Adenovirus type 5 / 3 for detection of circulating tumor cells

Abstract

The present invention relates to a chimeric adenovirus for detecting circulating tumor cells. The adenovirus type 5 / 3 for detecting circulating tumor cells of the present invention contains the knob domain of adenovirus type 3 and targets DSG2, thereby enabling the detection of EMT circulating tumor cells. It has excellent survival ability in the blood and can accurately detect and isolate circulating tumor cells from a larger number of carcinomas, enabling early cancer diagnosis and analysis of the type and stage of primary cancer. Genetic analysis of isolated circulating tumor cells can be used to predict cancer prognosis, monitor cancer progression, and analyze drug responsiveness and treatment effects, making it effective for personalized treatment.

Description

[Technical field]

[0001] The present invention relates to chimeric adenoviruses for the detection of circulating tumor cells. [Background technology]

[0002] The number of deaths from cancer (malignant neoplasms) in Korea in 2002 was 62,887, or 25.5% (29.6% of male deaths and 20.5% of female deaths) of the total deaths of 246,515 (crude death rate of 512 per 100,000 population), with cancer being the leading cause of death (130.7 per 100,000 population). The ranking of cancer deaths is lung cancer, stomach cancer, lung cancer, colon cancer and pancreatic cancer, with these five major cancers accounting for approximately 70% of all cancer deaths. The main causes of cancer deaths in men are lung cancer, stomach cancer, lung cancer, and colon cancer, with these four major cancer deaths (28,147 people) accounting for 70% of the total number of male cancer deaths (40,177 people), while the main causes of cancer deaths in women are stomach cancer, lung cancer, lung cancer, colon cancer, and pancreatic cancer, with these five major cancer deaths (13,630 people) accounting for 60% of the total number of female cancer deaths (22,710 people). There are dozens of types of cancer that have been identified so far, and they are mainly classified according to the location of the diseased tissue. Cancer grows very quickly, invades surrounding tissues, and causes metastasis, threatening life. Such types of cancer include brain and spinal cord tumors, head and neck cancer, lung cancer, breast cancer, thymoma, esophageal cancer, gastric cancer, colon cancer, lung cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, lung cancer, testicular cancer, germ cell tumors, ovarian cancer, cervical cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma, and skin cancer, etc. In addition, such cancers may be classified according to their pathogenesis or morphology using different classification systems.

[0003] Cancer metastasis occurs when cancer cells from the primary tumor spread throughout the body via blood vessels and lymphatic vessels, and some of these cells attach to and grow in other organs. This occurs due to circulating tumor cells (CTCs) that travel in the blood. It has been reported that the number of CTCs in the blood is related to cancer metastasis and prognosis, and that measuring the number of CTCs is useful for predicting the prognosis and treatment effect of metastatic cancer, such as metastatic breast cancer. Recently, various studies have been conducted to use CTCs as a representative marker for cancer diagnosis. The test method for detecting CTCs corresponds to liquid biopsy. Unlike tissue biopsy used in existing cancer diagnosis, liquid biopsy utilizes biological fluids such as blood, urine, and saliva. Tissue biopsy has the disadvantage that it cannot be performed frequently and is a burden on patients. Liquid biopsy can diagnose cancer early in a relatively simple manner and is attracting attention as a new alternative. However, in contrast to the billions of red blood cells and millions of white blood cells in the same volume of blood, CTCs are present in less than a few dozen numbers, making them extremely difficult to detect. The reality is that technology is needed to accurately locate and isolate CTCs, which exist in extremely small quantities in blood (1 in 109 red blood cells) without losing any cells.

[0004] In modern medicine, the same medicine has been prescribed within the established dosage range for the same disease, regardless of each individual's genetic traits. However, recently, various dielectric projects have laid the foundation for understanding human diseases at the genetic level, and as a result, the era of personalized medicine has arrived, where individualized disease diagnosis, treatment, and prevention are performed by comparing each individual's genetic traits and drug responses, rather than universal treatment tailored to each patient with a different genetic environment. At this time, in order to grasp individual differences, it is necessary to know the genetic traits of each individual, and therefore, it is necessary to first collect the individual's body tissue in order to analyze the genetic traits of each individual. However, in the past, the only way to collect biological tissue was through invasive tissue biopsy, which involves drilling holes in the body to collect biological tissue (especially diseased tissue such as cancer tissue). This is not only an expensive method, but also has the disadvantages of being painful for the patient during the collection process as invasive instruments must be invaded into the patient's body, and in the case of cancer cells, the invasive process can be dangerous for the patient because it is necessary to collect cancer cells from the bone marrow. However, collecting circulating tumor cells (CTCs) is simple and safe because it only requires drawing blood from the patient and no invasive instruments are used in the patient's body. Furthermore, blood can be drawn in a short time, which saves time and shortens the overall process of selecting the right anticancer drug for each patient based on the results of the anticancer drug response, making it economical.

[0005] Meanwhile, the adenovirus genome is a linear double-stranded DNA of about 36 kb in size, and in particular, each end contains an inverted repeat (ITR), and is composed of an encapsidation sequence (psi), an early gene, and a late gene. The early genes are contained in the E1, E2, E3, and E4 regions, and in particular, the E1 gene of adenovirus is the gene that is expressed earliest after infection of a target cell, and is composed of two transcription units, namely, E1A and E1B. The protein expressed by the E1A gene actively advances the cell cycle in the infected cell and promotes the transcription of the E1B gene and other early genes to regulate the replication and growth of the virus. However, in cells infected with adenovirus, as a defense mechanism to prevent this, excessive progression of the cell cycle or induction of cell death are suppressed (Shenk, T. Adenviridae: the viruses and their replication 3rd ed. New York: Lippincott-Raven 2111-2148 (1996) and Shenk, T. et al. Adv Cancer Res 57: 47-85 (1991)). On the other hand, the E1B gene encodes the E1B 19 kDa protein and the E1B 55 kDa protein. Of these, the E1B 19 kDa protein plays a role in suppressing cell death induced by the E1A protein, and is similar in base sequence and function to Bcl-2 (Imazu, T. et al. Oncogene 18: 4523-4529 (1999)). The E1B 55 kDa protein binds to the p53 protein to suppress cell death induced by the p53 protein and various other mechanisms, and functions to move adenoviral mRNA into the cytoplasm to promote protein synthesis (Babiss, L. et al. Mol Cell Biol 5:2552-2558 (1985) and Leppard, K. et al. EMBO J 8:2329-2336 (1989)). Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide an adenovirus for the detection of circulating tumor cells (CTCs).

