undergone epithelial-mesenchymal transition can be detected, and in early cancer or oligo recurrence that could not be detected by conventional methods, tumor cells floating in the body fluid can be detected. Can be detected.
  • tumor cells floating in a body fluid that has undergone epithelial-mesenchymal transition and tumor cells that are suspended in a body fluid that has not undergone epithelial-mesenchymal transition can be detected by phenotype, and in the detected body fluid Floating tumor cells not only as cancer diagnostic markers, but also for predicting or determining treatment success / resistance, histopathological malignancy and cancer stem cell acquisition, and prognosis and recurrence prediction markers Can be used.
  • the present invention will be described in detail.
  • FIG. 1 is a diagram showing a comparison between EMT-CTC and non-EMT-CTC among patients in which CTC is detected.
  • FIG. 2 shows the number of CTCs in patients with different histological grades.
  • FIG. 3 is a diagram showing the transition of CTC before and after administration of the anticancer agent.
  • FIG. 4 is a diagram showing the detection result of CD133 positive cells.
  • FIG. 5 is a diagram showing the detection results of EMT-CTC in cases of lung metastasis recurrence after esophageal cancer surgery, lung metastasis recurrence after gastric cancer surgery, rectal cancer, and renal cancer.
  • the present invention uses a recombinant virus growth controlled by a telomerase reverse transcriptase promoter to detect the growth of the recombinant virus, a step of using an epithelium-derived cell detection reagent, and a leukocyte detection reagent
  • a method for detecting a cell comprising a step. Further, the present invention uses the detection result detected by the above method as an index to determine the presence or absence of cancer in the subject, to predict or determine the success and / or resistance of cancer treatment, or to determine the prognosis or recurrence of cancer.
  • a method for testing or diagnosing cancer characterized by predicting.
  • the present invention is also a cell detection kit comprising a recombinant virus controlled by a telomerase reverse transcriptase promoter, a reagent for detecting epithelial cells, and a reagent for detecting leukocytes. Furthermore, the present invention is a biomarker for cancer diagnosis, for determination or prediction of cancer therapeutic effect, or for prediction of cancer prognosis or recurrence, comprising tumor cells floating in a body fluid, which is detected by the above method. is there.
  • the present invention comprises a step of contacting a biological sample collected from a subject with a recombinant virus containing a human telomerase reverse transcriptase promoter to detect the growth of the recombinant virus,
  • a method for detecting tumor cells floating in a body fluid comprising a step of detecting an epithelium-derived cell using an epithelium-derived cell detection reagent and a step of detecting a leukocyte using a leukocyte detection reagent.
  • the present invention is a method for detecting a tumor cell floating in a body fluid, particularly a circulating tumor cell (CTC), wherein the following set (a) is applied to a biological sample collected from a subject: Detecting the expression of the reporter gene by contacting with a modified adenovirus, detecting the epithelial cell using the following reagent (b), and detecting leukocytes using the following reagent (c): It is the said method including the process to perform.
  • CTC circulating tumor cell
  • A a human telomerase reverse transcriptase promoter, a polynucleotide comprising an E1A gene, an IRES sequence and an E1B gene in this order, and miR-142, miR-15, miR-16, miR-21, miR-126, miR-181,
  • a polynucleotide comprising a target sequence for at least one microRNA selected from the group consisting of miR-223, miR-296, miR-125, miR-143, miR-145, miR-199 and let-7 is an adenovirus genome
  • a replication cassette incorporated into the E1 region of A labeling cassette in which a reporter gene and a promoter capable of controlling the expression of the gene are incorporated in the E3 region of the adenovirus genome;
  • B Reagent for detecting epithelium-derived cells
  • C White blood cell detection reagent
  • EMT is a phenomenon in which cancer cells lose their epithelial characteristics and acquire the characteristics of mesenchymal cells that easily move to surrounding tissues. Cancer progression (invasion / metastasis, etc.), malignancy, resistance to treatment, Involvement in the acquisition of cancer stemness has been pointed out. It was expected to some extent that the CTC phenotype detected by a conventional CTC detection method typified by CellSearch system and a CTC detection method using a recombinant adenovirus differed due to the difference in principle.
  • the CTC phenotype detected by the CTC detection method using recombinant adenovirus was revealed for the first time. That is, in a conventional CTC detection method such as CellSearch system, at least EpCAM-positive cells are determined as CTCs, so that only CTCs that do not cause epithelial-mesenchymal transition (referred to as “non EMT-CTC”) are detected. However, in the CTC detection method using recombinant adenovirus, it was clarified in Examples described later that CTC that caused epithelial-mesenchymal transition (referred to as “EMT-CTC”) was mainly detected. .
  • EMT-CTC epithelial-mesenchymal transition
  • EMT-CTC may be useful as a key biomarker because an increase in EMT-CTC is seen when resistance to treatment is shown after starting chemotherapy for breast cancer with high histological grade. It has been reported.
  • the method of detecting CTC using only an antibody has a problem of detecting not only living cancer cells but also dead cancer cells, and the FISH method is enormous in measuring per sample. Costs, people and time.
  • the present invention can not only detect CTC with high sensitivity and specificity, and thus with high viscosity, but also detect CTC in a simple and short time. This is also advantageous.
  • CTC detected by this invention is useful for the diagnosis of cancer regardless of a disease stage.
  • Conventional CTC detection methods and tumor markers are also used for cancer diagnosis, but there is a problem that detection sensitivity of early cancer is low or cannot be detected.
  • CTC of non-small cell lung cancer is detected by CellSearch system, there is a report that CTC was identified in advanced cancer of stage III B, but a report that CTC was identified in earlier cancer Has not been.
  • the CTC detection method of the present invention has made it possible to identify CTC with high sensitivity and high specificity even from patients with very early stage IA in non-small cell lung cancer in which CTC detection is difficult. . Therefore, the CTC detected by the present invention is particularly useful for early detection of cancer.
  • non-small cell lung cancer tends to make EMT of the primary tumor (LungCancer.2013 Jul; 81 (1): 117-22.).
  • the reason why the CTC detection method of the present invention was able to detect CTC with high sensitivity and high specificity in non-small cell lung cancer, particularly in early non-small cell lung cancer, is also that of EMT-CTC. This is thought to be due to the difference in detection capability.
  • EMT is produced in various solid cancers (Cell Mol Life Sci. 2011 September; 68 (18): 3033-3046.). Therefore, according to the present invention, not only non-small cell lung cancer but also solid cancer in which at least a part of a tumor is converted to EMT, CTC can be detected with higher sensitivity and specificity than conventional CTC detection methods. Application development to docks and examinations can be expected.
  • EMT-CTC and non-EMT-CTC can be detected by phenotype, so that the detected CTC is not only a marker for cancer diagnosis but also treatment success / resistance, histopathological malignancy. It has been found that it is useful for predicting or determining the degree of acquisition of cancer degree and cancer stemness, and as a marker for predicting prognosis and recurrence. As shown in the Example mentioned later, about the case where non EMT-CTC was detected before the start of chemotherapy, all non EMT-CTC disappeared in one week after the start of chemotherapy.
  • the present invention includes a step of detecting the growth of a recombinant virus utilizing the growth of the recombinant virus controlled by a telomerase reverse transcriptase promoter, a step of using a reagent for detecting epithelium-derived cells, and leukocyte detection.
  • a method for detecting a cell comprising a step of using a reagent for use.
  • the present invention comprises a step of contacting a biological sample collected from a subject with a recombinant virus containing a human telomerase reverse transcriptase promoter to detect the growth of the recombinant virus,
  • a method for detecting tumor cells floating in a body fluid comprising a step of detecting epithelial cells using a detection reagent and a step of detecting leukocytes using a leukocyte detection reagent.
  • the present invention is a method for detecting tumor cells floating in a body fluid, particularly circulating tumor cells (CTC), wherein the following set of (a) is applied to a biological sample collected from a subject: Detecting the expression of the reporter gene by contacting with a modified adenovirus, detecting the epithelial cell using the following reagent (b), and detecting leukocytes using the following reagent (c): It is the said method including the process to perform.
