Aptamers specifically binding to cancer stem cells and uses thereof

Abstract

The present invention relates to an aptamer that specifically binds to cancer stem cells. The aptamer according to the present invention specifically binds to cancer stem cells, reduces cell adhesion, cell proliferation, anticancer drug resistance and cell migration that are characteristics of cancer stem cells, and has an excellent anticancer effect. Therefore, the aptamer can be used in the fields of cancer diagnosis, prognosis prediction and treatment in various ways.

Description

Technical Field

[0001] The present invention relates to aptamers that specifically bind to cancer stem cells. Background Art

[0002] Cancer stem cells are also present in cancer tissues, which can maintain and regenerate cancer tissues like normal organs. It is reported that cancer stem cells form spheres in in vitro suspension culture, and sphere-forming cancer stem cells have stronger tumor initiation ability than the original cell line. In addition, since cancer stem cells are involved in the development of cancer, the occurrence, recurrence or metastasis of cancer, and resistance to anticancer drugs by regenerating cancer cells that have been reduced after conventional cancer treatment, it is important to prevent the proliferation of cancer stem cells in cancer treatment.

[0003] Currently, in developed countries, information on cancer stem cells is available in areas such as breast cancer, liver cancer, colorectal cancer, and pancreatic cancer. Therefore, research on cancer stem cells in organs and tissues is needed, which may be different from the research directions of these developed countries.

[0004] In the case of cancer, heterogeneity is very strong even within a single tissue, and during anticancer treatment, cancer tissues express resistance to anticancer agents, so this is considered to be the main reason for the failure of anticancer treatment. Cancer stem cells are known to be the cells that show this heterogeneity characteristic at the highest level and are closely related to resistance to anticancer agents.

[0005] Although it is relatively easy to remove cancerous tissues through general anticancer treatment, due to the characteristics of cancer stem cells contained in tissues or obtained after anticancer treatment, despite the existence of various targeted treatment technologies, cancer cells continue to express drug resistance, which has become a new issue that must be overcome in the treatment of cancer patients. In order to develop anticancer treatment technologies targeting cancer stem cells, it is necessary to develop antibodies or aptamers that can specifically bind to the markers expressed in cancer stem cells.

[0006] On the other hand, CD166 was first reported as an activated leukocyte adhesion molecule (ALCAM), and its expression has been found in thyroid cancer, cervical cancer, lung cancer, and liver cancer. In particular, it has been reported that the expression of CD166 is associated with the metastatic potential of prostate cancer, the invasiveness of bile duct cancer, and the avoidance of apoptosis in breast cancer, and it has recently been reported that CD166 has the potential to serve as a signature marker for cancer stem cells. In addition, it has been reported that CD166 is also expressed together with CD40, CD90, and CD105 in mesenchymal stem cells, which are normal stem cells.

[0007] In addition, aptamers are single-stranded nucleic acid (DNA, RNA or modified nucleic acid) molecules with a stable tertiary structure, which have the characteristics of being able to bind to target molecules with high affinity and specificity. Aptamers are usually composed of 40mer to 120mer and can bind to target molecules with inherent high affinity (usually pM level) and specificity. Aptamers are being actively studied in the field of clinical cancer diagnosis and cancer treatment for the following reasons.

[0008] 1) No immune rejection reaction will occur in the body.

[0009] 2) Due to its very small size, its in vivo penetration and binding are efficient.

[0010] 3) Since aptamers are produced by chemical synthesis, they can be produced in a short time and at low cost.

[0011] 4) Aptamers have higher stability than antibodies. Aptamers can be stored or transported at room temperature and have high temperature stability. Therefore, aptamers are particularly useful for diagnostics that require long-term and repeated use.

[0012] 5) Aptamers are nucleic acid molecules that can be modified in a variety of ways, thus making them useful in a variety of studies and experiments.

[0013] Because of the advantages of aptamers, aptamers can be applied in many ways in the field of clinical cancer diagnosis and cancer treatment. The first aptamer-related drug developed was approved by the U.S. Food and Drug Administration in 2005. Summary of the invention

[0014] Technical issues

[0015] Therefore, the present inventors developed an aptamer that specifically binds to CD166 expressed in cancer stem cells, and confirmed that the aptamer reduces the characteristics of cancer stem cells and has no effect on the proliferation and function of mesenchymal stem cells, which are normal stem cells expressing CD166. Based on the above results, the present inventors completed the present invention.

[0016] Therefore, an object of the present invention is to provide an aptamer that specifically binds to cancer stem cells, and a composition comprising the same, the aptamer comprising at least one nucleic acid sequence selected from the group consisting of nucleic acid sequences represented by SEQ ID NOs: 1 to 6 or a fragment thereof.