[0007] It is also an object of the present invention to provide a composition for the detection of circulating tumor cells.

[0008] It is also an object of the present invention to provide a method for detecting circulating tumor cells.

[0009] It is also an object of the present invention to provide a method that provides the information necessary for the diagnosis of primary cancer.

[0010] It is also an object of the present invention to provide a method for providing information for personalized treatment.

[0011] Another object of the present invention is to provide a method for selecting an individualized anticancer drug.

[0012] It is also an object of the present invention to provide an application of adenovirus for the detection of circulating tumor cells.

[0013] It is also an object of the present invention to provide a diagnostic use of adenovirus for primary cancer.

[0014] Additionally, it is an object of the present invention to provide a method for diagnosing primary cancers utilizing adenoviruses for the detection of circulating tumor cells. [Means for solving the problem]

[0015] To achieve the above object, the present invention provides an adenovirus for detecting circulating tumor cells.

[0016] The present invention also provides a composition for detecting circulating tumor cells, comprising a peptide or antibody that specifically binds to DSG2, a nucleic acid encoding same, or a vector containing said nucleic acid.

[0017] The present invention also provides a method for detecting circulating tumor cells.

[0018] The present invention also provides methods that provide the information necessary for the diagnosis of primary cancer.

[0019] The present invention also provides methods for providing information for individualized therapy.

[0020] The present invention also provides a method for selecting an individualized anticancer drug.

[0021] The present invention also provides the use of adenovirus for the detection of circulating tumor cells.

[0022] The present invention also provides the use of adenovirus in the diagnosis of primary cancer.

[0023] Additionally, the present invention provides a method for diagnosing primary cancers utilizing adenoviruses for the detection of circulating tumor cells. Effect of the Invention

[0024] The adenovirus for detecting circulating tumor cells of the present invention targets DSG2 by including the knob domain of adenovirus type 3, and therefore can detect EMT circulating tumor cells. It has excellent survival ability in blood and can accurately detect and separate circulating tumor cells from a larger number of carcinomas, enabling early diagnosis of cancer and analysis of the type and stage of primary cancer. Furthermore, through genetic analysis of isolated circulating tumor cells, it is possible to predict cancer prognosis, monitor the progress of cancer, and analyze drug responsiveness and treatment effects, which can be used for personalized treatment. [Brief description of the drawings]

[0025] [Figure 1]This figure shows the production and confirmation of adenovirus for isolation / detection of CTCs. (A) is a schematic diagram of recombinant adenovirus serotype 5 / 3 encoding hTERT and GFP. (B) is a schematic diagram showing the CTC detection process. (C) is a fluorescent image confirming the detectability of EpCAM(+)CTCs and EpCAM(-)CTCs (white arrow: CTC). (D) is a diagram showing the CTC detection sensitivity and specificity of Ad5 / 3. [Diagram 2] Comparison of GFP expression levels after Ad5 / 3 infection in various cancer cell lines: (A) Images of A549, H460 and SK-OV-3 cell lines 24 and 48 hours after viral infection, (B) Pixel-based GFP intensity 24 and 48 hours after viral infection, (C) Change in percentage of GFP-positive cells 24 and 48 hours after viral infection. [Diagram 3] This figure confirms the expression levels of molecules that may be involved in Ad5 / 3 infection and replication. (A) shows the protein expression levels of viral receptors DSG2, CD46, CAR, and enzyme hTERT upon infection with Ad5 / 3 from each cancer cell line, (B) shows the protein expression level of DSG2 upon infection with Ad5 / 3 from each cancer cell line, and (C) shows the protein expression level of hTERT upon infection with Ad5 / 3 from each cancer cell line. (D) shows the Pearson correlation between the infection rate and the protein expression level of DSG2 24 hours after Ad5 / 3 infection, (E) shows the Pearson correlation between the infection rate and the protein expression level of DSG2 48 hours after Ad5 / 3 infection, (F) shows the Pearson correlation between the infection rate and the protein expression level of hTERT 24 hours after Ad5 / 3 infection, and (G) shows the Pearson correlation between the infection rate and the protein expression level of hTERT 48 hours after Ad5 / 3 infection. (H) is a graph showing GFP expression levels in DSG2 knockdown HeLa cells 24 hours after Ad5 / 3 infection, and (I) is a graph showing GFP expression levels in DSG2 overexpressing DU145 cells 24 hours after Ad5 / 3 infection. [Figure 4]This figure shows the number of CTCs before and after surgery in patients with renal cell carcinoma (RCC) and prostate cancer (PCa) clinically confirmed using Ad5 / 3 (each color line represents each patient). (A) is a collection image of a patient's blood sample, (B) is a figure showing the number of CTCs before and after surgery in RCC patients, (C) is a figure showing the number of CTCs before and after surgery in patients with localized RCC (n=4) and locally advanced RCC (n=9), (D) is a figure showing the number of CTCs before and after surgery in patients with non-metastatic RCC (n=7) and metastatic RCC (n=6), (E) is a figure showing the number of CTCs before and after surgery in patients with prostate cancer (n=20), and (F) is a figure showing the prostate-specific antigen (PSA) value before and after surgery in patients with prostate cancer (n=11). [Diagram 5] This figure shows that somatic mutations in CTCs isolated using Ad5 / 3 were detected, and that the detected CTCs were derived from primary cancers based on the characteristics of gene copy number. (A) shows the process of forming a gene library after single CTC selection and isolation and analyzing the entire gene sequence, (B) shows the process of analyzing the sequenced genome, (C) shows the number of somatic mutations present in the primary tumors and CTCs of each patient, (D) shows the gene copy number mutations identified in chromosomes 1 and 8 derived from the primary tumors of each patient, and (E) shows the gene copy number mutations identified in chromosomes 1 and 8 derived from CTCs. [Figure 6] FIG. 1 shows a process of collecting a blood sample from a patient, infecting it with Ad5 / 3 of the present invention, specifically isolating CTCs, and analyzing the genetic information of the isolated CTCs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiment is presented as an example of the present invention, and if a detailed description of a technology or configuration well known to those skilled in the art is deemed to unnecessarily obscure the gist of the present invention, the detailed description may be omitted and the present invention will not be limited thereby. The present invention is subject to various modifications and applications within the scope of the claims described below and the scope of equivalents analyzed therefrom.