  • CTC circulating tumor cells
  • A a human telomerase reverse transcriptase promoter, a polynucleotide comprising an E1A gene, an IRES sequence and an E1B gene in this order, and miR-142, miR-15, miR-16, miR-21, miR-126, miR-181,
  • a polynucleotide comprising a target sequence for at least one microRNA selected from the group consisting of miR-223, miR-296, miR-125, miR-143, miR-145, miR-199 and let-7 is an adenovirus genome
  • a replication cassette incorporated into the E1 region of A labeling cassette in which a reporter gene and a promoter capable of controlling the expression of the gene are incorporated in the E3 region of the adenovirus genome;
  • B Reagent for detecting epithelium-derived cells
  • C White blood cell detection reagent [1
  • telomerase reverse transcriptase (TERT) promoter those derived from mammals can be used.
  • a TERT promoter derived from human, mouse, rat, cow or the like can be used.
  • the mammalian TERT promoter including humans has been cloned, and its sequence information is available from databases such as NCBI and GenBank.
  • the human telomerase reverse transcriptase promoter (hTERT promoter) is preferred.
  • telomerase reverse transcriptase promoter examples include adenovirus, adeno-associated virus (AAV), herpes virus, retrovirus, lentivirus, Examples include baculovirus, and adenovirus is preferable.
  • AAV adeno-associated virus
  • baculovirus baculovirus
  • adenovirus is preferable.
  • a recombinant adenovirus a replication cassette containing the hTERT promoter, E1A gene, IRES sequence, E1B gene and microRNA target sequence is incorporated into the E1 region of the adenovirus genome, and the expression of the reporter gene and the gene is controlled.
  • a recombinant adenovirus comprising a gene encoding an adenoviral fiber protein that binds to CD46 and is incorporated into the E3 region of the adenoviral genome, and a labeling cassette comprising a promoter and a microRNA target sequence is preferred ( WO2013 / 27427).
  • the recombinant virus will be described.
  • the recombinant adenovirus will be described as a recombinant virus.
  • the replication cassette comprises a polynucleotide comprising a human telomerase reverse transcriptase (hTERT) promoter, an E1A gene, an IRES sequence and an E1B gene in that order, and a microRNA target sequence.
  • the recombinant adenovirus used in the present invention can be specifically propagated in cancer cells, and can suppress the growth of the virus in cells expressing the target miRNA.
  • the target sequence of miRNA contained in the replication cassette is the target sequence of miRNA that is specifically expressed in blood cells
  • the recombinant adenovirus used in the present invention is specific for cancer cells that express hTERT. And the growth is suppressed by blood cells.
  • the human telomerase reverse transcriptase (hTERT) promoter is a promoter of reverse transcriptase that is a component of human telomerase.
  • the downstream gene can be expressed specifically in cancer cells.
  • the virus can be specifically propagated in cancer cells expressing hTERT.
  • many transcription factor binding sequences have been confirmed in the region of 1.4 kbp upstream of its 5 ′ end, and this region is considered to be the hTERT promoter.
  • the 181 bp sequence upstream of the translation initiation site is an important core region for downstream gene expression.
  • any core containing this core region can be used without limitation.
  • an upstream sequence of about 378 bp that completely contains this core region is preferably used as the hTERT promoter.
  • This sequence of about 378 bp has been confirmed to have the same gene expression efficiency as compared to the case of the 181 bp core region alone.
  • the nucleotide sequence of the 455 bp long hTERT promoter is shown in SEQ ID NO: 1.
  • a polynucleotide that hybridizes under stringent conditions with a DNA comprising a nucleotide sequence complementary to the DNA comprising SEQ ID NO: 1 and has hTERT promoter activity are also included.
  • Such a nucleotide can be obtained by known hybridization methods such as colony hybridization, plaque hybridization, Southern blotting and the like using a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 or a fragment thereof as a probe. It can be obtained from a genomic library.
  • a method for preparing a cDNA library “Molecular Cloning, A Laboratory Manual 2nd ed.” (Cold Spring Harbor Press (1989)) can be referred to.
  • Commercially available cDNA libraries and genomic libraries may also be used. Examples of stringent conditions in the above hybridization include 1 ⁇ SSC to 2 ⁇ SSC, 0.1% to 0.5% SDS, and 42 ° C.
  • the E1A gene and the E1B gene are genes included in the adenovirus E1 gene.
  • the E1 gene is an early gene (early: E) or late gene (late: L) related to DNA replication of the virus.
  • the E1A protein encoded by the E1A gene of adenovirus activates transcription of a group of genes (E1B, E2, E4, etc.) necessary for infectable virus production.
  • the E1B protein encoded by the adenovirus E1B gene helps the late gene (L gene) mRNA to accumulate in the cytoplasm of the infected host cell and inhibits host cell protein synthesis, thereby replicating the virus. Promote.
  • the base sequences of E1A gene and E1B gene are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
  • the base sequences of the E1A gene and E1B gene are those shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of SEQ ID NO: 2 and SEQ ID NO: 3.
  • a base sequence encoding a protein that hybridizes and has E1A and E1B activities, respectively, is also included.
  • the hybridization method and stringent conditions are the same as those for the hTERT promoter.
  • the IRES (Internal Ribosome Entry Site) sequence is a protein synthesis initiation signal specific to the Picornaviridae family, and since it has a sequence complementary to the 3 ′ end of 18S ribosomal RNA, it is considered to play a role as a ribosome binding site. It has been. It is known that mRNA derived from the virus of the Picornaviridae family is translated through this sequence. The translation efficiency from the IRES sequence is high, and protein synthesis is performed independent of the cap structure even in the middle of mRNA. Therefore, in the virus used in the present invention, both the E1A gene and the E1B gene downstream of the IRES sequence are independently translated by the hTERT promoter.
  • the expression control of the hTERT promoter extends independently to the E1A gene and the E1B gene by using the IRES sequence, the growth of the virus is larger than when either the E1A gene or the E1B gene is controlled by the hTERT promoter. Can be more strictly limited to cells having telomerase activity.
  • the IRES sequence between the E1A gene and the E1B gene it is possible to increase the ability of the virus to grow in the host cell.
  • the base sequence of the IRES sequence is shown in SEQ ID NO: 4.
  • the base sequence of the IRES sequence encodes a protein that hybridizes with a DNA consisting of a base sequence complementary to the DNA consisting of SEQ ID NO: 4 under stringent conditions and has IRES activity.
  • the base sequence to be included is also included.
  • the hybridization method and stringent conditions are the same as those for the hTERT promoter.
  • miRNA is generally said to be a short single-stranded RNA of about 15 to 25 bases, and is considered to control translation of various genes by binding to a target sequence present in mRNA.
  • the expression of the target gene is suppressed in the cell.
  • the miRNA target sequence is not particularly limited as long as the target gene expression can be suppressed, but it is preferably inserted into the untranslated region of the target gene, and more preferably inserted downstream of the target gene.
  • the target sequence of miRNA used in the present invention includes a target sequence of miRNA expressed in non-cancer cells.
  • Non-cancer cells mean cells that are not malignant tumor cells, and include, for example, normal cells, benign tumor cells and the like.
  • Normal cells include, for example, normal blood cells, normal endothelial cells, normal fibroblasts, normal stem cells and the like.
  • circulating tumor cells are considered to be cells derived from malignant tumors, and are included in malignant tumor cells in the present invention.
  • the target sequence of miRNA used in the present invention includes a target sequence of miRNA that is specifically expressed in blood cells.
  • blood cells may include not only normal blood cells but also cancerous blood cells. That is, miRNA specifically expressed in blood cells may be specifically expressed in normal blood cells, or may be specifically expressed in both normal blood cells and cancerous blood cells.
  • RNA specifically expressed in blood cells miRNA that is expressed in normal blood cells but not in cancerous blood cells is more preferable.
  • blood cells are not limited, but include leukocytes (neutrophils, eosinophils, basophils, lymphocytes (T cells and B cells), monocytes, dendritic cells), CD34. Examples include positive cells, hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and peripheral blood mononuclear cells (PBMC).