[0017] Another object of the present invention is to provide a method for diagnosing cancer, which comprises reacting a biological sample with an aptamer that specifically binds to cancer stem cells.

[0018] Another object of the present invention is to provide a method for treating cancer, which comprises administering to a subject an aptamer that specifically binds to cancer stem cells.

[0019] Problem Solution

[0020] To achieve the above object, the present invention provides an aptamer that specifically binds to cancer stem cells, the aptamer comprising at least one nucleic acid sequence selected from the group consisting of nucleic acid sequences represented by SEQ ID NOs: 1 to 6 or a fragment thereof.

[0021] Furthermore, the present invention provides a composition for diagnosing cancer, the composition comprising an aptamer that specifically binds to cancer stem cells.

[0022] Furthermore, the present invention provides a kit for diagnosing cancer, the kit comprising a composition for diagnosing cancer.

[0023] Furthermore, the present invention provides a method for diagnosing cancer, the method comprising reacting a biological sample with an aptamer that specifically binds to cancer stem cells.

[0024] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising an aptamer that specifically binds to cancer stem cells.

[0025] Furthermore, the present invention provides an anticancer adjuvant composition comprising an aptamer that specifically binds to cancer stem cells.

[0026] Furthermore, the present invention provides a food composition for preventing or alleviating cancer, the food composition comprising an aptamer that specifically binds to cancer stem cells.

[0027] Furthermore, the present invention provides a composition for cancer-specific drug delivery, the composition comprising an aptamer that specifically binds to cancer stem cells.

[0028] Furthermore, the present invention provides a method for treating cancer, the method comprising administering to a subject an aptamer that specifically binds to cancer stem cells.

[0029] Effects of the Invention

[0030] The aptamer according to the present invention specifically binds to cancer stem cells, reduces cell adhesion, cell proliferation, drug resistance and cell migration (all of which are characteristics of cancer stem cells), and thus has excellent anti-cancer effects. Therefore, the aptamer can be used in cancer diagnosis, prognosis prediction and treatment fields in a variety of ways. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The results of RT-PCR analysis confirming CD166 expression in A2780 ovarian cancer cells (A2780-AD) cultured by attaching A2780 ovarian cancer cells to a cell culture dish as a general cell culture method, and in the A2780-SP ovarian cancer stem cell line cultured by three-dimensionally culturing cells to selectively amplify cancer stem cells.

[0032] Figure 2 The graph shows the results of confirming the expression of CD166 in ovarian cancer stem cell line A2780-SP cells by flow cytometry.

[0033] Figure 3 The figures represent the results obtained by confirming CD166 expression in CD166-expressing cells and non-expressing cells isolated from the ovarian cancer cell line A2780.

[0034] Figure 4 The figures represent the results obtained by confirming the expression of cancer stem cell-related genes in CD166-expressing cells and non-expressing cells isolated from the ovarian cancer cell line A2780.

[0035] Figure 5 The figures represent the results obtained by confirming the cell proliferation ability of CD166-expressing cells and non-expressing cells isolated from the ovarian cancer cell line A2780.

[0036] Figure 6 The figures represent the results obtained by confirming the ability to form spherical cancer spheres by cancer cell proliferation during 3D cell culture of CD166-expressing cells and non-expressing cells isolated from the ovarian cancer cell line A2780.

[0037] Figure 7 Represents the results obtained by confirming the drug sensitivity of CD166-expressing and non-expressing cells isolated from the ovarian cancer cell line A2780.

[0038] Figure 8 Represents the results obtained by confirming the cell migration of CD166-expressing cells and non-expressing cells isolated from the ovarian cancer cell line A2780.

[0039] Fig. 9 The figures show the results obtained by confirming the expression of cancer stem cell-related genes in the ovarian cancer cell line A2780 in which the expression of CD166 was suppressed.

[0040] Figures 10 to 13 The cell proliferation ability of ovarian cancer cell line A2780 was confirmed by confirming that the expression of CD166 was inhibited ( Fig.10 ), drug resistance ( Fig.11 ), cancer sphere formation ability ( Fig.12 ) and cell migration ( Fig.13 ) and the results obtained.

[0041] Fig.14 The results obtained by confirming the inhibitory activity of the focal adhesion signaling pathway in cells in which CD166 expression was suppressed are shown.

[0042] Figures 15 to 17 represents the results obtained by confirming the influence of CD166 expression on tumor formation, more specifically, the formed tumor ( Fig.15 ), tumor size ( Fig.16 ) and tumor weight ( Fig.17 )’s observation results.

[0043] Fig.18 and 19 The results show that the CD166 aptamer binds to the ovarian cancer cell line A2780-AD ( Fig.18 ) or the ovarian cancer stem cell line A2780-SP ( Fig.19 ) is the result obtained by the affinity between them.

