KR20140017129A - Composition for prevention or treating recurrent or metastatic cancer having immunoresistance - Google Patents

Abstract

The present invention relates to a composition for preventing or treating recurrent or metastatic cancer having immune resistance, wherein the composition comprises an inhibitor against at least one gene or protein selected from the group consisting of NANOG, T cell lecukemia/Lymphoma 1 (TCL1), AKT, and myeloid cell leukemia sequence 1 (MCL1) and wherein the inhibitor is siRNA, antisense-oligonucleotide, shRNAi, miRNA, or a vector or antibody including one thereof. Further, the present invention relates to an anticancer screening composition using an NANOG-overexpressing cell line. According to the present invention, the NANOG gene as a pathological marker can be used to diagnose, inhibit, or mitigate the recurrent or metastatic cancer having immune resistance, and can be used to screen agents for treating the recurrent or metastatic cancer having immune resistance, so that an effective therapeutic agent against cancers is expected to be developed.

Description

Composition for prevention or treating recurrent or metastatic cancer having immunoresistance

The present invention relates to a composition for preventing or treating relapse of cancer and cancer metastasis. More specifically, the present invention provides NANOG / TCL-1 / AKT / as a pathological marker for the treatment of cancers that have become resistant to chemotherapy such as immunotherapy, and cancers that have recurred or metastasized after chemotherapy such as immunotherapy. Selection of the MCL-1 molecular axis effectively diagnoses cancer recurrence and metastasis observed in cancers resistant to anticancer immunity and further targets the anticancer immunity-related molecular axis of NANOG / TCL1 / AKT / MCL-1. It relates to a composition capable of preventing or treating resistant cancer.

According to the analysis of the Korea National Statistical Office, cancer is the number one cause of death in Korea, with 31.7% of males and 22.5% of females, and 31.7% of the entire population die of cancer.

Our body is made up of tiny cells and maintains homeostasis by eliminating abnormal cells that proliferate or die on their own or that have genetic abnormalities through immune surveillance. However, when many genes overlap, the cell loses homeostasis that maintains proliferation and death and leads to abnormal cell proliferation, leading to cancer. Therefore, cancer is an immunological disease caused by overlapping gene abnormalities and avoiding death by immune cells, and can be said to be a chronic disease caused by overlapping abnormalities for a long time.

Cancer is divided into a benign tumor and a malignant tumor. The benign tumor has a relatively slow growth rate and is not a metastasis that is transferred to another tissue from the original site of the tumor. Tumors are invasive to other tissues leaving the primary and rapidly growing, which is a life-threatening and very important cause of death.

To treat cancer, surgical removal or radiation therapy, chemotherapy, immunotherapy, etc., but there are many studies, more than 50% of cancer patients are reported to eventually die without healing. The reason for this is that even after surgical resection, the cancer cannot recur due to the removal of finely metastasized cancer cells, or despite the development of various anticancer drugs, the cancer cells are not induced to die or the early death of the cancer cells when the cancer is treated with anticancer drugs. This is because the tumor cells appear to decrease in response, but cancer cells that are resistant to anticancer drugs proliferate during or after treatment. In addition, radiation therapy is also difficult to cure because it shows resistance to cancer cell radiation. Anti-cancer immunotherapy is a method of removing cancer cells containing antigens by using immune cells by inducing an immune response using cancer-specific antigens or cancer-related antigens. It is very insignificant. Importantly, cancers that have acquired resistance to these chemotherapy tend to have a significant increase in cancer metastasis with relapse. Therefore, for better cancer treatment, studies to overcome cancer cell resistance to immunotherapeutics including chemotherapy and radiation therapy are necessary, and at the same time, the identification of related genes is very important. In addition, cancer treatment methods that target identified genes should be researched, and early diagnosis of cancer is required to improve the quality of life of patients due to rapid cancer treatment.

On the other hand, NANOG was expressed in embryonic stem cells as one of the factors necessary for the maintenance of pluripotent cells. Research on the mechanism of action of NANOG proteins is currently not fully understood, but it has been reported to work with transcription factors such as OCT4 and SOX2, to inhibit the electronic activity of NF-kB and to maintain stem cell characteristics in cooperation with STAT3. . In addition, recently, several papers have reported that the expression of NANOG is increased not only in embryonic stem cells but also in various carcinomas (Jeter CR et al. Functional evidence that the self-renewal gene NANOG regulates human tumor development.Stem Cells 2009; 27 (5): 993-1005) There are no reports of chemoimmune tolerance, and in particular, there is no report that this increase in NANOG is associated with the recurrence and metastasis of chemoimmune cancer.

As a background technology of the present invention, Korean Unexamined Patent Publication No. 10-2010-0115254 discloses api by confirming the expression of nanog, Oct4, or Sox2, which is an undifferentiated therapeutic composition comprising apisidine and a gene representing stem cellity. A method for screening a seedine co-administrator is disclosed.

However, increased NANOG is an essential molecule for immune tolerance in tumor cells that acquire anticancer immunity, whereby NANOG directly regulates TCL1, an AKT activating factor, to increase gene expression, and by increased TCL-1. AKT activation has been shown to increase the expression of MCL-1, an anti-apoptotic factor, and that the molecular axis related to the anticancer immunity acquisition of NANOG / TCL1 / AKT / MCL-1 acquires anticancer immunity. It can be used for diagnosis as a pathological marker for recurring cancer, resistant cancer, or cancer metastasis after chemotherapy such as immunotherapy, and further targeting the anticancer immunity related molecular axis of NANOG / TCL1 / AKT / MCL-1. There is no known composition for effectively preventing or treating cancer-resistant cancer.

Republic of Korea Patent Publication 10-2010-0115254 (2010.10.27)

Noh, Kyung-Hee, AKT's role in tumor-resistant tumors, 2007. Jeter CR et al. Functional evidence that the self-renewal gene NANOG regulates human tumor development. Stem Cells. 2009; 27 (5): 993-1005

An object of the present invention is a pathological marker for cancers that have become resistant to chemotherapy, such as immunotherapy, and cancers that have recurred or metastasized after chemotherapy, such as immunotherapy, such as human NANOG, TCL1 (T cell lecukemia / Lymphoma 1). ), AKT or MCL1 (Myeloid cell leukemia sequence 1) using a gene related to the acquisition of anticancer immunity to provide a pharmaceutical use for the treatment of cancer recurrence and metastasis.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the invention, the invention comprises an inhibitor against one or more genes or proteins selected from the group consisting of NANOG, T cell lecukemia / Lymphoma 1 (TLC1), AKT and Myeloid cell leukemia sequence 1 (MCL1),

Preferably, the inhibitor provides siRNA, antisense-oligonucleotide, shRNAi, miRNA or a vector comprising them, or an antibody, which provides a composition for preventing or treating recurrence and metastasis of cancer-resistant cancer.

According to another aspect of the invention, the invention comprises a probe for detecting at least one expression selected from the group consisting of NANOG, T cell lecukemia / Lymphoma 1 (TLC1), AKT and Myeloid cell leukemia sequence 1 (MCL1) Provided is a composition for diagnosing relapse and metastasis of anticancer immune cancer.