  • leukocytes neutrils, eosinophils, basophils, lymphocytes (T cells and B cells), monocytes, dendritic cells
  • CD34 examples include positive cells, hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • cancerous blood cells include leukemia cells and lymphoma cells.
  • “specifically expressed” in a certain cell not only means that it is expressed only in that cell, but also means that the expression level is higher in that cell than in other cells.
  • “specifically expressed in blood cells” not only means that it is expressed only in blood cells, but also means that the expression level is higher in blood cells than in cells other than blood cells.
  • miRNAs specifically expressed in blood cells include miR-142, miR-15, miR-16, miR-21, miR-126, miR-181, miR-223, miR-296, and the like.
  • miR-142, miR-15 and miR-16 are examples of the expression of blood cells.
  • miRNA is a single-stranded RNA, but the target sequence of the single-stranded RNA on either side may be used as long as the expression of the target gene can be suppressed among the double-stranded RNAs of the premature.
  • miR-142 includes miR-142-3p and miR-142-5p, and either target sequence may be used in the present invention. That is, in the present invention, “miR-142” includes both miR-142-3p and miR-142-5p, preferably miR-142-3p.
  • miR-15 includes a sense strand (denoted as “miR-15S”) and an antisense strand (denoted as “miR-15AS”) among the double-stranded RNAs of premature. included.
  • miR-15S sense strand
  • miR-15AS antisense strand
  • the miR-142-3p gene is present in a site where translocation occurs in B cell leukemia and is expressed in hematopoietic tissues (bone marrow, spleen, thymus, etc.), but other tissues It is known that it is not expressed.
  • MiR-142-3p is also expressed in mouse fetal liver (fetal hematopoietic tissue) and is considered to be involved in the differentiation of the hematopoietic system (Chang-Zheng Chen, et al., Science, 2004).
  • specific gene expression in cancer cells is performed by the action of the hTERT promoter, and gene expression in blood cells is controlled by the action of miRNA, so that two-stage selective gene expression control is performed.
  • the target sequence of miRNA used in the present invention includes a target sequence of miRNA whose expression is suppressed in cancer cells.
  • miRNAs whose expression is suppressed in cancer cells include miR-125, miR-143, miR-145, miR-199, let-7, and the like.
  • specific gene expression in cancer cells is doubly controlled by the action of the hTERT promoter and miRNA.
  • miRNAs were originally discovered from nematodes, yeasts, etc., but are currently found in hundreds of humans and mice. These sequences are known and have access to public DBs (eg miRBBase sequence database (http://microrna.sanger.ac.uk/sequences/index.shtml, http://www.mirbase.org/)). Thus, sequence information and the like can be acquired.
  • one unit of miRNA target sequence consists of a sequence complementary to all or part of miRNA, the base length is 7 to 30 bases, preferably 19 to 25 bases, more preferably Is 21 to 23 bases in length.
  • one unit of a miRNA target sequence refers to a base sequence having a minimum length that can be targeted by a certain miRNA.
  • the entire target sequence incorporated into the recombinant adenovirus may include a plurality of one unit of the target sequence in order to effectively exert the interaction between the miRNA and the target sequence.
  • the length of the entire target sequence to be incorporated into the recombinant adenovirus is not particularly limited as long as it can be incorporated into the virus genome.
  • 1 unit of the target sequence may be contained in 1 to 10 copies, preferably 2 to 6 copies, more preferably 2 or 4 copies (John G. Doench, et al., Genes Dev. 2003 17: 438). -442).
  • An oligonucleotide having an appropriate length may be included between the sequence of one unit of the target sequence contained in the entire target sequence.
  • the length of the oligonucleotide having an appropriate length is not particularly limited as long as the entire target sequence can be integrated into the recombinant adenovirus genome, and may be, for example, an oligonucleotide having a length of 0 to 8 bases. .
  • the target sequence of each unit may be a target sequence for the same miRNA or a target sequence for different miRNAs. Furthermore, even when the target sequence for the same miRNA is included, the length of the target sequence of each unit and the base sequence of the target sequence may be different.
  • the target sequence of miRNA contained in the polynucleotide (or replication cassette containing the same) used in the present invention other sequences present in the recombinant adenovirus when the polynucleotide is incorporated into the recombinant adenovirus. In order to distinguish from the target sequence of miRNA, it can also be referred to as the “target sequence of the first microRNA”.
  • examples of the target sequence include sequences including the following sequences.
  • a miRNA target sequence is arranged downstream of the hTERT promoter-E1A gene-IRES sequence-E1B gene construct, and the hTERT promoter, E1A gene, IRES sequence, E1B gene, and miRNA target sequence are arranged in this order.
  • a nucleotide referred to as a replication cassette
  • expression of the E1 gene can be suppressed and virus growth can be suppressed in cells expressing the miRNA.
  • a miRNA target sequence downstream of the E1B gene or a reporter gene described later expression of the gene present upstream thereof is suppressed.
  • miRNA-RISC RNA-induced silencing complex
  • miRNA-RISC recruits poly A-degrading enzyme in the same manner as normal miRNA, and as a result of poly A being degraded, mRNA stability is lowered and gene expression is suppressed.
  • poly A poly A-degrading enzyme
  • the gene contained in the replication cassette can be obtained by ordinary genetic engineering techniques. For example, a nucleic acid synthesis method using a DNA synthesizer generally used as a genetic engineering technique can be used.
  • the target sequence of miRNA can be obtained by designing and synthesizing the target sequence for each unit of the target sequence so as to be complementary to all or part of the base sequence of miRNA.
  • the target sequence of miR-142-3p can be obtained by synthesizing DNA so as to be complementary to the base sequence of miR-142-3p. Thereafter, the genes obtained as described above are ligated in a predetermined order. First, each of the above genes is cleaved with a known restriction enzyme or the like, and the cleaved DNA fragment of the gene is inserted into a known vector according to a known method and ligated.
  • a known vector for example, a pIRES vector can be used.
  • the pIRES vector contains an IRES (Internal Ribosome Entry Site) sequence of encephalomyocarditis virus (ECMV) and is a vector capable of translating two open reading frames (ORFs) from one kind of mRNA.
  • IRES Internal Ribosome Entry Site
  • ORFs open reading frames
  • DNA ligase can be used for DNA ligation. If necessary, the CMV promoter contained in a known vector such as pShuttle was removed with a known restriction enzyme, and the site was excised from the hTERT promoter-E1A-IRES-E1B-miRNA target sequence using an appropriate restriction enzyme. Sequences can be inserted. By expressing the E1 gene necessary for the growth of adenovirus under the control of the hTERT promoter, the virus can be propagated specifically in cancer cells.
  • a reporter gene is incorporated into a recombinant virus, which can be used for detection of the growth of the recombinant virus described below.
  • a gene construct containing a reporter gene is referred to as a “tag cassette”.
  • the present invention relates to a recombinant adenovirus in which the replication cassette is incorporated into the E1 region of the adenovirus genome and a labeling cassette is further incorporated into the E3 region.
  • the label cassette includes a reporter gene and a promoter capable of controlling the expression of the gene, and may further include a miRNA target sequence.
  • An adenovirus death protein (ADP) of 11.6 kDa is present in the adenovirus E3 region, and ADP has a function of promoting cell damage and virus spread.
  • ADP adenovirus death protein
  • the viral genomic region encoding a protein having a function of promoting cell damage and viral spread such as the E3 region containing ADP has been removed, the timing of cell death Therefore, it is easy to identify cancer tissues by expressing GFP or the like. This is also effective in that circulating tumor cells can be detected alive for a long time.
  • the reporter gene contained in the label cassette of the recombinant adenovirus used in the present invention is not limited.
  • a gene encoding a fluorescent protein, a luminescent substance or a chromogenic substance is generated by an enzymatic reaction.
  • examples include genes encoding enzyme proteins, genes encoding antibiotics, genes encoding tag fusion proteins, genes encoding proteins that are expressed on the cell surface and bind to specific antibodies, genes encoding membrane transport proteins, etc. .