[0044] Fig. 20 Shows the results obtained by comparing the affinity of the CD166 aptamer and the antibody to the ovarian cancer stem cell line A2780-SP.

[0045] Figures 21 to 24 The cell adhesion ability of CD166 aptamer to ovarian cancer stem cells was confirmed ( Fig.21 ), cell proliferation ability ( Fig. 22 ), drug resistance ( Fig.23 ) and cell migration ( Fig.24 )’s influence.

[0046] Fig.25 Represents the results obtained by confirming the effect of CD166 aptamer on focal adhesion signaling pathway in ovarian cancer stem cells.

[0047] Figures 26 to 28 represents the results obtained by confirming the effect of CD166 aptamer on tumor formation of ovarian cancer stem cells, more specifically, the formed tumors ( Fig.26 ), tumor size ( Fig. 27 ) and tumor weight ( Fig.28 )’s observation results.

[0048] Fig.29 The figure shows the results of confirming the expression of CD166 in patient-derived ovarian cancer stem cells by flow cytometry.

[0049] Fig.30 The present invention represents the results obtained by confirming the effect of CD166 expression inhibition on the focal adhesion signaling pathway in patient-derived ovarian cancer stem cells.

[0050] Figures 31 to 35 This study confirmed the cell proliferation capacity of ovarian cancer stem cells derived from patients in which CD166 expression was suppressed ( Fig.31 ), drug resistance ( Fig.32), cancer sphere formation ability ( Fig.33 ), cell migration ( Fig.34 ) and cell adhesion ability ( Fig.35 ) and the results obtained.

[0051] Fig.36 A represents the results obtained by confirming the affinity of the CD166 aptamer for patient-derived ovarian cancer stem cells, Fig.36 B shows the results obtained by confirming the cell adhesion ability of the CD166 aptamer to patient-derived ovarian cancer stem cells.

[0052] Fig.37 The present invention shows the results obtained by confirming the effect of CD166 aptamer on the drug resistance of patient-derived ovarian cancer stem cells.

[0053] Fig.38 The figures represent the results obtained by confirming the effect of CD166 aptamer on cell migration in patient-derived ovarian cancer stem cells.

[0054] Fig.39 The present invention represents the results obtained by confirming the influence of CD166 aptamer on the focal adhesion signaling pathway of patient-derived ovarian cancer stem cells.

[0055] Fig.40 The results obtained by comparing the affinity of the CD166 aptamer and the antibody to mesenchymal stem cells, which are normal stem cells.

[0056] Figure 41 to Figure 43 This indicates that the cell proliferation ability of mesenchymal stem cells, which are normal stem cells, was confirmed by confirming that the CD166 aptamer Fig.41 ), cell migration ( Fig.42 ) and cell adhesion ability ( Fig.43 )’s influence.

[0057] Fig.44 The present invention shows the results obtained by confirming the influence of CD166 aptamer and CD166 expression inhibition on the focal adhesion signaling pathway in mesenchymal stem cells which are normal stem cells. DETAILED DESCRIPTION

[0058] Hereinafter, the present invention will be described in detail.

[0059] According to one embodiment of the present invention, the present invention provides an aptamer that specifically binds to cancer stem cells, the aptamer comprising at least one nucleic acid sequence selected from the group consisting of nucleic acid sequences represented by SEQ ID NOs: 1 to 6 or a fragment thereof.

[0060] In the present invention, "aptamer" refers to a small single-stranded oligonucleic acid that can specifically recognize a target substance with high affinity. In this regard, when the aptamer is RNA, T can be recognized as U in the nucleotide sequence.

[0061] The aptamer that specifically binds to cancer stem cells of the present invention preferably includes a nucleic acid sequence or a fragment thereof having at least 80% homology to SEQ ID NOs: 1 to 6, and most preferably includes at least one nucleic acid sequence or a fragment thereof selected from the group consisting of nucleic acid sequences represented by SEQ ID NOs: 1 to 6. "Nucleic acid sequence having at least 80% homology" refers to a nucleic acid sequence in which one to several nucleotides are added, deleted or substituted to have at least 80% and less than 100% sequence in common and show similar CD166 binding ability.

[0062] In the present invention, "nucleotide" refers to a unit constituting a nucleic acid. Unless otherwise specified herein, nucleotides A, T (U), G and C are defined as 2'-deoxyadenosine, 2'-deoxythymidine (or 2'-deoxyuridine), 2'-deoxyguanosine and 2'-deoxycytidine, respectively. In addition, 'N' is defined as 5-(N-naphthylcarboxamide)-2'-deoxyuridine (NapdU), which is represented by the following formula 1.