[0027] In addition, the terminology used in this specification is used to appropriately express the preferred embodiment of the present invention, and may vary depending on the intention of the user or operator, or the practice of the field to which the present invention belongs. Therefore, the definition of the term must be based on the entire contents of this specification. In the entire specification, when a part "includes" a certain element, this does not mean that other elements are excluded, but that other elements may be further included, unless otherwise specified.

[0028] All technical terms used in the present invention are used as generally understood by those of ordinary skill in the art in the relevant field of the present invention unless otherwise defined. In addition, although preferred methods and samples are described in this specification, similar or equivalent methods and samples are also included in the scope of the present invention. The contents of all publications mentioned as references in this specification are incorporated herein by reference.

[0029] In one aspect, the present invention relates to an adenovirus for detecting circulating tumor cells (CTCs), comprising a human telomerase promoter (hTERT) operably linked to E1A and E1B of an endogenous gene of the adenovirus, a marker gene inserted into the E3 region of the endogenous gene of the adenovirus, and an adenovirus type 3 knob domain protein.

[0030] In one embodiment, the adenovirus may be produced by expressing in a cell a recombinant adenovirus vector comprising a human telomerase promoter (hTERT) operably linked to E1A and E1B of an endogenous adenovirus gene, a marker gene inserted into the E3 region of an endogenous adenovirus gene, and a nucleic acid encoding the knob domain of adenovirus type 3, and the adenovirus vector may comprise a base sequence represented by SEQ ID NO: 15.

[0031] In one embodiment, the adenovirus may be a recombinant adenovirus vector comprising the adenovirus type 3 knob domain protein.

[0032] In one embodiment, the knob domain of adenovirus type 3 may bind to DSG2 (desmoglein-2), which may comprise the amino acid sequence of SEQ ID NO: 1 or may be encoded by the nucleic acid of SEQ ID NO: 2 or NM_001943.

[0033] In one embodiment, the adenovirus (Ad) may be a group C adenovirus.

[0034] In one embodiment, the adenovirus may comprise a fiber comprising the tail domain of adenovirus type 5, the shaft domain of adenovirus type 5 and the knob domain of adenovirus type 3, or may be a chimeric adenovirus (Ad5 / 3) in which the knob domain of the adenovirus 5 virus is genetically replaced with the corresponding domain of adenovirus-3, thereby comprising a fiber sequentially comprising the tail domain of Ad5, the shaft domain of Ad5 and the knob domain of Ad3.

[0035] In one embodiment, the tail domain of Ad5 may comprise the amino acid sequence of SEQ ID NO:3, the shaft domain of Ad5 may comprise the amino acid sequence of SEQ ID NO:4, and the knob domain of Ad3 may comprise the amino acid sequence of SEQ ID NO:5.

[0036] In one embodiment, the fiber of Ad5 / 3 may comprise the amino acid sequence of SEQ ID NO:6.

[0037] In one embodiment, the tail domain of Ad5 may be encoded by the base sequence of SEQ ID NO:7, the shaft domain of Ad5 may be encoded by the base sequence of SEQ ID NO:8, and the knob domain of Ad3 may be encoded by the base sequence of SEQ ID NO:9.

[0038] In one embodiment, the Ad5 / 3 fiber may be encoded by the base sequence of SEQ ID NO:10.

[0039] In one embodiment, the adenovirus has a 5'ITR-C1-C2-C3-C4-C5-3'ITR structure, where C1 includes E1A and E1B operably linked to the hTERT promoter, or includes only E1A operably linked to the hTERT promoter, or includes only E1B operably linked to the hTERT promoter, or includes only the hTERT promoter, C2 optionally includes E2B-L1-L2-L3-E2a-L4, C3 includes E3 including a marker gene, C4 optionally includes L5, and C5 optionally includes E4.

[0040] In one embodiment, hTERT may be encoded prior to the E1 region that drives expression of E1A and E1B genes linked to an internal ribosome entry site (IRES).

[0041] In one embodiment, the marker gene may be inserted into the E3 region under the cytomegalovirus (CMV) promoter of an adenovirus vector.

[0042] In one embodiment, the hTERT promoter may include the nucleotide sequence of SEQ ID NO:11, E1A may include the nucleotide sequence of SEQ ID NO:12, and E1B may include the nucleotide sequence of SEQ ID NO:13.

[0043] In one embodiment, an IRES sequence may further be included between E1A and E1B, and the IRES may include the base sequence of SEQ ID NO:14.

[0044] In one embodiment, the label may be a fluorescent substance, a probe, or a tag. The label may also be a chemical moiety that can be detected by a method known in the art. Examples of detectable labels may include a moiety that can be detected by spectroscopy, photochemistry, or by biochemical, immunochemical, or chemical means. A suitable method for labeling a nucleic acid probe may be selected depending on the type of label and the location of the label and the probe. Examples of labels may include enzymes, enzyme substrates, isotope substances, fluorescent dyes, chromophores, chemiluminescent labels, electrochemical luminescent labels, ligands with specific binding partners, and other labels that can react with each other to increase, modify, or decrease the intensity of a detection signal.