  • fluorescent protein examples include green fluorescent protein (GFP) derived from luminescent jellyfish such as Aequorea victoria, EGFP (enhanced-humanized GFP), rsGFP (renewed GFP), -Shift GFP), yellow fluorescent protein (YFP), indigo fluorescent protein (CFP), blue fluorescent protein (BFP), Renilla reGF
  • GFP green fluorescent protein
  • EGFP encoded-humanized GFP
  • rsGFP renewed GFP
  • YFP yellow fluorescent protein
  • CFP indigo fluorescent protein
  • BFP blue fluorescent protein
  • Renilla reGF Renilla reGF
  • the enzyme protein that generates a luminescent substance or a coloring substance by an enzymatic reaction include ⁇ -galactosidase, luciferase, and the like.
  • ⁇ -Galactosidase produces a blue coloring substance from 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactopyranoside (X-gal) by an enzymatic reaction.
  • Luciferase also reacts with luciferin to produce a luminescent substance.
  • luciferase firefly luciferase, bacterial luciferase, Renilla luciferase and the like are known, and those skilled in the art can appropriately select from known luciferases.
  • the expression of the reporter gene can be detected as fluorescence or color caused by the protein expressed from the reporter gene.
  • a protein expressed from the reporter gene is detected by Western blot, ELISA, mass spectrometry, or the mRNA expressed from the reporter gene is detected in situ hybridization. It can be detected by reverse transcription polymerase chain reaction, nucleic acid hybridization, electrophoresis, Northern blotting, mass spectrometry and the like.
  • the promoter capable of controlling the expression of the gene may be any promoter as long as it is suitable for the virus used for the expression of the target gene.
  • CMV promoter examples thereof include, but are not limited to, CMV promoter, hTERT promoter, SV40 late promoter, MMTV LTR promoter, RSV LTR promoter, SR ⁇ promoter, ⁇ actin promoter, PGK promoter, EF-1a promoter and the like.
  • CMV promoter or hTERT promoter can be used.
  • the target sequence of the miRNA incorporated into the label cassette may be the same as or different from the target sequence of the miRNA incorporated into the replication cassette.
  • expression of the reporter gene can be suppressed by placing the target sequence of the miRNA in the untranslated region of the reporter gene, preferably downstream of the gene.
  • the label cassette preferably includes a promoter capable of controlling a reporter gene, a reporter gene, and a target sequence of microRNA in this order.
  • the target sequence of miRNA contained in the replication cassette is referred to as “target sequence of first microRNA”.
  • the target sequence of the miRNA incorporated into the label cassette is referred to as the“ target sequence of the second microRNA ”.
  • the method for obtaining the recombinant gene contained in the labeling cassette, the purification method, the sequencing method and the like are the same as in the case of the replication cassette.
  • the recombinant adenovirus used in the present invention can include a gene encoding an adenovirus fiber protein that binds to CD46.
  • a commonly used adenovirus vector is prepared using a type 5 (or type 2) adenovirus belonging to Subgroup C among 57 serotype human adenoviruses as a basic skeleton.
  • Adenovirus type 5 is widely used due to its excellent gene transfer characteristics, but it can be infected by binding to the target cell Coxsackievirus and adenovirus receptor (CAR), so that it can infect cells with low CAR expression. There is a problem that it is difficult.
  • adenovirus having fiber protein of type 5 adenovirus is highly malignant cancer cell. May not be able to be infected.
  • CD46 is expressed in almost all cells except erythrocytes in humans, and is also expressed in cancer cells with high malignancy. Therefore, a recombinant adenovirus containing a gene encoding an adenovirus fiber protein that binds to CD46 can also infect cancer cells that are CAR-negative and highly malignant.
  • type 34 and type 35 adenoviruses bind to CD46 as a receptor and infect cells (Marko Martilla, et al., J. Virol. 2005, 79 (22): 14429-36).
  • CD46 is expressed in almost all cells except erythrocytes in humans
  • 34 and 35 adenoviruses can infect a wide range of cells including CAR negative cells. is there.
  • the adenovirus fiber consists of a knob region, a shaft region, and a tail region, and adenovirus infects cells by binding to the receptor, so that at least the fiber knob region of the fiber protein is type 5.
  • adenovirus-derived one By replacing an adenovirus-derived one with a type 34 or 35-type adenovirus, the virus can infect CAR negative cells via CD46.
  • adenovirus belonging to group B has been reported to bind to CD46.
  • Examples of adenoviruses belonging to Group B include adenoviruses of type 3, type 7, type 11, type 16, type 21, type 21 and type 50, in addition to type 34 and type 35 adenoviruses.
  • the adenovirus fiber protein that binds to CD46 is preferably an adenovirus fiber protein belonging to Group B, which is type 3, type 7, type 34, type 35, type 11, type 16, type 21, and type 50.
  • Type adenovirus fiber protein is more preferable, and type 34 and type 35 adenovirus fiber proteins are more preferable.
  • the nucleotide sequence of the gene encoding the fiber protein of type 34, type 35, type 3, type 7, type 11, type 16, type 21 or type 50 adenovirus, respectively, is GenBank of NCBI (The National Center for Biotechnology Information). And can be obtained from known gene information databases.
  • the base sequence of the gene encoding the fiber protein of type 34, type 35, type 3, type 7, type 11, type 16, type 21, type 21 or type 50 adenovirus is as follows.
  • a base sequence encoding a protein that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence and has a binding activity to CD46 is also included.
  • the binding activity to CD46 can be evaluated by measuring the infectivity of a recombinant adenovirus having a DNA containing the nucleotide sequence to a CD46-expressing cell.
  • the infectivity of the recombinant adenovirus can be measured using a known method, for example, detecting GFP expressed by a virus infected with a CD46-expressing cell with a fluorescence microscope or flow cytometry.
  • the hybridization method and stringent conditions are the same as described above.
  • the recombinant adenovirus used in the present invention may contain all or a part of the fiber protein of adenovirus that binds to CD46, and at least the fiber knob region of the fiber protein binds to CD46. Anything is acceptable.
  • the adenovirus fiber protein that binds to CD46 only needs to contain at least the fiber knob region of the adenovirus fiber protein belonging to Group B, and is a 34 type, 35 type, 3 type, 7 type. More preferably, it comprises at least a fiber knob region of an adenovirus fiber protein of a type selected from the group consisting of type 11, type 16, type 21 and type 50, and at least a type 34 or type 35 adenovirus fiber protein. More preferably, it includes a fiber knob region.
  • the technical idea of the present invention is not limited to fiber protein as long as it binds to CD46, but extends to various proteins that can bind to CD46 and proteins having a motif that can bind to CD46.
  • the fiber protein that binds to CD46 may include a region consisting of a fiber knob region and a fiber shaft region of an adenovirus fiber protein belonging to Group B. More preferably, it comprises a region comprising a fiber knob region and a fiber shaft region of an adenovirus fiber protein of a type selected from the group consisting of type 7, type 11, type 16, type 21, type 21 and type 50, type 34 Alternatively, it is more preferable to include a region consisting of a fiber knob region and a fiber shaft region of a fiber protein of type 35 adenovirus.
  • the fiber protein that binds to CD46 is of a type other than the above type (for example, type 2 and type 5) as long as it contains at least the fiber knob region of the fiber protein of the adenovirus fiber protein belonging to group B. It may include a fiber shaft region or fiber tail region of an adenovirus fiber protein.
  • fiber proteins include fiber knobs of adenovirus fiber proteins of the type selected from the group consisting of 34 type, 35 type, 3 type, 7 type, 11 type, 16 type, 21 type and 50 type. Examples include, but are not limited to, a fiber protein comprising a region and a fiber shaft region and a region consisting of a fiber tail region of a type 5 adenovirus fiber protein.
  • the base sequences of the gene encoding the fiber knob region of the type 34 adenovirus fiber protein, the gene encoding the fiber shaft region, and the gene encoding the region consisting of the fiber knob region and the fiber shaft region are shown in SEQ ID NOs: 29 and 30, respectively. And 31.
  • the base sequence of the gene encoding the region consisting of the fiber knob region and fiber shaft region of type 34 adenovirus fiber protein and the fiber tail region of type 5 adenovirus fiber protein is represented by SEQ ID NO: 32.
  • the base sequence of the gene hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence in addition to the base sequence represented by SEQ ID NO: 29-32.