According to another aspect of the present invention, the present invention provides an anticancer immunity cancer comprising at least one gene selected from the group consisting of NANOG, TCL1 (T cell lecukemia / Lymphoma 1), AKT and MCL1 (Myeloid cell leukemia sequence 1). Provided are compositions for screening relapse and metastasis therapeutics.

According to still another aspect of the present invention, the present invention provides a composition comprising a cell line expressing at least one selected from the group consisting of NANOG, T cell lecukemia / Lymphoma 1 (TLC1), AKT and Myeloid cell leukemia sequence 1 (MCL1). Treating candidate cancer therapeutic agents for relapse and metastasis of anticancer immune cancer; And

Measuring the expression level of NANOG, TCL1 (T cell lecukemia / Lymphoma 1), AKT or MCL1 (Myeloid cell leukemia sequence 1) in the candidate cell line (experimental group); And methods for screening metastatic therapies.

According to the present invention as described above it is possible to effectively prevent or treat cancers that developed resistance to chemotherapy, such as immunotherapy, and cancers that recurred or metastasized after chemotherapy, such as immunotherapy. In other words, by using the new Nanog / Tcl1 / pAkt / Mcl1 immunoresistant molecular axis as a pathological marker, it can be used for diagnosis, inhibition and alleviation of immunoresistant cancer, recurrent cancer and metastatic cancer, as well as therapeutic agents for recurrence and metastasis of anti-cancer immune cancer. It can be used for the screening of cancer, which is expected to develop an effective treatment for cancer.

1 is a result confirming the expression of human NANOG protein of the present invention in cell lines of various human carcinomas.
Figure 2 is a result showing the expression index (A and B) and the survival rate (C) of the progress of the cancer of the human Nanog / Tcl1 / pAkt protein of the present invention according to the progression stage of human cervical cancer.
Figure 3 shows the results confirming the expression of NANOG protein in anti-cancer immune TC-1 P3 cell line. (A) Western blot, (B) Immunofluorescein left: fluorescence image right: Percentage of Nanog expressing (Nanog +) cells.
Figure 4 shows the results of confirming the proliferation of cells (A) and the cell cycle (B) and the expression of cell cycle-related proteins (C) in the anti-cancer immunity-resistant TC-1 P3 cell line and the parental TC-1 P0 cell line without immune tolerance.
FIG. 5 shows in vitro (A: in vitro tumor cell formation capacity) and in vivo tumorigenicity (B and C: number of injected cells) in anti-cancer immunoresistant TC-1 P3 cell line and parental TC-1 P0 cell line without immune tolerance. According to tumor formation).
Figure 6 shows the NANOG dependent cell proliferation and cell formation capacity of the present invention. Decreased expression of NANOG in anti-cancer immunoresistant TC-1 P3 cell lines reduced cell proliferation (A), changes in cell cycle, decreased expression of cell cycle-related factors CyclinA (B), and decreased in vitro tumor cell formation. Conversely, induction of Nanog expression in parental TC-1 P0 cell line without immune tolerance resulted in increased cell proliferation (D), cell cycle change, increased expression of cell cycle-related factor CyclinA (E), and increased in vitro tumor cell formation. The result is confirmed.
7 is a result confirming the induction of resistance to anti-cancer immunity of NANOG using a cancer cell line (TC-1 / NANOG) overexpressing NANOG of the present invention. Increased resistance to Granzyme B induced apoptosis by NANOG expression (A) and increased resistance to E7 antigen-specific cytotoxic cells (E7 CTL) mediated apoptosis (B) and E7 antigen-specific cytotoxic cells In vivo anticancer immunity increase of NANOG overexpressing tumor after intravenous injection (C).
8 is a result confirming that the increased expression of NANOG of the present invention is shown through anticancer immunity. Only when inducing antigen-specific anti-cancer immunity, it was confirmed that cancer cell lines with high expression of NANOG (TC-1 P1, P2, P3) were selected (AC).
9 is a result of confirming in human cancer cells that the increase in expression of NANOG of the present invention through anti-cancer immunity. Immunoselection description of the CaSki Db used in the present invention (A), the results of confirming that the immune tolerance increases as the immune screening process (B), the percentage of Nanog expressing cells increases as the number of immune screening increases It is a result (C).
10 is a result confirming that the tumor formation ability is increased not only in vitro (AC) but also in vivo (D and E) in the human cervical cancer cell line (CaSki Db P3) obtained anti-cancer immunity.
FIG. 11 shows the results of confirming tumor formation ability (AD) and anticancer immunity neutralization (E) upon inducing reduction of NANOG expression using NANOG targeting siRNA in human cervical cancer cell line (CaSki Db P3).
Figure 12 using the NANOG and NANOG mutants of weakened transcriptional activity of the present invention, the activation of AKT-dependent transcriptional activity (B), cell cycle regulation (C and D), tumorigenicity (E), anticancer immunity of Nanog The correlation between resistance (F) and induction of expression of anti-apoptosis related protein MCL-1 (G) was examined.
FIG. 13 shows tumorigenic capacity (B and E) and anticancer immunity (C) when neutralizing AKT activation mechanisms (AKT inhibitors (AC), AKT targeting siRNA (DF)) in human cervical cancer overexpressing NANOG of the present invention. And F).
Figure 14 shows the expression of Nanog-dependent Tcl1a (A) and expression of pAkt and CyclinA and Mcl-1 by TCL1A (B) and Nanog-mediated tumorigenicity (C: cell proliferation) in human cervical cancer overexpressing NANOG of the present invention. , D: in vitro tumor cell formation) and anticancer immunity regulation (E).
15 is a result of investigating that NANOG of the present invention can physically bind to promoter-938 to -721 portions of TCL1A to regulate transcription.
16 is a result confirming the decrease in tumor volume due to a decrease in the immune resistance of tumors of HCT116 SCTE7, a human colon cancer cell line having high expression of Nanog, when siRNA NANOG is delivered in vivo using chitosan nanoparticles using NANOG of the present invention. These results demonstrate that NANOG can be used as a good target molecule in immunotherapy of human cancer.
17 is a result of in vitro experiments to determine the cell mobility (A) and invasion capacity (B) after the expression of the NANOG of the present invention using siRNA in the mouse anti-cancer immune cell line TC-1 P3 .
FIG. 18 shows the results of confirming the NANOG of the present invention using CaRNA / Db P3, a human anti-cancer immunity-resistant cell line, by using siRNA in vitro in vitro experiments on cell migration ability (B) and invasion ability (C). . The results in FIGS. 17 and 18 demonstrate that NANOG can be used as a diagnostic and target as an important factor in cancer metastasis of anticancer immune cancer cells.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention includes inhibitors for one or more genes or proteins selected from the group consisting of NANOG, T cell lecukemia / Lymphoma 1 (TLC1), AKT and Myeloid cell leukemia sequence 1 (MCL1),

The inhibitor provides siRNA, antisense-oligonucleotide, shRNA, miRNA or a vector comprising one, or an antibody, which provides a composition for preventing or treating recurrence and metastasis of cancer-resistant cancer.