[0063] [Formula 1]

[0064]

[0065] In one embodiment of the present invention, the aptamer that specifically binds to cancer stem cells preferably specifically binds to CD166 expressed in cancer stem cells.

[0066] In one embodiment of the present invention, the size of the aptamer that specifically binds to cancer stem cells is preferably 70mer to 80mer, more preferably 74mer to 78mer, even more preferably 76mer.

[0067] In the present invention, "cancer" is a general term for diseases caused by cells having the following characteristics: invasive characteristics, in which cells divide and grow regardless of normal growth limits; invasive characteristics, in which cells infiltrate surrounding tissues; and metastatic characteristics, in which cells spread to other parts of the body. The cancer is preferably selected from at least one of the group consisting of gastric cancer, breast cancer, lung cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue tumors, urethral cancer, prostate cancer, bronchial cancer and myeloma, but is not limited thereto.

[0068] In the present invention, "cancer stem cells" refer to cells that have the ability to produce tumors. Cancer stem cells have the same characteristics as normal stem cells, and specifically, have the ability to produce all cell types found in a specific cancer sample. That is, the tumorigenicity of cancer stem cells is different from that of cancer cells that do not form tumors. Cancer stem cells produce tumors in various cell types through self-renewal and differentiation capabilities (which are characteristics of stem cells). In addition, cancer stem cells are different from other populations in tumors and cause recurrence and metastasis by producing new tumors. Therefore, the development of specific treatments for cancer stem cells may increase the survival rate of cancer patients.

[0069] The aptamer specifically binding to cancer stem cells of the present invention has the effect of reducing cell adhesion ability, cell proliferation, drug resistance and cell migration, which are characteristics of cancer stem cells, and can therefore be used in the fields of cancer diagnosis, prognosis prediction and treatment in various ways.

[0070] According to another embodiment of the present invention, the present invention provides a composition for diagnosing cancer and a kit for diagnosing cancer, which comprise an aptamer that specifically binds to cancer stem cells.

[0071] In the present invention, "diagnosis" refers to confirming the presence or characteristics of pathological symptoms. Diagnosis includes not only confirming the onset of the disease, but also confirming the prognosis, the course of cancer, the stage, etc.

[0072] By using the composition for diagnosing cancer and the kit for diagnosing cancer of the present invention, cancer stem cells in biological samples, i.e. cells overexpressing CD166, are detected to confirm the presence of cancer. In addition, the prognosis, course of disease, stage, etc. of cancer and the presence of cancer can be confirmed.

[0073] In the present invention, "biological sample" refers to any sample obtained from an object in which the expression of the gene or protein of the present invention can be detected. The biological sample is any one selected from the group consisting of whole blood, serum, plasma, cells, sputum, tissue, saliva, biopsy, liquid culture, feces and urine, but is not particularly limited thereto. In addition, it can be processed and prepared by a method commonly used in the technical field of the present invention.

[0074] In one embodiment of the present invention, the kit for diagnosing cancer can be a kit of various forms, depending on the method used. For example, it includes a PCR kit, a DNA chip kit, a protein chip kit or a microarray kit, but is not limited thereto.

[0075] The kit for diagnosing cancer may include reagents for measuring gene expression or its protein expression level, and tools, reagents, etc. commonly used in the art for immunological analysis. Examples of the tools or reagents include, but are not limited to, suitable carriers, labeled substances capable of producing detectable signals, chromophores, solubilizers, detergents, buffers, stabilizers, etc. When the labeled substance is an enzyme, it may include substrates and reaction terminators capable of measuring enzyme activity. Carriers include soluble carriers and insoluble carriers, examples of soluble carriers include physiologically acceptable buffers known in the art, such as PBS, and examples of insoluble carriers include polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesins, cross-linked dextran, polysaccharides, polymers such as magnetic particles coated with metal on latex, other papers, glass, metals, agarose, and combinations thereof.

[0076] Since the kit of the present invention includes the above-mentioned composition as components, redundant descriptions are omitted to avoid overly complicating the present specification.

[0077] According to another embodiment of the present invention, the present invention provides a method for diagnosing cancer, the method comprising reacting a biological sample with an aptamer that specifically binds to a cancer stem cell.

[0078] In one embodiment of the present invention, the method can provide information on cancer diagnosis by reacting a biological sample with an aptamer that specifically binds to cancer stem cells and measuring the degree of reaction (ie, the degree of expression).

[0079] According to another embodiment of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising an aptamer that specifically binds to cancer stem cells.

[0080] Furthermore, the present invention provides a method for treating cancer, the method comprising administering to a subject an aptamer that specifically binds to cancer stem cells.

[0081] When the composition of the present invention is used as a pharmaceutical composition, the pharmaceutical composition of the present invention can be prepared and used in various forms according to conventional methods. For example, it can be formulated into oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions and syrups, and can be formulated into the form of external preparations, suppositories, and sterile injections.