  • the recombinant adenovirus used in the present invention can be prepared by excising a polynucleotide containing a replication cassette and a labeling cassette using an appropriate restriction enzyme and inserting it into an appropriate virus expression vector.
  • the viral expression vector is preferably an adenoviral vector, more preferably a type 5 adenoviral vector, which contains a gene encoding an adenoviral fiber protein that binds to CD46 (for example, a 34 or 35 type adenoviral fiber protein).
  • Type adenovirus vectors are particularly preferred.
  • the recombinant adenovirus can be obtained, for example, by the following method.
  • pHMCMV5 (Mizuguchi H. et al., Human Gene Therapy, 10; 2013-2017, 1999) is subjected to restriction enzyme treatment to insert the miRNA target sequence, and a vector having the miRNA target sequence is prepared.
  • pSh-hAIB (WO 2006/036004) containing the hTERT promoter-E1A-IRES-E1B construct was subjected to restriction enzyme treatment, and the resulting fragment containing the hTERT promoter-E1A-IRES-E1B construct was treated with the miRNA.
  • pHMCMVGFP-1 pHMCMV5 into which the EGFP gene has been inserted
  • pHMCMVGFP-1 pHMCMV5 into which the EGFP gene has been inserted
  • a restriction enzyme to obtain a fragment containing the CMV promoter and the EGFP gene.
  • This fragment is inserted into a vector having the miRNA target sequence, and CMV- A vector containing a construct of the EGFP-miRNA target sequence is obtained.
  • a hTERT promoter-E1A-IRES-E1B-miRNA target sequence-containing vector and a CMV-EGFP-miRNA target sequence-containing vector are each subjected to restriction enzyme treatment and ligated, whereby the hTERT promoter is added to the E1 deficient region of the adenovirus genome.
  • -A vector in which an E1A-IRES-E1B-miRNA target sequence is incorporated and a CMV-EGFP-miRNA target sequence is incorporated in the E3-deficient region can be obtained.
  • the hTERT promoter-E1A-IRES- is inserted into the E1 deficient region of the adenovirus genome.
  • a vector containing a gene encoding an adenovirus fiber protein that incorporates a CMV-EGFP-miRNA target sequence in the E3 deficient region and binds to CD46, as well as the E1B-miRNA target sequence is incorporated.
  • This vector is linearized with a known restriction enzyme and then transfected into cultured cells such as 293 cells, whereby infectious recombinant adenovirus can be produced.
  • a person skilled in the art can easily produce all viruses used in the present invention by slightly modifying the above production method.
  • Reagent for detection of epithelial cells The reagent for detecting epithelial cells is not particularly limited as long as epithelial cells can be detected, and can be appropriately selected according to the purpose.
  • Epithelium-derived cells include the “epidermis” that covers the body surface, the “epithelium (narrow definition)” that constitutes the mucous membrane of luminal organs, “acinar cells” that constitute exocrine glands, and “gland cells” that constitute endocrine glands. means.
  • cell surface markers known to be specifically expressed in epithelial cells can be used, such as EpCAM, cytokeratin, E-cadherin (E- cadherin), Occludin, ZO-1, and other epithelial cell adhesion molecules.
  • the reagents used in the present invention include antibodies against the above cell surface markers, such as anti-EpCAM antibodies, anti-cytokeratin antibodies, anti-E-cadherin antibodies, anti-Occludin antibodies, anti-ZO-1 antibodies and other epithelial cell adhesion molecules.
  • An antibody is mentioned. These antibodies are commercially available and can be easily obtained.
  • immunohistochemical staining is common. For example, an antibody is added to a test sample to perform an antigen-antibody reaction, and a secondary antibody for labeling is added to check for positive or negative.
  • epithelium-derived cells can be detected by methods other than staining.
  • beads or magnetic particles to which the antibody is bound in advance may be used.
  • cell surface markers can be detected by Western blot, ELISA, mass spectrometry, etc.
  • cell surface marker mRNA can be detected by in situ hybridization, reverse transcription polymerase chain reaction, nucleic acid hybridization, electrophoresis, Northern blotting, It can be detected by mass spectrometry or the like.
  • the presence of epithelium-derived cells in the test sample means that the cancer of the epithelium-derived cells may have been released from the primary or metastatic focus in test samples such as blood and lymph that originally do not contain epithelial-derived cells.
  • the reagent for detecting leukocytes is not particularly limited as long as leukocytes can be detected, and can be appropriately selected according to the purpose.
  • a cell surface marker known to be specifically expressed in leukocytes can be used, and examples thereof include CD45.
  • examples of the reagent used in the present invention include an antibody against the cell surface marker, such as an anti-CD45 antibody. These antibodies are commercially available and can be easily obtained.
  • an immunohistochemical staining method is common. For example, an antibody is added to a test sample to perform an antigen-antibody reaction, and a secondary antibody for labeling is added to check for positive or negative.
  • the reagent for detecting leukocytes include Giemsa solution (a mixture of eosin and Azure B). Morphological identification of white blood cells that can be falsely positive by Giemsa's solution is also performed. Hereinafter, typical stained images of white blood cells will be described.
  • the detection of white blood cells can also be performed by methods other than staining, and for example, beads or magnetic particles to which the antibody is bound in advance may be used.
  • cell surface markers can be detected by Western blot, ELISA, mass spectrometry, etc.
  • cell surface marker mRNA can be detected by in situ hybridization, reverse transcription polymerase chain reaction, nucleic acid hybridization, electrophoresis, Northern blotting, It can be detected by mass spectrometry or the like.
  • Detecting white blood cells in a test sample means determining whether a CTC candidate detected by TelomeScan F35 or a reagent for detecting epithelium-derived cells is a false positive due to white blood cells.
  • the cell to be detected is not particularly limited as long as it is a eukaryotic cell, but is preferably an animal cell, more preferably a human cell. Although it does not specifically limit as an animal cell, A tumor cell etc. are mentioned, Especially, it is preferable that it is a tumor cell which floats in a bodily fluid.
  • the tumor cells floating in the body fluid are not particularly limited as long as they are tumor cells released from the primary lesion or metastasis.
  • CTC and tumor cells floating in the body cavity fluid are considered CTC and tumor cells floating in the body cavity fluid.
  • floating means a state of moving away from a tissue and entering a body fluid, and includes at least the meanings of release and circulation.
  • tumor cells that float in the body fluid do not always need to be separated from the tissue. If the tumor cells remain temporarily on the tissue or if the body fluid is collected by a general method of blood collection or puncture, It also includes the case of floating in a body fluid with agitation and suction.
  • the body fluid is a general term for fluids existing outside the cells in the animal body, and examples thereof include blood, lymph fluid, tissue fluid, intercellular fluid, spinal fluid, and body cavity fluid.
  • the body cavity fluid means fluid stored in the four body spaces of the left and right thoracic cavities, the abdominal cavity and the pericardial cavity, and examples thereof include pleural effusion, ascites and pericardial effusion.
  • a body cavity washing liquid thoracic cavity washing liquid, abdominal cavity washing liquid, pericardial cavity washing liquid obtained by washing the body cavity is also included in the body cavity fluid.
  • the type of cancer or tumor to be detected or diagnosed as a tumor cell is not limited, and any type of cancer cell can be used.
  • solid cancer or blood tumor can be mentioned, but solid cancer is preferable.
  • solid cancer examples include brain tumor, cervical cancer, esophageal cancer, tongue cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, small intestine cancer, duodenal cancer, colon cancer, bladder cancer, kidney cancer, liver cancer, prostate cancer,
  • Examples include uterine cancer, cervical cancer, ovarian cancer, thyroid cancer, gallbladder cancer, pharyngeal cancer, sarcoma, and melanoma, with lung cancer being preferred.
  • lung cancer include non-small cell lung cancer, small cell cancer, carcinoid, columnar tumor, and mucoepidermoid cancer, and non-small cell lung cancer is preferred.
  • Non-small cell lung cancer includes lung squamous cell carcinoma, lung adenocarcinoma, lung large cell carcinoma, etc., preferably lung squamous cell carcinoma and lung adenocarcinoma, more preferably lung adenocarcinoma.