In the present specification, "primary cancer" refers to a conventional cancer, and "immune-resistant cancer" refers to a cancer obtained resistance to chemoimmune therapy, and anti-cancer immunotherapy induces an immune response to a cancer-specific antigen or a cancer-associated antigen. Cancer specificity refers to the integration of all systems of removing cancer cells by toxic immune cells. Immune induction methods for cancer antigens include genes, proteins, viruses, dendritic cells and the like. "Recurrence cancer" refers to cancer that has reoccurred after conventional cancer treatment, and "resistant cancer" refers to cancer that is resistant to the cancer treatment. Here, conventional cancer treatments include, for example, surgery, chemotherapy, radiation therapy, hormonal therapy, biological therapy, immunotherapy and the like. Examples of the cancer include brain tumors, spinal cord tumors, retinal cell tumors, oral cancers, nasal cancers, sinus cancers, pharyngeal cancers, laryngeal cancers, head and neck cancers including cervical cancers, skin cancers, breast cancers, thyroid cancers, breast cancers including malignant adrenal tumors Gastrointestinal cancer, kidney cancer, bladder cancer, prostate cancer, including lung cancer, pleural tumor, respiratory cancer, esophageal cancer, stomach cancer, small intestine malignant tumor, colon cancer, anal cancer, liver cancer, biliary tract cancer or pancreatic cancer Urinary cancer including testicular cancer or penile cancer, cervical cancer, cervical cancer, gynecological cancer including choriocarcinoma or ovarian cancer, hematologic cancer including acute or chronic leukemia, malignant lymphoma or multiple myeloma, bone tumor or soft tumor Bone or soft tumors, and pediatric cancer including pediatric leukemia or pediatric solid tumors. In addition, the term "cancer metastasis or metastatic cancer" refers to a metastasis of a primary or recurring cancer that occurs at a specific site to another site.

Since the NANOG protein of the present invention exhibits not only anticancer immunity but also anticancer agent and radiation resistance, and is highly expressed in high metastatic cancer cells, the relapse of anticancer immunity cancer is administered by administering an inhibitor that inhibits the expression or activity of the gene or protein of the protein. And metastasis as well as recurrence, resistant cancer or cancer metastasis can be used for therapeutic purposes.

The prevention or treatment of the recurrence and metastasis of anticancer immune cancer of the present invention includes the prevention, inhibition or alleviation of the recurrence and metastasis of anticancer immune cancer.

As the NANOG gene inhibitor (expression inhibitor) of the present invention, siRNA, antisense, or shRNAi that specifically binds to the mRNA of the NANOG gene and inhibits expression may be mentioned. Since the NANOG gene exhibits anticancer immunity resistance, anticancer agent and radiation resistance, and is a gene related to cancer metastasis, it is possible to prevent or reduce recurrence and metastasis of anticancer immune cancer by inhibiting NANOG gene expression by siRNA, antisense, or shRNAi. Can be.

As used herein, the term "siRNA" refers to a double-stranded RNA that induces RNA interference through cleavage of the mRNA of a target gene. The term " siRNA " It consists of the RNA strand of the sequence.

The siRNA may include a form expressed by inserting the siRNA itself or a base sequence encoding the siRNA synthesized in vitro into the expression vector.

Said siRNA is preferably SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 and 41 It may have one or more sequences selected from the group consisting of.

More specifically, the siRNA sequence set forth in SEQ ID NO: 1 has 830-852 nt of NANOG (GenBank Accession No. NM_028016) of mouse as the target site.

The siRNA sequence set forth in SEQ ID NO: 3 has 865-887 nt of NANOG of mouse as the target site.

The siRNA sequence set forth in SEQ ID NO: 5 has 92-114 nt of NANOG of the mouse at the target site.

The siRNA sequence set forth in SEQ ID NO: 7 has 327-349 nt of NANOG of the mouse at the target site.

The siRNA sequence set forth in SEQ ID NO: 9 has 529-551 nt of NANOG of mouse as the target site.

The siRNA sequence set forth in SEQ ID NO: 11 has 165-187 nt of human NANOG (GenBank Accession No. NM_024865) as a target site.

The siRNA sequence set forth in SEQ ID NO: 13 has 857-879 nt of human NANOG at the target site.

The siRNA sequence set forth in SEQ ID NO: 15 has 810-832 nt of human NANOG at the target site.

The siRNA sequence set forth in SEQ ID NO: 17 has 313-335 nt of human NANOG at the target site.

The siRNA sequence set forth in SEQ ID NO: 19 has 811-833 nt of human NANOG at the target site.

The siRNA sequence set forth in SEQ ID NO: 21 has 1007-1029 nt of human AKT1 (GenBank Accession No. NM_005163) and 1010-1032 nt of AKT2 (GenBank Accession No. NM_001626) as target sites.

The siRNA sequence set forth in SEQ ID NO: 23 has 96-118 nt of human AKT1 at the target site.

The siRNA sequence set forth in SEQ ID NO: 25 has 853-875 nt of human AKT1 at the target site.

The siRNA sequence set forth in SEQ ID NO: 27 has 72-94 nt of human AKT1 at the target site.

The siRNA sequence set forth in SEQ ID NO: 29 has 527-549 nt of human AKT1 at the target site.

The siRNA sequence set forth in SEQ ID NO: 31 has 429-451 nt of human AKT2 at the target site.

The siRNA sequence set forth in SEQ ID NO: 33 has 96-118 nt of human AKT2 at the target site.

The siRNA sequence set forth in SEQ ID NO: 35 has 1218-1240 nt of human AKT2 at the target site.

The siRNA sequence set forth in SEQ ID NO: 37 has 858-880 nt of human AKT2 at the target site.

The siRNA sequence set forth in SEQ ID NO: 39 has 104-126 nt of human TCL1 (GenBank Accession No. NM_021966) at the target site.

The siRNA sequence set forth in SEQ ID NO: 41 has 275-297 nt of human TCL1 at the target site.

In addition, the antisense has a sequence complementary to all or part of the mRNA sequence transcribed from the NANOG, AKT1, AKT2, TCL1 gene or fragments thereof, and may bind to the mRNA to inhibit the expression of the cancer metastasis gene or fragment.

In addition, the shRNAi (short hairpin RNAi) can be used according to a conventional method by targeting the shRNAi common sequence region on human or mouse.

The inhibitor of the NANOG protein may be a substance that inhibits the expression of the proteins or a substance that inhibits the function. Specifically, the inhibitor may be a monoclonal antibody or a polyclonal antibody of the NANOG protein.

In addition, the composition for the prevention or treatment of recurrence and metastasis of the anticancer immune cancer of the present invention containing the mRNA or protein inhibitor of the NANOG gene as an active ingredient may further comprise one or more anticancer agents.