[0082] The composition of the present invention may comprise one or more active ingredients known to have preventive or therapeutic effects on cancer and an aptamer that specifically binds to cancer stem cells.

[0083] The composition of the present invention may further include pharmaceutically acceptable additives, wherein starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, toffee, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, etc. can be used as pharmaceutically acceptable additives. Based on the composition, the content of the pharmaceutically acceptable additive according to the present invention is preferably 0.1 to 90 parts by weight, but is not limited thereto.

[0084] During actual clinical application, the compositions of the present invention can be used in various oral or parenteral formulations. When preparing the compositions, it can be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc., preferably using suitable agents known in the art.

[0085] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and solid preparations are prepared by mixing at least one or more excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. In addition, liquid preparations for oral administration include suspensions, oral preparations, emulsions, syrups, etc. In addition to water and liquid paraffin (which is a commonly used simple diluent), various excipients such as wetting agents, sweeteners, spices, preservatives, etc. may also be included.

[0086] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), injectable esters (such as ethyl oleate) etc. can be used as non-aqueous solvents and suspending agents. Witepsol, polyethylene glycol (macrogol), Tween 61, cocoa butter, laurel fat, glycerin gelatin etc. can be used as the base of suppositories.

[0087] The dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method of the pharmaceutical composition, the mode of administration, the time of administration and / or the route of administration, etc. In addition, it may vary depending on various factors, including: the type and degree of response achieved by administering the pharmaceutical composition, the type, age, weight and general health of the subject of administration, the symptoms or severity of the disease, sex, diet, excretion, other components of the drug or composition used together with the subject at the same time or at different times, and similar factors well known in the medical field, and those skilled in the art can easily determine and prescribe the effective dosage for the desired treatment.

[0088] The route and mode of administration of the pharmaceutical composition of the present invention can be independently and are not particularly limited, and any route and mode of administration can be selected as long as the pharmaceutical composition can reach the desired site.

[0089] The pharmaceutical composition of the present invention can be used for preventing or treating cancer alone or in combination with methods using surgery, radiotherapy, hormone therapy, chemotherapy and biological response modifiers.

[0090] According to another embodiment of the present invention, the present invention provides an anti-cancer adjuvant composition comprising an aptamer that specifically binds to cancer stem cells.

[0091] The anticancer adjuvant composition of the present invention may be in the form of a pharmaceutical composition or a food composition, and more specifically, may be an anticancer drug adjuvant or an anticancer food adjuvant.

[0092] In the present invention, "anticancer adjuvant" refers to an agent that can be used as an adjuvant to enhance the effect of cancer therapeutic agents commonly used in the art, and the effect of cancer therapeutic agents or anticancer therapy can be enhanced by using the adjuvant according to the present invention.

[0093] According to another embodiment of the present invention, the present invention provides a food composition for preventing or alleviating cancer, the food composition comprising an aptamer that specifically binds to cancer stem cells.

[0094] When the composition of the present invention is used as a food composition, the food composition of the present invention refers to a food having the effect of preventing or alleviating cancer and diseases caused by cancer, and should be harmless to the human body when taken for a long time.

[0095] There is no particular restriction on the type of food. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products (including ice cream), various soups, beverages, teas, drinks, alcoholic beverages, multivitamins, etc., and include all health foods in the ordinary sense.

[0096] In one embodiment of the invention, food composition of the present invention can be a food additive. This food additive can be used like the aptamer that adds and cancer stem cell specificity combines or can be used together with other food or food ingredients, and can be used appropriately according to ordinary method. The mixed amount of active ingredient can be appropriately determined according to the purpose of use (preventive, health care or curative treatment).

[0097] In one embodiment of the present invention, the food composition of the present invention can be a health drink composition. In addition to the aptamer that specifically binds to cancer stem cells, the health drink composition can also contain various flavoring agents or natural carbohydrates as additional components like conventional beverages. Monosaccharides (such as glucose and fructose), disaccharides (such as maltose and sucrose), natural sweeteners (such as dextrin and cyclodextrin), synthetic sweeteners (such as saccharin and aspartame), etc. can be used as the above-mentioned natural carbohydrates. The ratio of natural carbohydrates is generally about 0.01g to 10g per 100ml of the present composition, preferably about 0.01g to 0.1g.

[0098] In addition to the above, the composition of the present invention may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectin acid and its salt, alginic acid and its salt, organic acid, protective colloid thickener, pH regulator, stabilizer, preservative, glycerine, alcohol, the carbonating agent used in carbonated beverages etc. In addition, the composition of the present invention may include the pulp used to produce natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used alone or in combination. The ratio of these additives is not critical, but is usually selected in the range of 0.01 to 0.1 weight portion per 100 weight portions of the present composition.