  • Examples of hematological tumors include leukemia, lymphoma and multiple myeloma (MM).
  • the miRNA target sequence contained in the adenovirus used in the present invention is preferably a target sequence of miRNA expressed specifically in normal blood cells.
  • Most cancer cells derived from human tissues (85% or more) show an increase in telomerase activity, and the present invention can generally detect cancer cells that express such telomerase.
  • cancer stem cells can be detected.
  • cancer stem cells refer to cells (stem cells) that are the origin of cancer cells. Cancer stem cells include cells with drug resistance and dormant cells.
  • the biological sample is not limited as long as it contains a body fluid, and examples thereof include blood, lymph fluid, tissue fluid, intercellular fluid, spinal fluid, body cavity fluid, etc., which reduce the burden on the subject. Blood is preferred in that it can be done.
  • CTCs are thought to circulate in blood or lymph. However, infiltration, migration, and leakage into tissue fluid, cerebrospinal fluid, or body cavity fluid may occur due to circulatory processes or cell / tissue damage. .
  • blood and lymph not only blood and lymph but also tissue fluid, intercellular fluid, spinal fluid, and body cavity fluid can be used as a biological sample in detecting CTC.
  • the amount of the biological sample to be collected is not particularly limited.
  • 1 to 50 mL is preferable, 5 to 10 mL is more preferable, and 7 to 8 mL is particularly preferable.
  • the number of tumor cells floating in the body cavity fluid per volume is larger than the number of CTCs contained in the blood.
  • Cells are detected as follows. Recombinant virus growth controlled by the TERT promoter is used to detect the growth of the recombinant virus. “Detecting the growth of a recombinant virus” refers to detecting that a recombinant virus is infected and propagated in cells in a biological sample by bringing the recombinant virus into contact with the biological sample.
  • Examples include whole recombinant virus, virus-derived components (proteins, nucleic acids, lipids, sugars, etc.), and foreign gene-derived components (proteins, nucleic acids, lipids, sugars, etc.) incorporated into recombinant viruses. It is preferable that it is a component derived from.
  • the foreign gene is not particularly limited, but is preferably a reporter gene.
  • the recombinant adenovirus used in the present invention incorporates a label cassette containing a reporter gene. By bringing the recombinant adenovirus into contact with the biological sample, the reporter gene is expressed in the cell when the recombinant adenovirus infects and proliferates in the cell in the biological sample.
  • a step of contacting a biological sample collected from a subject with a recombinant virus containing a human telomerase reverse transcriptase promoter to detect the growth of the recombinant virus, a reagent for detecting epithelial cells A step of detecting epithelium-derived cells using the method, and a step of detecting leukocytes using a reagent for detecting leukocytes.
  • a biological sample collected from a subject is contacted with the recombinant adenovirus of (a) to detect the expression of the reporter gene, and the epithelium using the reagent of (b) A step of detecting a derived cell, and a step of detecting leukocytes using the reagent of (c).
  • Contacting means that a biological sample can be infected with the recombinant virus, and includes a step of adding or inoculating the biological sample with the recombinant virus.
  • the order of the step of detecting the growth of the recombinant virus, the step of using the reagent for detecting epithelial-derived cells, and the step of using the reagent for detecting white blood cells is arbitrary for the collected biological sample. Or may be done in a different order.
  • the step of detecting an epithelium-derived cell using a reagent for detecting an epithelium-derived cell it is judged positive if an epithelium-derived cell is detected, and negative if it is not detected.
  • the step of detecting leukocytes using a leukocyte detection reagent it is judged positive when leukocytes are detected, and negative when no leukocytes are detected.
  • a reagent for detecting dead cells is used in addition to the detection step using the virus or reagent described above.
  • a dead cell staining step of detecting dead cells may be added.
  • the order of the steps for detecting dead cells is arbitrary, and may be performed after the step for detecting the growth of recombinant virus, the step for using a reagent for detecting epithelial cells, and the step for using a reagent for detecting leukocytes. , You may go in the middle or first.
  • the determination of (ii) includes a step of detecting using a dead cell detection reagent, it can be summarized as shown in (iii) below.
  • (Iii) When the detection result of the recombinant adenovirus growth is negative, the detection result of the epithelium-derived cell is positive, the detection result of the leukocyte is negative, and the detection result of the dead cell is negative, the CTC is non EMT-CTC It is determined that it is alive.
  • the reagent for detecting dead cells is not particularly limited as long as dead cells can be detected, and can be appropriately selected according to the purpose.
  • Examples thereof include reagents for staining dead cells.
  • reagents for staining dead cells For detection of dead cells, techniques such as observation by trypan blue staining or fluorescence observation by propidium iodide staining or 7-aminoactinomycin D staining are generally employed.
  • LIVE / DEAD Fixable Blue Dead Cell Stain Kit can be suitably used.
  • These reagents are commercially available and can be easily obtained.
  • a living cell detection reagent may be used instead of a dead cell detection reagent, and it may be determined that the cell is alive when the detection result using the living cell detection reagent is positive.
  • the relationship between EMT-CTC or non EMT-CTC and cancer can be evaluated as follows.
  • the early cancer in the present invention means a cancer at a stage up to stage II, preferably stage I, and more preferably stage IA.
  • TNM classification category
  • category TNM Classification of Alignment Tumors, 7th edition Union for International Cancer Control publication
  • TNM classification TNM Classification of Alignment Tumors, 7th edition Union for International Cancer Control publication
  • TNM classification TNM Classification of Alignment Tumors, 7th edition Union for International Cancer Control publication
  • TNM classification TNM Classification of Alignment Tumors, 7th edition Union for International Cancer Control publication
  • TNM classification T represents the degree of progression of the primary tumor by T0 to T4
  • N represents the presence or absence and range of lymph node metastasis by N0 to N4
  • M represents the presence or absence of metastasis by M0 or M1.
  • stage II in lung cancer corresponds to T1 to T2b, N1, M0 or T2b to T3, M0, N0 in TNM classification
  • stage I corresponds to T2a, N0, M0 in TMN classification.
  • the early cancer in the present invention means a cancer at an earlier stage than T2b, N1, M0 or T3, N0, M0, regardless of the type of cancer, and is preferably earlier than T2a, N0, M0, More preferably, T1, N0, M0.
  • “Relapse” refers to a new lesion in which cancer cells originating from the primary lesion grow again in the same place or other organs / tissues after the cancer treatment has made it impossible to confirm the tendency to shrink or the presence of cancer.
  • cancer cells derived from the primary lesion are orthotopic.
  • Judgment is "relapsed” when a new appearance appears in the target / ectopic position.
  • Oligo recurrence means that one or a few distant metastases or recurrences occur in one or more organs in a situation where the primary lesion is controlled after resection (Jpn. Clin. Oncol. 2010; 40 (2) -107-111).
  • Oligo recurrence is a concept proposed by Niibe et al. In 2006, revising “oligometastasis”, a concept proposed by Hellman and Nemoral in 1995. Cancer recurrence is known to have a poor prognosis, but it has been pointed out that local treatment such as surgery and radiation therapy may be effective for oligo recurrence.
  • the number of distant metastasis or recurrence organs is preferably one.
  • the number of distant metastasis or recurrence is preferably 5 (location) or less, more preferably 2 or less, and particularly preferably 1 (single shot).
  • Prognosis means the overall survival of a cancer patient.
  • “Response success and / or recurrence” refers to RESIST (Response Evaluation Criteria in Solid Tumors), 1.1th Edition (New response evaluation criteria in solidJUMO LEUNEULE). ) Judgment is made according to the criteria of 228-247).
  • the CTC detected as described above can be used as a biomarker for cancer diagnosis.
  • the detected CTC can be used as a biomarker for determining or predicting the therapeutic effect of cancer, or for predicting the prognosis or recurrence of cancer.
  • CTC is detected for each phenotype in a predetermined number of subjects (primary population), and each phenotype (EMT-CTC or non) is obtained from the obtained detection result.