Inhibitors or antibodies of NANOG proteins can be used with chemotherapeutic agents well known to those skilled in the art, for example cyclophosphamide, aziridine, alkylalconsulfonates, nitrosoureas, dacarbazine, carboxes Alkylating agents such as boplatin, cisplatin and the like, antibiotics such as mitomycin C, anthracycline, doxorubicin (adriamycin), antimetabolitic agents such as methotrexate, 5-fluorouracil, cytarabine, vinca alkaloids Plant-derived drugs such as hormones and the like.

In addition, the composition for the prevention or treatment of recurrence and metastasis of the anticancer immune cancer of the present invention containing the mRNA or protein inhibitor of the NANOG gene as an active ingredient is a pharmaceutically acceptable carrier in addition to the active ingredient described above for administration It may be preferably formulated into a pharmaceutical composition containing one or more.

Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added.

In addition, the composition for the prevention or treatment of recurrence and metastasis of the anticancer immunity cancer of the present invention containing the mRNA or protein inhibitor of the NANOG gene as an active ingredient using a pharmaceutically acceptable and physiologically acceptable adjuvant in addition to the active ingredient The auxiliary agent may be a solubilizer such as an excipient, a disintegrating agent, a sweetening agent, a binder, a coating agent, an expanding agent, a lubricant, a lubricant, or a flavoring agent.

In addition, the composition for the prevention or treatment of recurrence and metastasis of the anticancer immunity cancer of the present invention is added to the addition of diluents, dispersants, surfactants, binders and lubricants, injectable formulations, pills, capsules such as aqueous solutions, suspensions, emulsions, etc. It can be formulated into granules or tablets. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In addition, the composition for preventing or treating the recurrence and metastasis of the anticancer immune cancer of the present invention is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, nasal, inhalation, topical, rectal Administration can be in conventional manner via the oral, intraocular or intradermal route.

The dose of the composition for preventing or treating recurrence and metastasis of the anticancer immune cancer is meant an amount required to achieve an effect of suppressing the recurrence and metastasis of the anticancer cancer resistant cancer. Thus, the type of disease, the severity of the disease, the type and amount of the active and other ingredients contained in the composition, the type of formulation and the age, weight, general health, sex and diet, sex and diet, time of administration, route of administration and composition of the patient. It can be adjusted according to various factors including the rate of secretion, the duration of treatment, and the drug used concurrently. In adults, when the inhibitor of NANOG gene is administered once or several times per day, 0.01 ng / kg to 10 mg / kg for siRNA, 0.1 ng / kg to 10 mg / kg for compound, and monoclonal antibody It is preferable to administer at a dose of 0.1ng / kg-10mg / kg.

The invention also relates to the recurrence of anticancer immunity cancers comprising immune cells containing epitopes of at least one protein selected from the group consisting of NANOG, TCL1 (T cell lecukemia / Lymphoma 1), AKT and Myeloid cell leukemia sequence 1 (MCL1). And to compositions for preventing or treating metastasis.

The composition for the treatment of recurrence and metastasis of anticancer immunity cancer of the present invention comprises immune cells containing an epitope of NANOG protein and enhances immune response in vivo, or induces immune response by inducing an immune response. It may be a vaccine composition capable of preventing or alleviating the recurrence and metastasis of sexual cancer.

The immune cell comprising the epitope of the NANOG protein may be a sensitized epitope according to a conventional method, or transformed into a recombinant vector expressing the epitope through a transformation method.

The present invention is for diagnosing the recurrence and metastasis of cancer-resistant cancer comprising a probe for detecting one or more expression selected from the group consisting of NANOG, TCL1 (T cell lecukemia / Lymphoma 1), AKT and MCL1 (Myeloid cell leukemia sequence 1) It relates to a composition.

As used herein, the term "probe" is a concept that encompasses antibodies, PCR primers, nucleic acid probes, and the like that can specifically bind to and recognize NANOG genes or proteins.

The composition for diagnosing recurrence and metastasis of anticancer immune cancer of the present invention is for detecting a pathological marker of recurrence and metastasis of anticancer immune cancer in a physiological sample.

NANOG gene is shown to specifically increase the expression inside and outside of cancer cells with recurrence and metastatic cancer, and the tumorigenic ability of the cell increases in proportion to its expression level, NANOG gene in cancer cells or tissues, or By quantitatively analyzing the expression level of the protein encoded therefrom, it is possible to diagnose the recurrence and metastasis of the cancer-resistant cancer.

The NANOG is derived from human or mouse, and known sequences, such as GenBank Accession No. NM_024865, GenBank Accession No. NM_028016 and the like, or may include a variant that can perform the same function even if the deletion, substitution, addition of a base or amino acid.

The composition for diagnosing recurrence and metastasis of anticancer immune cancer of the present invention may include a monoclonal antibody or a polyclonal antibody that specifically binds to an epitope of NANOG protein with the probe.

Diagnosis of recurrence and metastasis of cancer-resistant cancers using the composition for diagnosing recurrence and metastasis of cancer-resistant cancers of the present invention comprises the steps of: obtaining a protein sample expressed from a mammalian tissue or cell sample; And identifying and quantifying NANOG protein in the obtained sample.

Alternatively, the diagnosis of recurrence and metastasis of the anticancer immunity cancer may include obtaining a protein sample expressed from a mammalian tissue or cell sample; And reacting the obtained sample with a monoclonal antibody of NANOG protein to confirm and quantify the expression of NANOG protein.

The diagnostic method includes ELISA (Enzyme Linked Immunosorbent assay) on a substance (such as protein extracts or plasma of cancer cells or tissues) discharged from a living body of a diagnostic composition for recurrence and metastasis of an anticancer immune cancer containing a monoclonal antibody of NANOG protein. Quantitative analysis by color reaction using the method or by the use of NANOG protein specific immunostaining (histoimmunostaining) in cancer tissues or cells discharged from the living body can diagnose the recurrence and metastasis of the anti-cancer immune resistant cancer. Alternatively, it is possible to diagnose the recurrence and metastasis of anticancer immune cancer by comparing the amount of NANOG protein expression at the protein level with the normal control group using Western blot analysis on cancer tissues or cells discharged from the living body.

Alternatively, the diagnosis of recurrence and metastasis of the anticancer immunity cancer may include attaching a monoclonal antibody against NANOG protein to a gel-like support to prepare an immunoaffinity column; Quantifying NANOG protein in a material (eg, protein extract or plasma of cancer cells or tissues) released from the living body using the immunoaffinity column of the above step by HPLC method; And diagnosing the degree of recurrence and metastasis of anticancer resistant cancer by comparing and analyzing the quantitative results.

The monoclonal antibody of the NANOG protein may be produced and used through a monoclonal antibody production method common in the art, and may be commercially available.

Monoclonal antibodies of the NANOG protein are generally developed using a secondary antibody conjugated with an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) and a substrate thereof. It can also be quantitatively analyzed by reacting or quantitatively using the conjugate of AP or HRP enzyme or the like directly to NANOG protein monoclonal antibody.

In addition, a polyclonal antibody that recognizes NANOG protein may be used in place of the NANOG protein monoclonal antibody, which may be manufactured and used through a conventional antiserum manufacturing method in the art.