[0045] In one embodiment, the labeling substance may be a fluorescent substance, and the fluorescent substance may be green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), or modified red fluorescent protein (mRFP).

[0046] In one embodiment, the circulating tumor cells may be blood circulating tumor cells.

[0047] The terms "specifically bind" and "specifically recognize" in the present invention have the same meaning as commonly known to those skilled in the art, that is, an antigen and an antibody specifically interact with each other and react immunologically.

[0048] The term "probe" in the present invention means a nucleic acid fragment such as RNA or DNA, which can specifically bind to mRNA and is as short as a few bases or as long as several hundred bases, and is labeled to confirm the presence or absence of a specific mRNA. The probe may be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, etc. In the present invention, hybridization is performed using a probe complementary to the AFP, HMMR, NXPH4, PITX1, THBS4 and / or UBE2T gene, and the degree of expression of the gene can be diagnosed based on the presence or absence of hybridization. The selection of an appropriate probe and the hybridization conditions can be modified based on what is known in the art, and are not particularly limited in the present invention.

[0049] The primers or probes of the present invention may be chemically synthesized using phosphoramidite solid support methods or other widely known methods. Such nucleic acid sequences may also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more analogues of natural nucleotides, and internucleotide modifications, such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

[0050] In the present invention, the suitable conditions for hybridizing the probe with the cDNA molecule may be determined in a series of steps by optimization procedures. Such procedures are carried out in a series of steps by those skilled in the art to establish a protocol for laboratory use. For example, conditions such as temperature, concentration of components, hybridization and washing time, buffer components and their pH and ionic strength depend on various factors such as the length and GC content of the probe and the target nucleotide sequence. Detailed conditions for hybridization can be found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); and MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. NY (1999). For example, among the stringent conditions, the highly stringent conditions refer to hybridization at 65° C. in 0.5 M NaHPO4, 7% SDS (sodium dodecyl sulfate), and 1 mM EDTA, followed by washing at 68° C. in 0.1×SSC (standard saline citrate) / 0.1% SDS. Alternatively, the highly stringent conditions refer to washing at 48° C. in 6×SSC / 0.05% sodium pyrophosphate. The low stringent conditions refer to washing at 42° C. in 0.2×SSC / 0.1% SDS.

[0051] The term "for the detection of circulating tumor cells" as used in the present invention may be used interchangeably or interchangeably with "for the isolation of circulating tumor cells" and "for the identification of circulating tumor cells".

[0052] The terms "detected," "detect" or "detection" as used herein may describe the general practice of finding or identifying or specifically observing a detectably labeled composition.

[0053] In one aspect, the invention relates to a method for producing an adenovirus for detection of circulating tumor cells, comprising expressing in a cell a recombinant adenovirus vector comprising a human telomerase promoter (hTERT) operably linked to E1A and E1B of an endogenous gene of an adenovirus, a marker gene inserted into the E3 region of the endogenous gene of the adenovirus, and a nucleic acid encoding the knob domain of adenovirus type 3, and purifying the adenovirus in the cell.

[0054] In one aspect, the invention relates to a composition for the detection of circulating tumor cells comprising an adenovirus or an adenoviral vector of the invention.

[0055] In one aspect, the present invention relates to a kit for the detection of circulating tumor cells comprising the composition for the detection of circulating tumor cells of the present invention.

[0056] In one aspect, the present invention relates to a method for detecting circulating tumor cells, comprising the step of treating a sample isolated from an individual with a composition for detecting circulating tumor cells of the present invention.

[0057] In one embodiment, the sample may be blood, serum, plasma, amniotic fluid, saliva, perforation, bone marrow, leak, bile, lung lavage fluid, cerebrospinal fluid, pleural fluid, synovial fluid, lymph, semen, urine, or a solution of protein extracted from tissue biopsy or cells, and is most preferably blood.

[0058] In one embodiment, the method may further include determining that marker gene-expressing cells in the sample are circulating tumor cells.

[0059] The methods for detecting circulating tumor cells of the present invention include methods for detecting and isolating and identifying circulating tumor cells present in the blood of an individual.

[0060] In one aspect, the present invention relates to a method for providing information necessary for diagnosing a primary cancer, comprising the steps of: 1) treating a sample isolated from an individual with a composition for detecting circulating tumor cells of the present invention; 2) isolating marker gene-expressing cells in the sample; 3) culturing the isolated cells; and 4) determining the primary cancer.

[0061] In an embodiment, the primary cancer may be any one or more selected from the group consisting of colon cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, brain cancer, head and neck cancer, melanoma, myeloma, leukemia, lymphoma, gastric cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, esophageal cancer, small intestine cancer, anal cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulva cancer, Hodgkin's disease, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvis cancer, bone cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, intraocular melanoma, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, glioblastoma multiforme, and pituitary adenoma.

[0062] In one aspect, the present invention relates to a method of providing information for personalized therapy, comprising the steps of: 1) treating a sample isolated from an individual with a composition for the detection of circulating tumor cells of the present invention; 2) isolating marker gene-expressing cells within the sample; and 3) performing genomic sequencing of the isolated cells to detect and analyze genetic variations. In one embodiment, genome sequencing may be performed by next-generation sequencing (NGS), and more preferably by whole genome sequencing (WGS).

[0063] In one embodiment, the method may further include a step of setting a gene in which the detected genetic variation occurs as a therapeutic target for the individual.

[0064] In one embodiment, the information for personalized therapy may be cancer diagnosis, cancer prognosis prediction, cancer progression monitoring, drug response, treatment effect, or a combination thereof. The term "individual" or "person" as used herein means any animal, including a monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig, including a human, that has or may have cancer. In one aspect, the present invention relates to a method for selecting an individualized anticancer drug, comprising the steps of: 1) treating a sample isolated from an individual with a composition for detecting circulating tumor cells of the present invention; 2) isolating cells expressing a marker gene in the sample; 3) culturing the isolated cells; 4) reacting the cultured cells with a candidate anticancer drug; 5) analyzing the response of the candidate anticancer drug; and 6) selecting an individualized anticancer drug using the response information of the analyzed candidate anticancer drug.