  • EMT-CTC is associated with cancer. Therefore, when detecting cancer of a subject individual, the data from the plurality of subjects is used as a population, and it is examined where the data of the subject subject of the individual is located in the population data. Thus, it is possible to know the possibility of cancer in the individual subject. In addition, when an individual test is performed independently, cancer can be tested or diagnosed by applying the test result using the population data as reference data.
  • CTCs detected by the present invention are those in which the presence of cancer cells themselves are detected, and are more reliable than tumor markers used for docking and screening. Therefore, it is considered that the present invention can be used for simple diagnosis of cancer in docking / examination in the future. 3.
  • the kit of the present invention contains a recombinant adenovirus, a reagent for detecting an epithelial cell, and a reagent for detecting a leukocyte, and can further contain a reagent for detecting a dead cell.
  • the recombinant adenovirus is treated as it is, for example, by freezing or the like, or is used as it is or a known pharmaceutically acceptable carrier such as an excipient, a bulking agent, a binder, or a lubricant, a known addition It can be produced by mixing with an agent (including a buffering agent, an isotonic agent, a chelating agent, a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetening agent, etc.).
  • the reagent for detecting an epithelial-derived cell may be any solid or liquid form as long as it can detect the epithelial-derived cell.
  • the leukocyte detection reagent only needs to be able to detect leukocytes, and can be in various solid and liquid forms.
  • the dead cell detection reagent only needs to be able to detect dead cells, and can be in various solid and liquid forms.
  • the kit of the present invention may contain a buffer solution, an enzyme solution, a secondary antibody, a dilution solution, instructions for use, and the like.
  • the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
  • Example 1 CTC detection method using a recombinant adenovirus (also referred to as “TeloScan F35” or simply “F35” in this example) Preparation of TelomeScan F35 (1) Preparation of pHMCMV5-miR-142-3pT A fragment obtained by treating pHMCMV5 (Mizukuchi H. et al., Human Gene Therapy, 10; 2013-2017, 1999) with NotI / KPnI was synthesized as follows. PHMCMV5-miR-142-3pT (pre) was prepared by ligation with a double-strand oligo prepared by annealing DNA.
  • miR-142-3pT-S1 miR-142-3pT-AS1: Next, the miR-142-3p target sequence was ligated with a double-strand oligo prepared by annealing the following synthetic oligo DNA with the pHMCMV5-miR-142-3pT (pre) -treated fragment. PHMCMV5-miR-142-3pT having a 4 times repeat sequence was obtained.
  • miR-142-3pT-S2 miR-142-3pT-AS2: (2) Preparation of pHM5-hAIB-miR-142-3pT which is E1 shuttle plasmid pSh-hAIB (WO 2006/036004) was digested with I-CeuI / PmeI, and the digested product was electrophoresed using an agarose gel. A band of about 4.5 kbp (hAIB cassette) was cut from the gel, and the DNA fragment was purified and recovered using GENECLEAN II (Q-Biogene).
  • the purified DNA fragment (hAIB cassette) and pHMCMV5-miR-142-3pT were digested with NheI, then treated with Klenow Fragment, and further the fragment digested with I-CeuI was ligated, whereby the hTERT promoter, E1A gene, IRES ( pHM5-hAIB-miR-142-3pT having an internal ribosomal entry site) sequence, E1B gene and miR-142-3pT target sequence was obtained.
  • pHM13CMV-EGFP-miR-142-3pT which is an E3 shuttle plasmid pEGFP-N1 (Clontech) was digested with ApaI and NotI, and the resulting digested product was inserted into the ApaI / NotI site of pHMCMV5.
  • pHMCMVGFP-1 was obtained.
  • pHMCMVGFP-1 was digested with PmeI / HindIII, and the digested product was electrophoresed using an agarose gel. A band of about 750 bp (EGFP) was excised from the gel, and the DNA fragment was purified and recovered using GENECLEAN II.
  • PBSKS-EGFP was prepared by ligating the purified DNA fragment (EGFP) and the fragment obtained by digesting pBluescript II KS + with Hinc II / Hind III.
  • pBSKS-EGFP was digested with ApaI / XbaI, and the digested product was electrophoresed using an agarose gel. A band of about 750 bp (EGFP) was excised from the gel, and the DNA fragment was purified and recovered using GENECLEAN II.
  • PHMCMV5-EGFP-miR-142-3pT was obtained by ligating the purified DNA fragment (EGFP) and the fragment obtained by digesting pHMCMV5-miR-142-3pT with ApaI / XbaI.
  • pHMCMV5-EGFP-miR-142-3pT was digested with BglII, and the digested product was electrophoresed using an agarose gel.
  • a band of about 2 kbp (CMV-EGFP-miR-142-3pT) was excised from the gel, and the DNA fragment was purified and recovered using GENECLEAN II.
  • the purified DNA fragment (CMV-EGFP-miR-142-3pT) and pHM13 (Mizuguchi et al., Biotechniques, 30; 1112-1116, 2001) were digested with BamHI and then treated with CIP (Alkaline Phosphatase, Calf Intest).
  • pAdHM49 is a recombinant adenovirus in which the region containing the gene encoding the fiber knob and fiber shaft of the type 5 adenovirus fiber is replaced with the region containing the gene encoding the fiber knob and fiber shaft of the type 34 adenovirus fiber. Yes, it contains the base sequence (SEQ ID NO: 31) of the gene encoding the region consisting of the fiber knob region and fiber shaft region of the type 34 adenovirus fiber protein.
  • SEQ ID NO: 32 The base sequence of the gene encoding the fiber protein of pAdHM49 (fiber knob region and fiber shaft region of type 34 adenovirus fiber and fiber tail region of type 5 adenovirus fiber) is shown by SEQ ID NO: 32.
  • the base sequence of the gene encoding the fiber tail region of type 5 adenovirus fiber is the 1st to 132nd base sequence
  • the gene encoding the fiber shaft region of type 34 adenovirus fiber The base sequence of the gene is the 133rd to 402th base sequence
  • the base sequence of the gene encoding the fiber knob region of type 34 adenovirus fiber is the 403th to 975th base sequence.
  • the base sequence of the region derived from type 5 adenovirus fiber is the 1st to 132nd base sequence
  • the base sequence of the region derived from type 34 adenovirus fiber is the 133th to 975th base sequence. Is the base sequence.
  • a fragment obtained by digesting pAdHM49-hAIB142-3pT with Csp45I and a fragment obtained by digesting pHM13CMV-EGFP-miR-142-3pT with ClaI were ligated.
  • the hTERT promoter, E1A gene, IRES sequence, E1B gene and miR-142-3pT target sequence are incorporated into the E1 deletion region of the adenovirus vector, and the CMV promoter, EGFP and miR-142-3pT target sequence are incorporated into the E3 deletion region.
  • pAdHM49-hAIB142-3pT-CG142-3pT containing a gene encoding the fiber protein of type 34 adenovirus was obtained.
  • the subject's blood (7.5 mL) was collected in a vacuum blood collection tube containing a CPD (Citrate Phosphate Dextrose) solution and maintained at 15-25 ° C.
  • 120 mL of red blood cell lysis buffer ((ammonium chloride 8.9 g / L, potassium hydrogen carbonate 1.0 g / L, EDTA 0.042 g / L)) was applied to 7.5 mL of blood.
  • red blood cells were hemolyzed, and peripheral blood mononuclear cells (a white blood cell fraction containing PBMCs and CTC) were collected by centrifugation.
  • the recovered PBMCs were washed twice using a medium containing 10% serum.
  • the washed PBMCs were suspended in 1 mL of 10% serum-containing medium, 1 ⁇ 10 9 VP of TelomeScan F35 was added, and the cells were cultured at 37 ° C. for 24 hours, and then stained according to (i) or (ii) below.
  • (I) CD45 / Vimentin immunostaining Infected PBMCs were collected by centrifugation, blocked with PBS containing 10% serum for 10 minutes, and subjected to primary antibody reaction for 30 minutes with anti-CD45 antibody (BioLegend, 304002). It was. The primary antibody was removed by washing, and a fluorescently labeled secondary antibody (Invitrogen, A21235) was reacted for 30 minutes.