[0099] According to another embodiment of the present invention, the present invention provides a composition for cancer-specific drug delivery, the composition comprising an aptamer that specifically binds to cancer stem cells.

[0100] The aptamer that specifically binds to cancer stem cells specifically binds to CD166 that is overexpressed in cancer stem cells. By using it, drugs such as anticancer agents can be delivered to the site where cancer has already occurred.

[0101] Embodiments of the present invention

[0102] Hereinafter, the present invention will be described in more detail by way of examples. These examples are only used to illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention should not be construed as being limited by these examples.

[0103] Example 1. Confirmation of CD166 expression in ovarian cancer cells and ovarian cancer stem cells

[0104] RT-PCR was performed to detect the expression of CD166 in ovarian cancer cell line A2780 cells and ovarian cancer stem cell line A2780-SP cells isolated and established by three-dimensional culture of A2780 cells. After extracting mRNA from A2780 and A2780-SP cells, the expression of CD166 was determined by RT-PCR. Specifically, total RNA was isolated from ovarian cancer cell line A2780 and ovarian cancer stem cell line A2780-SP cells. For RT-PCR, 2 μg of total RNA and a reverse transcription cDNA synthesis kit (NanoHelix Co.) were used to synthesize the first-strand cDNA according to the manufacturer's instructions. Thereafter, 20 μl of the reaction solution containing Ready 2×Go pre-mix PCR kit (NanoHelix Co.) and 10 pM CD166-specific primers (CD166: 5′-CAGAACACGATGAGGCAGAC-3′, 5′-AGCAAGGAGGAGACCAACAA-3′) was used to amplify an equal amount of cDNA, and then the expression of CD166 was confirmed. The RT-PCR results are shown in Figure 1 shown.

[0105] like Figure 1 As shown, it was confirmed that A2780 cells did not express CD166, while A2780-SP cells expressed CD166.

[0106] In addition, flow cytometry (FACS) was performed to confirm the expression of CD166 in ovarian cancer cell line A2780 cells and ovarian cancer stem cell line A2780-SP cells isolated and established by three-dimensional culture of A2780 cells. Specifically, CD166 FACS antibody (PE Mouse Anti-Human CD166, Cat. No.: 559263, BD Pharminggen) was used to stain ovarian cancer cell line A2780 cells and ovarian cancer stem cell line A2780-SP cells, and flow cytometry was performed using FACS Canto II (BD Bioscience). The expression of CD166 in A2780 and A2780-SP cells was confirmed by flow cytometry. Figure 2 shown.

[0107] like Figure 2 As shown, it was confirmed that the expression level of CD166 was high in A2780-SP cells, which are ovarian cancer stem cell lines.

[0108] Example 2. Flow cytometry using CD166 in ovarian cancer cell lines and confirmation of expression of key genes in cancer stem cells

[0109] CD166 expressing cells and non-expressing cells were separated from ovarian cancer cell line A2780 using flow cytometry (ARIA3), and the expression of various cancer stem cell related genes in separated CD166 expressing cells was confirmed by RT-PCR. Specifically, RNA was isolated from ovarian cancer cell line A2780 and ovarian cancer stem cell line A2780-SP cells, and the expression levels of ABC transporters (ABCB1, ABCG2, ABCC6), ALDH and stem cell ability related genes (OCT4, SOX2), which are drug resistance genes, were confirmed by RT-PCR. For RT-PCR, 2 μg of total RNA and a reverse transcription cDNA synthesis kit (NanoHelix Co.) were used to synthesize the first-strand cDNA according to the manufacturer's instructions. The PCR products were expressed in 967 amino acids and 845 amino acids, respectively. The expression of 547 amino acids in the 100 μM PCR product was performed using a Ready 2×Go pre-mix PCR kit (NanoHelix Co.) and 10 μM of each primer (GAPDH: 5′-TCCATGACAACTTTGGTATCG-3′, 5′-TGTAGCCAAATTCGTTGTCA-3′; OCT4A: 5′-GATCGGATCCATGGCGGGACACCTGGCT-3′, 5′-CCTTCCCAAATAGAACCC-3′; SOX2: 5′-CAACATGATGGAGACGGAGC-3′, 5′-GTGCATCTTGGGGTTCTCCT-3′; ALDH1: 5′-CTCGAAATTAAGTACACCAA-3′, 5′-TCAGTAGA CCCTGTGAATGC-3'; ABCB1: 5'-CCATCAGTCCTGTTCTTG-3', 5'-CTGCTCCTCTTGCATTT-3'; ABCG2: 5'-TTCAGCCGTGGAACTCTTTG-3', 5'-CCACACTCTGACCTGCTGCT-3'; ABCC6: 5'-AGACAGACGCTGGGACCC-3', 5'-ACCATCTTGGCTTTGAAGAGTG-3') were added to a 20 μl reaction solution to amplify equal amounts of cDNA, and then the expression of CD166 was confirmed. The results of the confirmation of CD166 expression are shown in Figure 3 The results of the confirmation of cancer stem cell-related gene expression are shown in Figure 4 shown.