In addition, the probe may include a PCR primer or nucleic acid probe that specifically binds to mRNA of NANOG.

RT-PCR or quantitative RT-PCR using primers for the NANOG gene may be performed to compare the expression level of NANOG gene with the normal group to diagnose recurrence and metastasis of cancer-resistant cancer.

In addition, Northern blot analysis and the like using a nucleic acid probe for the NANOG gene may be performed to compare the expression level of the NANOG gene with a normal group to diagnose recurrence and metastasis of anticancer immune resistant cancer.

The PCR primer or nucleic acid probe is not particularly limited as long as it is a nucleotide capable of specifically binding to the mRNA of the NANOG gene.

Reaction and anti-cancer immunity-resistant cancer composition according to the present invention comprising a primer or nucleic acid probe for detecting the mRNA of the NANOG gene and a reaction (RT-PCR or quantitative) with a substance (RNA extracted from cancer cells or tissues) discharged from the living body RT-PCR or Northern blotting) can be compared with the normal group to carry out the diagnosis of recurrence and metastasis of anticancer immune cancer.

Diagnosis of recurrence and metastasis of the anticancer immunity cancer may include obtaining an RNA sample expressed from a mammalian tissue or cell sample; And reacting the obtained sample with a PCR primer or probe specifically binding to the NANOG gene to confirm and quantify the expression of the gene.

The present invention also relates to a composition for screening a therapeutic agent for relapse and metastasis of anti-cancer immunity cancer comprising at least one gene selected from the group consisting of NANOG, TCL1 (T cell lecukemia / Lymphoma 1), AKT and MCL1 (Myeloid cell leukemia sequence 1). It is about.

NANOG, TCL1 (T cell lecukemia / Lymphoma 1), AKT and MCL1 (Myeloid cell leukemia sequence 1) genes included in the composition of the present invention are the base or the fragments, or bases including polymorphisms derived from human or mouse Sequences and the like can be used. More specifically, the base sequence may be one or more selected from among nucleotide sequences including polymorphism of the entire nucleotide sequence, and included in a prokaryotic or eukaryotic expression vector system in the form of a therapeutic agent for relapse and metastasis of anticancer immune cancer. do. Although not limited thereto, the composition preferably includes a TC-1 / NANOG or CaskiDb / NANOG cell line comprising the plasmid expressing the NANOG. The cell line can be used as a positive control for NANOG / TCL-1 / AKT / MCL-1 anticancer immunity molecular axis screening.

The present invention also relates to a composition for screening a therapeutic agent for recurrence and metastasis of anticancer immunity cancer comprising at least one protein selected from the group consisting of NANOG, TCL1 (T cell lecukemia / Lymphoma 1), AKT and MCL1 (Myeloid cell leukemia sequence 1). It is about.

NANOG, TCL1 (T cell lecukemia / Lymphoma 1), AKT and MCL1 (Myeloid cell leukemia sequence 1) proteins included in the composition of the present invention are amino acids comprising the entire amino acid sequence or fragment thereof derived from human or mouse, or polymorphism May be a sequence. It may be at least one selected from NANOG polypeptide fragments exhibiting the same physiological activity as NANOG protein.

Since NANOG has an effect of promoting recurrence and metastasis of anticancer immune cancer, the composition for screening a therapeutic agent for recurrence and metastasis of anticancer immune cancer of the present invention is a substance that inhibits transcription and translation from NANOG gene sequence to mRNA and protein. Alternatively, a substance that inhibits the activity of NANOG protein can be screened and selected as a therapeutic agent for recurrence and metastasis of anticancer immune cancer.

In addition, the present invention provides a relapse of anti-cancer immunity cancer in a composition comprising a cell line expressing at least one selected from the group consisting of NANOG, TCL1 (T cell lecukemia / Lymphoma 1), AKT and MCL1 (Myeloid cell leukemia sequence 1). Treating the metastatic therapeutic candidate; And measuring the expression level of NANOG, TCL1 (T cell lecukemia / Lymphoma 1), AKT or MCL1 (Myeloid cell leukemia sequence 1) in the candidate cell treated cell line (experimental group). A method for screening relapse and metastases therapies.

The expression level measurement may be performed by a method of measuring the amount of transcript, a method of measuring the amount of protein or a method of measuring the amount of phosphorylated protein. In the case of AKT, the degree of activation can be measured by measuring the amount of phosphorylated protein. For example, the method of measuring the amount of the transcript is performed by reverse transcriptase (RT) -PCR, and the method of measuring the amount of protein and the amount of phosphorylated protein is measured by performing Western blotting. However, the present invention is not limited thereto and may be performed using a method known to those skilled in the art.

More specifically, in the screening method of the present invention, confirming the reaction between the composition containing the NANOG gene and the candidate material is a conventional method used to confirm the reaction between DNA-DNA, DNA-RNA, DNA-protein, and DNA-compound. Methods can be used. For example, in the living body outside (in a method for measuring the expression rate of the gene through quantitative PCR, quantitative real-time PCR, or the like, or a method for measuring the expression level of the gene by reacting mammalian cells with a candidate substance, A method of introducing a reporter gene into a cell, reacting with a candidate substance, and measuring the expression ratio of a reporter protein can be used.

In this case, the composition of the present invention may include distilled water or a buffer solution, in addition to the NANOG gene, to keep the structure of the nucleic acid stable.

Although not limited thereto, the cell line is a TC-1 / NANOG or CaskiDb / NANOG cell line comprising a plasmid expressing the NANOG.

In the screening method of the present invention, confirmation of the reaction between the composition comprising the NANOG protein and the candidate may use conventional methods used to confirm the reaction between the protein-protein and the protein-compound.

For example, a method for measuring activity after reacting a NANOG gene, or a protein thereof, with a candidate, a yeast two-hybrid, and a phage-displayed peptide clone that binds to a NANOG protein. High throughput screening, drug hit HTS, cell-based screening, or DNA arrays using natural products and chemical libraries. The screening method used etc. can be used.

In this case, the composition of the present invention may include a buffer or a reaction solution that stably maintains the structure or physiological activity of the protein, in addition to the protein expressed from the NANOG gene. In addition, the composition of the present invention may be administered in vivo ( in For the vivo experiment, cells expressing the protein, or a cell containing a plasmid expressing the protein under a promoter capable of regulating the transcription rate, and the like may be included.

In the screening method of the present invention, the candidate material is estimated to have the potential as a therapeutic agent for the recurrence and metastasis of anticancer immune cancer according to a conventional selection method or randomly selected individual nucleic acids, proteins, other extracts or natural products, compounds, etc. This can be

Candidates exhibiting activity that enhances gene expression or enhances protein function obtained through the screening method of the present invention, and vice versa, candidates exhibiting activity that inhibits gene expression or inhibits protein function, In this case, the drug may be a candidate for recurrence, metastasis, or cancer metastasis by developing an inhibitor for the candidate.