  • reaction was performed for 30 minutes using a dead cell staining reagent (Invitrogen, L-23105). After the reaction, blocking was performed for 10 minutes with PBS containing 10% serum, and a primary antibody reaction was performed for 30 minutes with an anti-CD45 antibody (BioLegend, 304002). The primary antibody was removed by washing, and a fluorescently labeled secondary antibody (Invitrogen, A21235) was reacted for 30 minutes. The secondary antibody was removed by washing, and a fluorescently labeled EpCAM antibody (Abcam, ab7504) was reacted for 30 minutes using a direct labeling reagent (Invitrogen, Z25005). After the antibody reaction, fixation was performed with 4% paraformaldehyde for 10 minutes. 3.
  • a dead cell staining reagent Invitrogen, L-23105
  • a primary antibody reaction was performed for 30 minutes with an anti-CD45 antibody (BioLegend, 304002).
  • the primary antibody was removed by washing, and a fluorescently labeled secondary antibody
  • EMT-CTC means those determined by TelomeScan F35 positive (GFP positive) / CD45 negative / EpCAM negative
  • non EMT-CTC means TelomeScan F35 negative (GFP negative) / CD45. It means that determined by negative / EpCAM positive / dead cell staining negative
  • the total CTC means the ratio of cases where one or more non-EMT-CTC or EMT-CTC determined by the combination is detected.
  • CTC could be identified with a positive rate of 39.4%.
  • the positive rate of EMT-CTC was 36.4%
  • the positive rate of non EMT-CTC was 12.5%
  • the positive rate of total CTC was 39.4%.
  • the total CTC including CTCs that are dead cells includes not only negative (live cells) but also positive (dead cells) in the non-EMT-CTC.
  • the positive rate of (live cells + dead cells) was 48.5% (16/33).
  • EMT-CTC was all cases of TelomeScan F35 positive (GFP positive) and at the same time all dead cell staining negative (100%), that is, live cells. It was confirmed. According to the present invention, it has been shown that both EMT-CTC and non-EMT-CTC can be detected from clinical specimens of cancer patients.
  • the positive rate of CTC in non-small cell lung cancer was higher in adenocarcinoma than in squamous cell carcinoma. As shown in Table 2, there was a tendency for the positive rate of CTC to increase as the stage progressed (stage I vs IV: 33.3% vs 75%).
  • AIS means adenocarcinomas in situ.
  • stage IA the CTC positive rate and the EMT-CTC positive rate were both 46.2%, which were significantly high values.
  • stage IV the positive rate of non EMT-CTC also increased at the same time.
  • CEA positive rate and “SLX positive rate” indicate the positive rates of CEA and SLX determined by serum test, respectively, CEA is 3.0 ng / ml or more, SLX is 38 ng / ml or more positive It is said.
  • stage IA CTC vs CEA: 46 .2% vs 23.1% the positive rate of CTC was significantly high in stage I (stage IA CTC vs CEA: 46 .2% vs 23.1%).
  • the positive rate of CTC was significantly higher than CEA and SLX.
  • the CTC positive rate according to the method of the present invention increases as the stage progresses, but there has been no CTC detection technique that can detect non-small cell lung cancer earlier than stage IIIA, and Considering that a significant difference was observed in the positive rate even when compared with tumor markers such as CEA and SLX as shown in Table 3, it was shown that the method of the present invention is particularly useful for early cancer diagnosis. It was done.
  • FIGS. 1 to 3 will be described.
  • the vertical axis in FIGS. 1 to 3 indicates the number of CTCs detected from one sample.
  • FIG. 1 is a diagram showing a result of comparing EMT CTC and non-EMT CTC among non-small cell lung cancer patients. As can be seen from FIG. 1, EMT-CTC was higher than non EMT-CTC.
  • FIG. 2 shows the number of CTCs in patients with different histological grades.
  • non-EMT-CTC can be used as an indicator of high therapeutic effect by chemotherapy.
  • EMT-CTC can be used as an indicator of low therapeutic effect by chemotherapy. It turned out to be.
  • FIG. 3 in cases where EMT-CTC still remained after 1 week from the start of chemotherapy, remote organ recurrence cases were observed at an early stage of treatment (no data).
  • Example 2 Detection of cancer stem cells 1 ⁇ 10 4 H69 (CD133 positive) was suspended in 7.5 mL of blood, and red blood cells were hemolyzed to recover PBMC. Thereto was added 1 ⁇ 10 9 VP of virus, and the mixture was infected at 37 ° C. for 24 hours while rotating with a rotator. Infected cells were collected, and immunostaining using CD45 antibody and CD133 antibody (Milltenyi 130-090-422) and nuclear staining with DAPI were performed.
  • Example 3 As a biological sample for detecting tumor cells floating in pleural effusion, 20 ml of pleural effusion collected from a patient confirmed to be lung cancer by a pathological specimen of lung tissue obtained by biopsy or surgery was used. In the same manner as in Example 1, collection of PBMC fraction and virus infection, (ii) CD45 / EpCAM / dead cell staining, and analysis of specimens were performed.
  • the number of TelomeScan F35 positive / EpCAM negative / CD45 negative cells and the number of TelomeScan F35 negative / EpCAM positive / CD45 negative / dead cell staining positive were about 34,000, respectively. They were 12,000, 300,000, 170, 500, 70, 64,000, 6,600, 23,000, 0, 0, 0. From these results, it was shown that tumor cells floating in pleural effusion can be detected in detail according to the present invention.
  • phenotypic analysis of tumor cells in body cavity fluid also shows usefulness of treatment response, tolerance, recurrence, malignancy, and prognosis for predicting local control of cancerous pleurisy, pericarditis, and peritonitis as well as count information Information.
  • Example 4 Lung metastasis recurrence after esophageal cancer surgery, lung metastasis recurrence after gastric cancer surgery, rectal cancer and renal cancer detection
  • EMT-CTC detection biological samples include pathological specimens of cancer tissue obtained by biopsy or surgery, esophageal cancer 7.5 ml of blood collected from patients with confirmed lung metastases after surgery, recurrence of lung metastases after surgery for gastric cancer, rectal cancer and renal cancer was used. Metastasis of lung metastases after surgery for esophageal cancer and lung metastasis after surgery for gastric cancer is single and is classified as oligo recurrence.
  • FIG. 5 shows photographs of TelomeScan F35 positive / EpCAM negative / CD45 negative cells detected in lung cancer recurrence after esophageal cancer surgery, lung metastasis recurrence after gastric cancer surgery, rectal cancer, and renal cancer. Photographs of rectal cancer cases were provided by Dr. Hiromitsu Komiyama, Juntendo University. Photographs of kidney cancer cases were provided by Dr. Haruna Kono at Juntendo University. From these results, it was shown by the present invention that EMT-CTC can be detected even in cancer types other than lung cancer. Moreover, it was shown by this invention that EMT-CTC can be detected in oligo recurrence.

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Abstract

L'objectif de la présente invention est de proposer une méthode pour la détection de cellules. La présente invention concerne une méthode pour la détection de cellules qui comprend une étape permettant d'utiliser la croissance d'un virus de recombinaison régulée par un promoteur de transcriptase inverse de la télomérase afin de détecter la croissance de ce virus de recombinaison ; une étape faisant intervenir un réactif pour détecter des cellules dérivées de l'épithélium ; et une étape faisant intervenir un réactif pour détecter des leucocytes.
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JP2017129584A (ja) * 2016-01-19 2017-07-27 東ソー株式会社 希少細胞を用いて癌患者の予後を予測する方法
WO2017126634A1 (fr) * 2016-01-19 2017-07-27 東ソー株式会社 Procédé de prédiction de pronostic de patient à l'aide de cellules rares
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WO2018190379A1 (fr) * 2017-04-13 2018-10-18 日立化成株式会社 Méthode de prédiction de l'efficacité d'un inhibiteur de point de contrôle immunitaire sur un sujet test
WO2018190382A1 (fr) * 2017-04-13 2018-10-18 日立化成株式会社 Méthode de détection de cellules cancéreuses pd-l1 positives
JP2021110664A (ja) * 2020-01-14 2021-08-02 学校法人杏林学園 癌被験体の予後を予測する方法

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