[0110] like Figure 3 As shown, it was confirmed that CD166-expressing cells (CD166+) isolated from the ovarian cancer cell line A2780 expressed CD166, while non-CD166-expressing cells (CD116-) did not express CD166.

[0111] like Figure 4 As shown, increased expression of ABC transporters (ABCB1, ABCG2, ABCC6), which are genes associated with drug resistance of cancer stem cells, ALDH, which is known as a marker for cancer stem cells, and genes associated with stem cell ability (OCT4, SOX2) were confirmed.

[0112] Example 3. Confirmation of the effect of CD166 expression on cell proliferation, drug resistance, spheroid formation ability and cell migration

[0113] 3-1. Confirmation of the effect of CD166 on cell proliferation

[0114] To confirm the effect of CD166 expression on cell proliferation rate, 1×10 CD166-expressing cells and 1×10 non-expressing cells were cultured. 4 The cells were attached to a 24-well plate, and then detached with 0.25% trypsin-EDTA at a certain time every day for 2 consecutive days. The number of cells was measured on a hemocytometer. The results were as follows: Figure 5 shown.

[0115] like Figure 5 As shown, it was confirmed that CD166-expressing cells have a faster proliferation rate than non-expressing cells.

[0116] 3-2. Confirmation of the effect of CD166 on spheroid formation ability

[0117] The self-renewal ability of CD166-expressing cells isolated from ovarian cancer cell lines was confirmed by sphere formation assay. In order to confirm the sphere formation ability according to the change of CD166 expression, CD166 expressing cells and non-expressing cells were separated from A2780 cells using CD166 FACS antibody (PE mouse anti-human CD166, Cat. No.: 559263, BD Pharminggen). Thereafter, 100 to 10,000 cells were seeded in a 24-well-Ultra-Low Attachment culture plate and cultured for 7 days. The results of the sphere formation assay are shown in Figure 2. Figure 6 shown.

[0118] like Figure 6 As shown, it was observed that CD166-expressing cells formed more spheres than non-expressing cells. The results indicate that CD166-expressing cells have a high self-renewal and non-adherent proliferation capacity, which is the most important characteristic of stem cells.

[0119] 3-3. Confirmation of the effect of CD166 on sensitivity to anticancer drugs

[0120] To measure drug sensitivity, 1 × 10 CD166-expressing and non-expressing cells were added. 4Cells were attached to a 96-well plate. The adherent cells were treated with paclitaxel, an anticancer agent, at concentrations of 0, 0.01 μM, 0.1 μM, and 1 μM and cultured for 48 hours. MTT solution (Sigma-Aldrich, Inc. St. Louis, MO) was added to the cultured cells and allowed to react for 4 hours. After the reaction was complete, the cells were treated with DMSO (sigma-Aldrich) and the absorbance at 570 nm was measured to analyze drug sensitivity. The results of drug sensitivity analysis are shown in Figure 2. Figure 7 shown.

[0121] like Figure 7 As shown, it was confirmed that CD166-expressing cells were resistant to the anticancer agent paclitaxel. The results indicate that CD166-expressing cells are cancer stem cells.

[0122] 3-4. Confirmation of the effect of CD166 on migration

[0123] In order to confirm whether the expression of CD166 promotes cell migration, the degree of cell migration was confirmed in vitro. Specifically, in order to confirm cell migration, CD166 FACS antibody (PE mouse anti-human CD166, Cat. No.: 559263, BD Pharminggen) was used to separate CD166 expressing cells and non-expressing cells from A2780 cells. Thereafter, cell migration was measured using a 96-well chemotaxis chamber (Neuro Probe, Gaithersburg, MD) according to the manufacturer's instructions. The results of confirming cell migration are as follows. Figure 8 shown.

[0124] like Figure 8 As shown, it was observed that CD166-expressing cells had higher migration ability than non-expressing cells.