[0065] In one aspect, the present invention relates to the use of an adenovirus comprising a human telomerase promoter operably linked to the E1A and E1B endogenous genes of the adenovirus, a marker gene inserted into the E3 region of the endogenous gene of the adenovirus, and an adenovirus type 3 knob domain protein for the detection of circulating tumor cells.

[0066] In one aspect, the present invention relates to a primary cancer diagnostic use of an adenovirus comprising a human telomerase promoter operably linked to E1A and E1B of an endogenous adenovirus gene, a marker gene inserted into the E3 region of the endogenous adenovirus gene, and an adenovirus type 3 knob domain protein.

[0067] In one aspect, the present invention relates to a method for diagnosing primary cancer utilizing an adenovirus for the detection of circulating tumor cells, comprising a human telomerase promoter operably linked to E1A and E1B of an endogenous gene of the adenovirus, a marker gene inserted into the E3 region of the endogenous gene of the adenovirus, and an adenovirus type 3 knob domain protein.

[0068] In an embodiment, the primary cancer may be any one or more selected from the group consisting of colon cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, brain cancer, head and neck cancer, melanoma, myeloma, leukemia, lymphoma, gastric cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, esophageal cancer, small intestine cancer, anal cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulva cancer, Hodgkin's disease, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvis cancer, bone cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, intraocular melanoma, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, glioblastoma multiforme, and pituitary adenoma.

[0069] The present invention will be described in more detail with reference to the following examples. However, the following examples are merely for the purpose of illustrating the content of the present invention, and the present invention is not limited thereto.

[0070] Example 1. Design and construction of adenovirus 5 / 3 (Ad5 / 3) chimeric vector for CTC detection

[0071] CAR (Coxsackievirus and adenovirus receptor), the binding receptor for adenovirus serotype 5, is known to be downregulated in aggressive tumors, and adenovirus serotype 3 fiber uses DSG2 (desmoglein-2) as its main receptor. Therefore, to target both CAR and DSG2 and specifically infect aggressive tumors and solid tumors, we designed and constructed an adenovirus 5 / 3 (Ad5 / 3) chimera (i.e., an adenovirus having a fiber that sequentially contains the Ad5 tail domain, Ad5 shaft domain, and Ad3 knob domain) that contains the adenovirus 3 knob domain in the fiber of adenovirus serotype 5. Specifically, the viral vector system Ad5 / 3; pAd1127, pAd1128, pAd1129 and pAd1130 were purchased from DO260 Inc., (ID, USA), and the viral backbone was constructed using Cosmid construction kit-2 according to the protocol (OD260). In addition, the E1 region, which drives the expression of E1A and E1B genes linked to an internal ribosome entry site (IRES), was designed to stop the expression of hTERT-specific binding transcription factors, and the green fluorescent protein (GFP) gene for fluorescent expression was inserted into the E3 region under the cytomegalovirus (CMV) promoter of the Ad5 / 3 genome, so that the virus would express GFP under the condition of human telomerase reverse transcriptase (hTERT).The recombinant virus was produced by transfecting the Pac-I (NEB, MA, USA)-cleaved full-length viral genome into 293A cells (Invitrogen, CA, USA) using the CalPhos™ Mammalian Transfection Kit (Clontech, Ltd. CA.USA), and was amplified and purified using the Adeno-X™ Mega Purification Kit (Clontech). The purified virus was stored at -80°C, and the infectious virus titer was confirmed and determined in human embryonic kidney 293 (HEK293; ATCC) cells using the Adeno-X™ Rapid Titer Kit (Clontech). The genetically modified virus replicates and expresses GFP only in cancer cells due to the specificity of the promoter, whereas Ad5 / 3 can infect both normal and cancer cells (Figure 1A). Using this characteristic, cells expressing GFP, i.e., CTCs, from patient samples can be observed by fluorescence microscopy (Figure 1B).

[0072] Example 2. Confirmation of specific detection ability of Ad5 / 3 CTCs

[0073] In order to confirm the specific infection of the virus Ad5 / 3 prepared in Example 1 to circulating tumor cells (CTCs), and the sensitivity and specificity of CTC detection, 100 cells each of cancer cell lines A549 and MCF7, and peripheral blood monocytic cells (PBMCs) were dispensed, and the adenovirus Ad5 / 3 was treated at 10 MOI (multiplicity of infection) and incubated for 24 hours. Then, fluorescence was observed using a Nikon DIAPHOT 300 (Nikon, Japan) with an illumination device to confirm GFP-positive cells and EpCAM-positive cells (stained with anti-EpCAM (Cell Signaling Technology Inc., CA, USA)), and the infection rate was calculated by dividing the number of GFP-expressing cells by the total number of cells. Each cell line was calculated using ImageJ (v2.0) software. Each experiment was repeated five times, and GFP-positive cells and EpCAM-positive cells were identified by fluorescence microscopy, and the percentage of GFP-positive cells was counted to calculate the sensitivity and specificity of the virus (repeated five times).

[0074] As a result, it was shown that the virus of the present invention can detect not only EpCAM(+) CTCs but also EpCAM(-) CTCs, and it was confirmed that the virus infected CTCs and expressed GFP regardless of the presence or absence of EpCAM expression in CTCs (FIG. 1C). In addition, while no GFP expression was observed in PBMCs, 96% of A549 cells and 82.5% of MCF7 cells were detected, confirming the sensitivity and specificity of CTC detection based on GFP-coding Ad5 / 3 (FIG. 1D).

[0075] This confirmed that Ad5 / 3 of the present invention can selectively detect CTCs among PBMCs, and can detect both EpCAM(+) and EpCAM(-) CTCs.