Such candidate drugs for the recurrence and metastasis of anti-cancer immunity cancer will act as the leading compound in the course of development of the next recurrence, resistant cancer or cancer metastasis treatment, and the leading material is the NANOG gene or the protein expressed therefrom. By modifying and optimizing its structure to show its inhibitory effect, new therapeutic agents can be developed for recurrent cancer, resistant cancer or cancer.

The material thus obtained has a partial or complete activity inhibitory effect on the NANOG gene or the protein expressed therefrom. Thus, by repressing NANOG gene expression or decreasing the function of the protein expressed therefrom, recurrence and metastasis of anticancer immune cancers, It can suppress the diseases caused.

(2001)) and Frederick et al . (Frederick et al ., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, M. Ausubel et al ., Current protocols in molecular biology volume 1, 2, 3, John Wiley & (1994)). ≪ / RTI >

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited to these embodiments in accordance with the gist of the present invention.

< Example  1> human NANOG  Expression vector production

Denatured step using a pSin-EF2-Nanog-Puro vector (Addgene plasmid 16578) as a template, XhoI-NANOG primer (5'-GCTCGAGATGAGTGTGTGGATCCAGCTTG-3 '), NANOG-EcoRI (5'-CGAATTCTCACACGTCTTGAGGTTG -3') primer 35 cycles were carried out (95 ° C., 30 sec), annealing step (56 ° C., 30 sec) and extension step (72 ° C., 40 sec) as one cycle, and amplified by PCR.

Retroviral vector expressing NANOG by treating XhoI and Eco RI (NEB, USA) to pMSCV vector (clontech, USA) and NANOG gene fragment amplified above and then ligating with ligation (Bionia) Was prepared. The vector was named pMSCV NANOG.

< Example  2> human NANOG Expressing CaSkiDb  Cell line manufacturing

Lipofectamine 2000 (lipofectamin 2000, Invitrogen, MD, USA), a vector prepared in Example 1, was mixed at a volume ratio of 1: 2 and added to a phoenix cell (clontech), a retrovirus packaging cell line. After the time treatment, the transformation reaction was stopped by adding a culture medium containing serum. After 24 hours of incubation, the transformed Phoenix cells were selectively cultured with a culture solution containing puromycin (Invitrogen, MD, USA). The selected cells were cultured for 3 to 4 days, and the virus obtained from the culture was centrifuged at 12000 rpm for 30 minutes using amicon ultra-15 (millipore), concentrated to 500 μl, and 4 μg of PEI (Polyethyleneimine; Db of MHC class I gene in mice a mixture mixed with human cervical cancer cell line CaSki cell line is transduced to CaSki Db (the Journal of experimental medicine 185: 453-459, 1997) to produce a CaSki Db / NANOG cell line expressing NANOG.

< Example  3> NANOG Expressing TC - 1 cell line  Produce

Each vector and the liposome preparation Lipofectamine 2000 prepared in Example 1 were mixed at a volume ratio of 1: 2, and after 3 hours of treatment, a culture medium containing serum was added to the retrovirus-packaged cell line to stop the transformation reaction. I was. Then, after 24 hours of incubation, the transformed phoenix cells were cultured with a culture solution containing puromycin. Incubate the selected cells for 3 to 4 days and centrifuge the virus obtained from the culture solution at 12000 rpm for 30 minutes using amicon ultra-15 (millipore). TC-1 (ATCC), a mouse lung cancer cell line, was transduced to prepare a TC-1 / NANOG cell line expressing NANOG.

< Example  4> In human cancer cell lines NANOG  Confirmation of protein expression

Human kidney cell line HEK293, cervical cancer cell line CaSki, CUMC6, breast cancer cell line MCF7, MDA321, MDA435, lung cancer cell line H1299, liver cancer cell line HepG2, ovarian cancer cell line OVCAR3, SKOV3, A2780, colorectal cancer cell line HT29, SNUC4, SW620, HCT116 cell line After placing in a buffer (Elpis, Korea) for 30 minutes on ice, centrifuged at 13000 rpm for 30 minutes in a refrigerated centrifuge, only the supernatant was taken and 50 μg of protein was loaded on an SDS-PAGE gel. After transferring the gel loaded in PVDF and blocking with 5% BSA for 1 hour, NANOG antibody (Millipore, USA) was diluted 1: 1000 and incubated. After HRP-attached secondary antibody reaction and washing process, the band of the protein was confirmed using ECL solution.

As shown in FIG. 1, NANOG protein was expressed in all human carcinomas.

< Example  5> In cervical cancer tissue NANOG  Confirmation of expression

The cervical cancer tissue slides were subjected to deparinization using xylene and dehydration using alcohol, and then the slides were immersed in a citrate buffer for 10 minutes in a microwave oven and cooled at room temperature for 1 hour. After washing with distilled water and reacting with 5% goat serum at room temperature for 1 hour, Nanog, Tcl1, and pAkt antibodies were diluted 1:50, 1:20, and 1:50, respectively, and applied to clinical samples. After HRP-attached secondary antibody was developed using a DAB kit.

As shown in FIG. 2, it was confirmed that the expression level of Nanog / Tcl1 / pAkt was increased as the stage of cervical cancer progressed in the tissue (A and B). In the long-term follow-up, the survival rate of patients with cancers with high Nanog / Tcl1 / pAkt expression was significantly lower (C).

< Example  6> Anti-cancer immunity

To investigate the anticancer immunity of the NANOG gene of the present invention, anticancer immunity resistance experiments were performed using the 293Db / NANOG, CaSki / Db NANOG, and TC-1 / NANOG cell lines prepared in Examples 2 and 3. .

First, CFSE (Carboxyfluorescein succinimidyl ester) was used to label TC-1 / no isnert and TC-1 / NANOG cell lines, and then mixed E7 specific CTL (cytotoxic T-lymphocyte) at a 1: 1 ratio. Active caspase-3 in TC-1 / no insert, TC-1 / NANOG cell line was observed through FACS.

After labeling on CaSki / Db no insert and CaSki / Db NANOG cell lines using CFSE (Carboxyfluorescein succinimidyl ester), 1ug / ml of E7 peptide (amino acids No. 49-57, RAHYNIVTF) was stimulated for 1 hour and then E7 specific FACS of CaSki / Db no insert and CaSki / Db NANOG cell lines with CFSE markers after FAC 1: 1 ratio of cytotoxic T-lymphocyte (CTL) It was.

As shown in FIG. 7, FIG. 12, and FIG. 13, the cell line overexpressing NANOG was found to be resistant to T cell attack.

Each siRNA sequence and target site are shown in Tables 1, 2, 3, 4 and 5. 6 and 11 show the mouse NANOG target site 830-852 (SEQ ID NO: 1) and the human NANOG target site 165-TC in mouse anticancer immunity cells TC-1 P3 and human anticancer immune cells CaSki / Db P3 It shows the expression of NANOG by the treatment of 187 (SEQ ID NO: 11).

Mouse NANOG-specific siRNA Target site siRNA sequence SEQ ID NO: 830-852 sense 5'-CCACAAGCCUUGGAAUUAU UU -3 ' One Antisense 5'-UU GGUGUUCGGAACCUUAAUA-3 ' 2 865-887 sense 5'-GACUCCACCAGGUGAAAUA UU-3 ' 3 Antisense 5'-UU CUGAGGUGGUCCACUUUAU -3 ' 4 92-114 sense 5'-CCCGAGAACUAUUCUUGCU UU-3 ' 5 Antisense 5'-UU GGGCUCUUGAUAAGAACGA-3 ' 6 327-349 sense 5'-CACUCAAGGACAGGUUUCA UU-3 ' 7 Antisense 5'-UU GUGAGUUCCUGUCCAAAGU-3 ' 8 529-551 sense 5'-CAGCAUCCAUUGCAGCUAU UU-3 ' 9 Antisense 5'-UU GUCGUAGGUAACGUCGAUA-3 ' 10

Human NANOG-specific siRNA Target site siRNA sequence SEQ ID NO: 165-187 sense 5'-CCUCCAUGGAUCUGCUUAU UU-3 ' 11 Antisense 5'-UU GGAGGUACCUAGACGAAUA -3 ' 12 857-879 sense 5'-CCACAAACCAUGGAUUUAU UU -3 ' 13 Antisense 5'-UU GGUGUUUGGUACCUAAAUA-3 ' 14 810-832 sense 5'-GGGAAGGCCUUAAUGUAAU UU-3 ' 15 Antisense 5'-UU CCCUUCCGGAAUUACAUUA-3 ' 16 313-335 sense 5'-CCAGCUGUGUGUACUCAAU UU-3 ' 17 Antisense 5'-UU GGUCGACACACAUGAGUUA-3 ' 18 811-833 sense 5'-GGAAGGCCUUAAUGUAAUA UU-3 ' 19 Antisense 5'-UU CCUUCCGGAAUUACAUUAU-3 ' 20

Human AKT1-specific siRNA Target site siRNA sequence SEQ ID NO: 1007-1029 sense 5'-GUGGUCAUGUACGAGAUGA UU-3 ' 21 Antisense 5'-UU CACCAGUACAUGCUCUACU-3 ' 22 96-118 sense 5'-GCACCUUCAUUGGCUACAA UU-3 ' 23 Antisense 5'-UU CGUGGAAGUAACCGAUGUU-3 ' 24 853-875 sense 5'-CGGGCACAUUAAGAUCACA UU-3 ' 25 Antisense 5'-UU GCCCGUGUAAUUCUAGUGU-3 ' 26 72-94 sense 5'-GCUACUUCCUCCUCAAGAA UU-3 ' 27 Antisense 5'-UU CGAUGAAGGAGGAGUUCUU-3 ' 28 527-549 sense 5'-GCCAUGAAGAUCCUCAAGA UU-3 ' 29 Antisense 5'-UU CGGUACUUCUAGGAGUUCU-3 ' 30

Human AKT2-specific siRNA Target site siRNA sequence SEQ ID NO: 1010-1032 sense 5'-GUGGUCAUGUACGAGAUGA UU-3 ' 21 Antisense 5'-UU CACCAGUACAUGCUCUACU-3 ' 22 429-451 sense 5'-GGGCUAAAGUGACCAUGAA UU-3 ' 31 Antisense 5'-UU CCCGAUUUCACUGGUACUU-3 ' 32 96-118 sense 5'-GCUCCUUCAUUGGGUACAA UU-3 ' 33 Antisense 5'-UU CGAGGAAGUAACCCAUGUU-3 ' 34 1218-1240 sense 5'-GGUUCUUCCUCAGCAUCAA UU-3 ' 35 Antisense 5'-UU CCAAGAAGGAGUCGUAGUU-3 ' 36 858-880 sense 5'-GCCACAUCAAGAUCACUGA UU-3 ' 37 Antisense 5'-UU CGGUGUAGUUCUAGUGACU-3 ' 38

Human TCL1-specific siRNA Target site siRNA sequence SEQ ID NO: 104-126 sense 5'-CCCUUAACCAUCGAGAUAA UU-3 ' 39 Antisense 5'-UU GGGAAUUGGUAGCUCUAUU-3 ' 40 275-297 sense 5'-CGCUUAGUGUACCACAUCA UU-3 ' 41 Antisense 5'-UU GCGAAUCACAUGGUGUAGU-3 ' 42

As shown in FIG. 11, the expression of NANOG protein was inhibited upon siRNA treatment in CaSki / Db P3 cells, which is a human anticancer immunity resistant cell line.

In addition, as shown in Figure 9, in the case of human anti-cancer immune cell line expressing NANOG, it showed resistance to T cell attack.

However, as a result of investigating the effect on the resistance effect when the expression of NANOG was reduced by treating siRNA, human anticancer immunity-resistant cell lines treated with siRNA of the control group, green fluorescent protein (GFP), had little effect on T cell attack. However, when treated with siRNA of NANOG, it was confirmed that the resistance to T cell attack decreases.

FIG. 13 shows that neutralization of T cells by the attack of the human AKT1 target site 1007-1029 (SEQ ID NO: 21) and the target site of human AKT2 1010-1032 (SEQ ID NO: 21) in the CaSki / Db NANOG cell line was neutralized. (SEQ ID NO: 21 simultaneously targets human AKT1 and AKT2).

Figure 14 shows that treatment of the human TCL1 target site 104-126 (SEQ ID NO: 39) in the CaSki / Db NANOG cell line reduces the amount of pAKT expression and at the same time neutralizes resistance to T cell attack.

< Example  7> cells Mobility  observe

In order to confirm the nutrition of the NANOG gene of the present invention on cell mobility, TC-1 P3 and CaSki / Db P3 cell lines overexpressing NANOG were used. SiRNA GFP was used as a control, and siRNA NANOG was used to reduce the expression of NANOG in TC-1 P3 and CaSki / Db P3 cells, and 1 x 10 ⁶ cells were laid on a 6-well plate for cell culture. . After wounding at a constant width, wound recovery was observed over 12 and 24 hours.

As shown in FIG. 17 and FIG. 18, it was confirmed that all cell lines overexpressing NANOG migrated in a faster time than the control group not containing NANOG. However, when siRNA NANOG was used to reduce the expression of NANOG in TC-1 P3 and CaSki / Db P3 cells, it was confirmed that the mobility of the cells was reduced.

< Example  8> cells Infiltration ability  observe

In order to confirm the invasion ability of the NANOG gene of the present invention, TC-1 P3 and CaSki / Db P3 cell lines overexpressing NANOG were used. SiRNA GFP was used as a control, and siRNA NANOG was used to reduce NANOG expression in TC-1 P3 and CaSki / Db P3 cells, and then mixed Matrigel with the cell culture medium at a ratio of 1: 3 and 8.0 μm. 50 μl was added to a cell culture membrane with a pore size (Cell culture insert membrane, Falcon, USA) and left to stand for 30 minutes in a 37 ° C. incubator. 500 μl of 10% FBS medium was added to a 24-well plate for cell culture, and a cell culture additive membrane containing hardened matrigel was added thereto. Each 1 × 10 ⁵ cells were mixed with 0.1% FBS medium. After 24 hours, the number of cells penetrating the cell membrane was observed.