[0076] Example 3. Confirmation of the detectability of Ad5 / 3 for various types of CTCs

[0077] In order to confirm the diagnostic effect of the adenovirus (Ad5 / 3) prepared in Example 1 on various types of CTCs, the GFP expression levels in various cancer cell lines were confirmed and compared. Specifically, 12 cancer cell lines, 253J-BV (human bladder cancer), ACHN (papillary renal cancer), Caki-2 (clear cell carcinoma), SN12C (human renal cancer), SN12PM6 (human renal cancer) (Korea Cell Line Bank, Seoul, Korea), Hep3B (hepatocellular carcinoma), A549 (lung adenocarcinoma), H460 (lung large cell carcinoma), SK-OV-3 (ovarian cancer), C4-2B (prostate cancer), HeLa (cervical cancer), and DU 145 (human prostate cancer) (ATCC; VA, USA), were cultured at 3×10 5 The cells were inoculated into a 6-well plate at a cell concentration of 100 μg / ml, cultured for 24 hours, and then replaced with fresh medium containing the CTC-specific adenovirus 5 / 3 (Ad5 / 3) of Example 1 at 15 MOIs. After 24 and 48 hours of incubation, the number of GFP-positive cells was counted under a fluorescent microscope. The infection rate was calculated by dividing the number of GFP-expressing cells by the total number of cells, and was calculated for each cell line using ImageJ (v2.0) software. To visualize the GFP intensity between the two time points, it was analyzed at the pixel level, the brightness was converted to a range of 0 to 250, and the cell number was analyzed by the number of pixels.

[0078] As a result, the infection rate and GFP intensity changed depending on the incubation time (Figure 2A), and generally, the number of cells (determined by pixel count) was relatively higher at 48 h than at 24 h (Figure 2B). In addition, the infection rate tended to increase over time in all cell lines, and the percentages at 24 h and 48 h differed for each cell line, especially the initial infection rate. In addition, the change in the percentage of GFP-positive cells varied among the cell lines (Figure 2C), confirming that the expression of the viral receptor and the replication rate differed among the cell lines.

[0079] Example 4. Correlation between Ad5 / 3 infection rate in cancer cells and DSG2

[0080] 4-1. Confirmation of changes in expression of adenovirus receptors due to adenovirus infection

[0081] In the adenovirus Ad5 / 3 of the present invention, the fiber knob of Ad3 is attached to the Ad5 structure, and the fiber knob of type 3 binds to DSG2 (desmoglein-2) expressed on the cell membrane, so the correlation between the expression levels of CAR (Coxsackievirus and adenovirus receptor), a virus receptor different from DSG2, CD46 (cluster of differentiation 46), and replication-related gene hTERT (human telomerase reverse transcriptase) and the infectivity of the adenovirus was confirmed by Western blot analysis. Specifically, the 12 cancer cell lines of Example 2 were infected with Ad5 / 3, and after 24 and 48 hours, they were disrupted with RIPA buffer (Cure Bio, Korea), respectively, and the same volume of protein disruptants were electrophoresed on SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was blocked with 5% w / v skim milk (in TBST) and then reacted with primary antibodies against Coxsackievirus and adenovirus receptor (1:1000 dilution, Bioss, MA, USA), CD46 (1:1000 dilution, Prosci, Inc, CA, USA), DSG2 (1:1000 dilution, Santa Cruz, TX, USA), hTERT (1:1000 dilution, Santa Cruz), and β-actin (Santa Cruz). The membrane was washed three times with TBST, reacted with secondary antibodies for 2 h, and washed three times with TBST. Proteins on the membrane were visualized with luminol reagent (Santa Cruz) and detected using an automatic X-ray film processor JP-33 (JPI, Korea). The detected Western blot bands of DSG2 and hTERT were analyzed semi-quantitatively and normalized to β-actin levels, and the correlation between the infection rates and protein expression levels of DSG2 at 24 and 48 h was confirmed through Pearson correlation analysis.

[0082] The results showed that the difference in expression of DSG2 and hTERT between cell lines may affect the infectivity of Ad5 / 3, and that the protein expression level of DSG2 or hTERT affected the infectivity (Figure 3A to Figure 3C). In addition, the correlation between receptor expression and the virus infection rate was analyzed, and the correlation coefficient was relatively high for DSG2 (Figure 3D and Figure 3E). In addition, hTERT, an enzyme responsible for virus replication, showed a correlation with GFP expression (Figure 3F and Figure 3G). In addition, the HeLa cell line showed a high infection rate of over 80% at both time points (24h and 48h) on the premise of high expression of DSG2 and hTERT (Figure 3B and Figure 3C). The Hep3B cell line showed relatively low DSG2 expression levels and high hTERT expression levels (Figure 3B and C), which may be related to the low GFP expression (25.3%) at 24 h and high infection (76.4%) at 48 h. The SN12CPM6 cell line showed a similar infection to the Hep3B cell line at 24 h (25.5%), and its GFP was significantly expressed at 48 h (33.5%), unlike the Hep3B cell line (76.4%). The relatively low infection at 48 h in SN12CPM6 was inferred to be related to the low hTERT protein expression.

[0083] 4-2. Confirmation of correlation between changes in DSG2 expression and viral infectivity

[0084] DSG2 expression was artificially suppressed or overexpressed using siRNA or an expression vector, and infection with the virus of the present invention was confirmed using a fluorescent microscope. 5 After dispensing 100 HeLa cells into a 6-well plate, 24 hours later, negative control (Thermofisher Scientific, Inc.) and siDSG2 (Bioneer, Korea) were added to each well using Lipofectamine. TMThe cells were transfected with 2000 (Thermofisher Scientific, Inc.). After 48 hours, the cells were treated with the adenovirus Ad5 / 3 of the present invention at 5 MOI, incubated for 24 hours, and then the fluorescence was confirmed by a fluorescence microscope. In addition, 1 × 10 5 DU145 cells were plated in 6-well plates and then transfected with pmCherry-DSG2 (Addgene, MA, USA) or its empty vector using Lipofectamine. TM 3000 (Thermofisher Scientific, Inc.) After 24 hours, the cells were treated with the adenovirus Ad5 / 3 of the present invention at 15 MOI, and after incubation for 24 hours, the fluorescence was confirmed by a fluorescence microscope.