As shown in FIG. 17 and FIG. 18, all the cell lines overexpressing NANOG were found to have significantly increased invasive ability compared to the control group without NANOG.

< Example  9> In vitro Tumor formation ability  Confirm( Tumor zone  Formation experiment)

Each 5 x 10 ³ cells were cultured in 1 ml DMEM-F12 medium containing 20 ng / ml EGF (epitdermal growth factor; Invitrogen, USA), 20 ng / ml basic fibroblast growth factor (Invitrogen), and 1X B27. Incubated in 24well-super-low adherence plates (Corning, Lowell, MA) for 2 weeks. At this time, 100ul of fresh medium was added every three days. The formed tumor cells were quantified by counting tumor spheres of 20-50 μm or more by measuring the diameter of the tumor spheres after obtaining a photographic image using an optical microscope.

< Example  10> In vivo Tumor formation ability  Confirm

Each cell was diluted in Opti-MEM I (GIBCO, USA) and injected with 1 x 10 ² , 1 x 10 ³ , 1 x 10 ⁴ , 1 x 10 ⁵ cells into the ventral epidermis of NOD / SCID mice, respectively. do. For each mouse, the presence of tumor was observed and recorded three times a week.

< Example  11> In vivo Anti-cancer immunity  Confirmation of neutralization

HCT116 / SCT E7 cells were injected into the abdominal epidermis of each NOD-SCID mouse group (female, 5 weeks old, n = 5, central experimental animals) by 1 X 10 ⁶ and chitosan nanoparticles loaded with siRNA NANOG on day 10 of injection. Was injected intravenously. At this time, the control group was confirmed by injecting chitosan nanoparticles loaded with siRNA GFP. Chitosan nanoparticles were injected three times at two-day intervals and two days later, E7 specific CD8 + T cells were injected intravenously with IL-2. After 2 days, chitosan nanoparticles 3 injections and E7 T cells were repeated twice. For each mouse, the tumor volume was measured by caliber (Mitutoyo, Japan), and the tumor volume was calculated by the formula (short axis x long axis ² ) / 2.

As shown in Figure 16, chitosan nanoparticles loaded with siRNA NANOG and CD8 + T cells were injected together, the tumor volume was significantly reduced compared to the control group. This demonstrates that the efficacy of anti-cancer immunotherapy is enhanced by nanog-dependent immunity neutralization by nanog targeting.

Although specific portions of the present invention have been described in detail, those skilled in the art will appreciate that these specific embodiments are merely examples of preferred embodiments and that the scope of the present invention is not limited thereto. It is therefore intended that the scope of the present invention be defined by the appended claims and their equivalents.

<110> KOREAN UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> Composition for prevention or treating recurrent or metastatic          cancer having immunoresistance <130> P11-120626-01 <160> 42 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 1 ccacaagccu uggaauuauu u 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 2 uugguguucg gaaccuuaau a 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 3 gacuccacca ggugaaauau u 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 4 uucugaggug guccacuuua u 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 5 cccgagaacu auucuugcuu u 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 6 uugggcucuu gauaagaacg a 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 7 cacucaagga cagguuucau u 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 8 uugugaguuc cuguccaaag u 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 9 cagcauccau ugcagcuauu u 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 10 uugucguagg uaacgucgau a 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 11 ccuccaugga ucugcuuauu u 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 12 uuggagguac cuagacgaau a 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 13 ccacaaacca uggauuuauu u 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 14 uugguguuug guaccuaaau a 21 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 15 gggaaggccu uaauguaauu u 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 16 uucccuuccg gaauuacauu a 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 17 ccagcugugu guacucaauu u 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 18 uuggucgaca cacaugaguu a 21 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 19 ggaaggccuu aauguaauau u 21 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 20 uuccuuccgg aauuacauua u 21 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 21 guggucaugu acgagaugau u 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 22 uucaccagua caugcucuac u 21 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 23 gcaccuucau uggcuacaau u 21 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 24 uucguggaag uaaccgaugu u 21 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 25 cgggcacauu aagaucacau u 21 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 26 uugcccgugu aauucuagug u 21 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 27 gcuacuuccu ccucaagaau u 21 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 28 uucgaugaag gaggaguucu u 21 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 29 gccaugaaga uccucaagau u 21 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 30 uucgguacuu cuaggaguuc u 21 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 31 gggcuaaagu gaccaugaau u 21 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 32 uucccgauuu cacugguacu u 21 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 33 gcuccuucau uggguacaau u 21 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 34 uucgaggaag uaacccaugu u 21 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 35 gguucuuccu cagcaucaau u 21 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 36 uuccaagaag gagucguagu u 21 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 37 gccacaucaa gaucacugau u 21 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 38 uucgguguag uucuagugac u 21 <210> 39 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 39 cccuuaacca ucgagauaau u 21 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 40 uugggaauug guagcucuau u 21 <210> 41 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 41 cgcuuagugu accacaucau u 21 <210> 42 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA <400> 42 uugcgaauca caugguguag u 21

Claims (7)

An inhibitor for one or more genes or proteins selected from the group consisting of NANOG, T cell lecukemia / Lymphoma 1 (TCL1), AKT and Myeloid cell leukemia sequence 1 (MCL1),
The inhibitor is a siRNA, antisense-oligonucleotide, shRNAi, miRNA or a vector comprising one of them, or a composition for preventing or treating recurrence and metastasis of cancer-resistant cancer, characterized in that the antibody. The method of claim 1,
The siRNA is from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 and 41 A composition for preventing or treating relapse and metastasis of at least one selected cancer-resistant cancer. A composition for diagnosing relapse and metastasis of cancer-resistant cancer comprising a probe for detecting one or more expressions selected from the group consisting of NANOG, TCL1 (T cell lecukemia / Lymphoma 1), AKT, and MCL1 (Myeloid cell leukemia sequence 1). A composition for screening a therapeutic agent for recurrence and metastasis of cancer-resistant cancer comprising at least one gene selected from the group consisting of NANOG, T cell lecukemia / Lymphoma 1 (TLC1), AKT and Myeloid cell leukemia sequence 1 (MCL1). According to claim 4, wherein the composition comprises a TC-1 / NANOG or CaskiDb / NANOG cell line comprising the plasmid expressing the NANOG composition for screening therapeutic agents for recurrence and metastasis of anti-cancer immunity cancer. Treating a composition comprising a cell line expressing one selected from the group consisting of NANOG, T cell lecukemia / Lymphoma 1 (TCL1), AKT, and MCL1 (Myeloid cell leukemia sequence 1), and treating candidates for the recurrence and metastasis of anticancer immunity cancer Making; And
Measuring the expression level of ANOG, TCL1 (T cell lecukemia / Lymphoma 1), AKT or MCL1 (Myeloid cell leukemia sequence 1) in the candidate cell line (experimental group); And metastasis screening methods. The method of claim 6, wherein the cell line is a TC-1 / NANOG or CaskiDb / NANOG cell line comprising the plasmid expressing the NANOG.

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