[0085] The results showed that downregulation of DSG2 expression in HeLa cells, where DSG2 is relatively highly expressed, reduced GFP expression at low MOI and early time points by low virus infection (Figure 3H). On the other hand, when DSG2 was overexpressed in the DU145 cell line, GFP expression was even higher than in the previous conditions (Figure 3I).

[0086] This confirmed that DSG2 is directly correlated with Ad5 / 4 virus infection of target cancer cells.

[0087] Example 5. Clinical application and validation of adenovirus for CTC detection

[0088] In general, it has been reported that the prognosis of patients in whom the number of CTCs decreases after primary tumor debulking and the number of CTCs remains stable even after surgery is poor, and it can be assumed that the main cause of CTCs is the primary tumor. Therefore, to verify whether the Ad5 / 3 adenovirus for detecting CTCs of the present invention specifically infects and detects CTCs in actual clinical specimens before and after surgery, blood samples were collected from 17 renal cell carcinoma (RCC) patients and 20 prostate cancer (PCa) patients (Advanced RCC; H-1811-087-986, Metastatic RCC; H-1805-186-950, Prostate cancer; H-1607-134-777) before and 30 days after surgery (interval between the two time points: 1 month) (Figure 4A). To count the numbers from each blood sample collected, 9 ml of blood was transferred to a heparin-coated tube at 4°C, and then the sample was mixed with RBC (red blood cell) disruption solution (Qiagen, Hilden, Germany) in a ratio of 1:3 and incubated at RT for 20 min. Then, each tube was centrifuged at RT for 5 min at 300 × g, and the cells remaining at the bottom of the tube were collected and washed twice with serum-free DMEM. The adenovirus of the present invention was injected at 3 × 10 9 Culture in DMEM containing VG / mL for 24 h at 37 °C and CO 2 After incubation in an incubator, CTCs were isolated from PBMCs and counted as in the previous example. The cancer grade was evaluated based on the pathological grade of each tumor cell after surgery.

[0089] The number of CTCs was confirmed in the pre- and post-operative samples, and it was shown that the number of CTCs in RCC patients decreased after surgery (Figure 4B). Then, the degree of cancer localization was classified into two groups using the T factor of the TNM stage system (local: T1 (n = 4), locally advanced: T2-3 (n = 9)) and the change in the number of CTCs was confirmed. It was shown that the number of CTCs was significantly decreased only in locally advanced RCC patients after surgery (Figure 4C). In addition, the number of CTCs was significantly decreased only in non-metastatic RCC patients after surgery by classifying them into non-metastatic and metastatic RCC patients (Figure 4D). In addition, it was shown that the number of CTCs was decreased in PCa patients after surgery (Figure 4E), and the level of PSA (prostate-specific antigen), a marker for prostate cancer, was also decreased after surgery (Figure 4F).

[0090] This confirmed that the CTC detection technique of the present invention using the adenovirus was accurate enough to observe the reduction in CTCs after surgery.

[0091] Example 6. Isolation and analysis of CTCs using Ad5 / 3

[0092] To confirm whether somatic mutations can be detected from CTCs collected by the method of the present invention, two to three CTCs were isolated from samples of two RCC patients using a micro-suction system with the virus Ad5 / 3 of the present invention, and whole genome sequencing (WGS) was performed on the CTCs and primary tumors (Figure 5A). Specifically, GFP-expressing CTCs were isolated from the PBMC layer, resuspended in serum-free medium, and single cell picking was performed using a Micro Pick and Placement system (Nepa Gene Co., Ltd., Japan). Then, genomic DNA of the CTCs was obtained and converted into an Illumina library using a PicoPLEX Gold single cell DNA-Seq kit (Takara Bio Inc., CA, USA) and a DNA HT Dual index kit (Takara Bio). One-sequencing data of FASTQ files with an average sequencing depth of more than 20X was obtained by Illumina Paired-End (PE) sequencing. Reads were mapped to the hg38 (human genome 38) reference genome using the Burrows-Wheeler Aligner (BWA)-MEM algorithm (0.7.17). Aligned SAM files were converted and aligned to BAM files using Samtools (v1.7), and duplicate reads were removed from each BAM file using Picard (v2.23.8). Somatic mutations were identified using the Mutect2 tool in GATK (v.4.1.8). The functional effects of mutations were annotated using VEP (v102), and the mutations were filtered for expected medium or high effect for consequence types. COSMIC (Catalog of Somatic Mutations in Cancer; v92) was used to annotate the COSV ID for each somatic mutation (Figure 5B).Comparison of copy number variations (CNVs) between primary tumors and CTCs was assessed using the FREEC algorithms (v11.6) with breakpointThreshold 0.6 and no other additional options. Because gene amplification by PCR can cause mutations, we typically only characterized the copy numbers of genes on chromosomes 1 and 8 between primary tumors and CTCs.

[0093] Mutect2 showed that the total number of somatic mutations present in the primary tumors and CTCs from the two patients was 13,545 and 14,844, respectively. Of these, 21 and 10 somatic nonsynonymous mutations causing amino acid mutations were shown to be present in the primary tumors and CTCs, respectively. In addition, 493 and 707 somatic mutations present in the primary tumors and CTCs of each patient were confirmed to be COSMIC (Figure 5C). In addition, the CNVs analysis showed that the isolated CTCs were derived from the primary cancer site (Figure 5D and Figure 3E).

[0094] As a result, it was confirmed that the CTC detection method using the adenovirus of the present invention can specifically separate / extract only CTCs from a large number of blood cells, particularly PBMCs, and analyze their characteristics, such as mutations, using NGS (Next Generation Sequencing), particularly WGS, and thus can be applied to personalized treatment by predicting drug responsiveness and therapeutic efficacy.

Claims

1. a human telomerase promoter (hTERT) operably linked to the endogenous adenovirus genes E1A and E1B; A marker gene inserted into the E3 region of an endogenous gene of an adenovirus; An adenovirus for detecting circulating tumor cells (CTC), comprising an adenovirus type 3 knob domain protein.

2. The adenovirus for detecting circulating tumor cells described in claim 1, wherein the knob domain of adenovirus type 3 comprises the amino acid sequence of SEQ ID NO:

5.

3. The adenovirus for detecting circulating tumor cells according to claim 1, wherein the knob domain of adenovirus type 3 binds to DSG2 (desmoglein-2).

4. The adenovirus for detecting circulating tumor cells according to claim 1, comprising a fiber comprising the tail domain of adenovirus type 5, the shaft domain of adenovirus type 5 and the knob domain of adenovirus type 3.

5. The adenovirus for the detection of circulating tumor cells according to claim 4, wherein the fiber comprises the amino acid sequence of SEQ ID NO:

6.

6. An adenovirus for detecting circulating tumor cells as described in claim 1, translated from an adenovirus vector comprising the base sequence represented by SEQ ID NO:

15.

7. The adenovirus for detecting circulating tumor cells according to claim 1, wherein the endogenous genes of the adenovirus have a 5'ITR-C1-C2-C3-C4-C5-3'ITR structure, C1 comprises E1A and E1B operably linked to an hTERT promoter, or comprises only E1A operably linked to an hTERT promoter, or comprises only E1B operably linked to an hTERT promoter, or comprises only an hTERT promoter, C2 optionally comprises E2B-L1-L2-L3-E2a-L4, C3 comprises E3 comprising a marker gene, C4 optionally comprises L5, and C5 optionally comprises E4.

8. The adenovirus for detecting circulating tumor cells according to claim 1, further comprising an IRES sequence between E1A and E1B.

9. The adenovirus for detecting circulating tumor cells according to claim 1 , wherein the label is a fluorescent substance, a probe or a tag.

10. The adenovirus for detecting circulating tumor cells according to claim 9, wherein the fluorescent substance is green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP) or modified red fluorescent protein (mRFP).

11. A composition for detecting circulating tumor cells, comprising the adenovirus of claim 1.

12. A kit for detecting circulating tumor cells comprising the composition for detecting circulating tumor cells according to claim 11.

13. A method for detecting circulating tumor cells, comprising the step of treating a sample isolated from an individual with a composition for detecting circulating tumor cells as described in claim 11.

14. The method for detecting circulating tumor cells according to claim 13, wherein the sample is blood, serum, plasma, amniotic fluid, saliva, perforated fluid, bone marrow, leaky fluid, bile, lung lavage fluid, cerebrospinal fluid, pleural effusion, synovial fluid, lymph, semen, urine, or a solution of protein extracted from tissue biopsy or cells.

15. The method for detecting circulating tumor cells according to claim 13, further comprising the step of determining that marker gene-expressing cells in the sample are circulating tumor cells.

16. 1) treating a sample isolated from an individual with a composition for detecting circulating tumor cells according to claim 11; 2) isolating cells expressing the marker gene within the sample; and 3) culturing the isolated cells; 4) determining the primary cancer.

17. Primary cancers include colon cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, brain cancer, head and neck cancer, melanoma, myeloma, leukemia, lymphoma, stomach cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, esophageal cancer, small intestine cancer, anal cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulva cancer, Hodgkin's disease, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvis cancer, bone cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, intraocular melanoma, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, central nervous system cancer, The method for providing information necessary for diagnosing a primary cancer according to claim 16, wherein the primary cancer is any one or more selected from the group consisting of CNS (central nervous system) tumor, primary CNS lymphoma, spinal cord tumor, glioblastoma multiforme, and pituitary adenoma.

18. 1) treating a sample isolated from an individual with a composition for detecting circulating tumor cells according to claim 11; 2) isolating cells expressing the marker gene within the sample; and 3) performing genomic sequencing of the separated cells to detect and analyze genetic variations, thereby providing information for personalized treatment.

19. The method for providing information for personalized therapy according to claim 18, wherein the genome sequencing is performed by next-generation sequencing (NGS).

20. The method for providing information for personalized therapy according to claim 18, further comprising a step of setting a gene in which the detected genetic variation occurs as a therapeutic target for the individual.

21. The method for providing information for personalized treatment according to claim 18, wherein the information for personalized treatment is cancer diagnosis, prediction of cancer prognosis, monitoring of the progress of cancer, drug response, treatment effect, or a combination thereof.

22. 1) treating a sample isolated from an individual with a composition for detecting circulating tumor cells according to claim 11; 2) isolating cells expressing the marker gene within the sample; and 3) culturing the isolated cells; 4) reacting the cultured cells with a candidate anticancer drug; 5) Analyzing the response of candidate anticancer drugs; 6) A method for selecting an individualized anticancer drug, comprising the step of selecting an individualized anticancer drug by utilizing the reaction information of the analyzed candidate anticancer drugs.

23. A use of an adenovirus comprising a human telomerase promoter operably linked to E1A and E1B of an endogenous gene of the adenovirus, a marker gene inserted into the E3 region of the endogenous gene of the adenovirus, and an adenovirus type 3 knob domain protein for detecting circulating tumor cells.

24. A primary cancer diagnostic use of an adenovirus comprising a human telomerase promoter operably linked to E1A and E1B of an endogenous gene of the adenovirus, a marker gene inserted into the E3 region of the endogenous gene of the adenovirus, and an adenovirus type 3 knob domain protein.

25. A method for diagnosing primary cancer utilizing an adenovirus for detecting circulating tumor cells, comprising a human telomerase promoter operably linked to E1A and E1B of an endogenous gene of the adenovirus, a marker gene inserted into the E3 region of the endogenous gene of the adenovirus, and an adenovirus type 3 knob domain protein.

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