[0125] Example 4. Confirmation of the effect of CD166 expression inhibition on stem cell-related gene expression, cell proliferation, drug resistance, sphere formation ability and cell migration of ovarian cancer stem cells

[0126] siRNA was used to inhibit the expression of CD166 in the ovarian cancer stem cell line A2780-SP, and the expression of various cancer stem cell-related genes in cells in which CD166 expression was inhibited was confirmed by the RT-PCR method of Examples 1 and 2. Specifically, in order to inhibit the expression of CD166, a control (si control) (5'-UUC UCC GAA CGU GUC ACG UTT-3'; 5'-ACG UGA CACGUU CGG AGA ATT-3') and si CD166 (5'-AAG CCC GAU GGC UCC CCA GUA UU-3'; 5'-AAU ACUGGG GAG CCA UCG GGC UU-3') were constructed. The expression of CD166 was inhibited by reacting the constructed siRNA with the ovarian cancer stem cell line A2780-SP cells. The confirmation results of the expression of cancer stem cell-related genes are shown in Figure 1. Fig. 9 shown.

[0127] like Fig. 9 As shown, it was confirmed that inhibition of CD166 expression using siRNA reduced the expression of ABC transporters (ABCB1, ABCG2, ABCC6), a drug resistance gene of cancer stem cells, the activity of ALDH (ALDH), a cancer stem cell marker, and stem cell ability-related genes (OCT4, SOX2).

[0128] Next, the proliferation, drug resistance, spheroid formation ability and cell migration of cells with suppressed CD166 expression were measured in the same manner as in Example 2. The results were as follows: Figures 10 to 13 shown.

[0129] like Figures 10 to 13 As shown, it was confirmed that cells in which CD166 expression was suppressed had reduced cell proliferation ability, increased drug sensitivity, and reduced sphere formation ability and migration. The results indicate that CD166 expression plays an important role in the stem cell function of ovarian cancer stem cells.

[0130] Example 5. Confirmation of the inhibition of focal adhesion signaling pathway in the process of suppressing cancer stem cell characteristics by inhibiting CD166 expression in ovarian cancer stem cells

[0131] siRNA was used to inhibit CD166 expression in ovarian cancer stem cell line A2780-SP, and the focal adhesion signaling pathway of cells in which CD166 expression was inhibited was confirmed by Western blotting. Specifically, in order to inhibit the expression of CD166, a control (si control) (5'-UUC UCC GAACGU GUC ACG UTT-3'; 5'-ACG UGA CAC GUU CGG AGA ATT-3') and si CD166 (5'-AAG CCC GAU GGC UCC CCA GUA UU-3'; 5'-AAU ACU GGG GAG CCA UCGGGC UU-3') were constructed. The expression of CD166 was inhibited by reacting the constructed siRNA with the ovarian cancer stem cell line A2780-SP cells, and the cells were cultured after the expression was inhibited. After 48 hours of culture, the cells were collected and the protein expression of FAK, SRC, Paxillin and CD44, which are known as the main genes of the focal adhesion signaling pathway, was confirmed by Western blotting. Antibodies against CD166 (ab109215, Abcam), CD44 (5640S, Cell Signaling Technology), p-PAXILLIN (2541S, Cell Signaling Technology), p-SRC (2101S, Cell Signaling Technology), p-FAK (3284S, Cell Signaling Technology), FAK (3285S, Cell Signaling Technology), and GAPDH (MAB374, EMD Milliore Corp, Billerica, MA) were used as primary antibodies. The results of confirmation of the inhibitory activity of the focal adhesion signaling pathway in cells with suppressed CD166 expression are shown in Figure 2. Fig.14 shown.

[0132] like Fig.14 As shown, it was confirmed that the expression of p-PAXILLIN and p-SRC involved in cell adhesion was suppressed in A2780-SP cells in which the expression of CD166 was suppressed. In particular, it was confirmed that the expression of p-FAK Y925 site, which is known to be involved in adhesion, among the FAX phosphorylation sites, was reduced in the cells.

[0133] Example 6. Confirmation of the effect of CD166 expression on tumor formation

[0134] To confirm the effect of CD166 expression on tumor formation, CD166-expressing cells and non-expressing cells were isolated from the ovarian cancer cell line A2780 using flow cytometry (ARIA3). 1×10 5The cells were subcutaneously inoculated into nude mice. At this time, CD166-expressing cells were inoculated on the left side of the nude mice, and non-CD166-expressing cells were inoculated on the right side of the nude mice. After inoculation, the size of tumor formation was measured from the 18th day. The nude mice were euthanized on the 38th day after inoculation. The tumors were isolated from the nude mice, and finally the weight and size of the tumors were measured. The observation results of the isolated tumors were as follows: Fig.15 The results of measuring tumor size are shown in Fig.16 The results of measuring tumor weight are shown in Fig.17 shown.

[0135] like Figures 15 to 17 As shown, it was confirmed that tumors were formed faster in CD166-expressing cells. Also, it was confirmed that tumors in CD166-expressing cells were heavier and larger in size than tumors in non-CD166-expressing cells.

[0136] Example 7. Construction of an aptamer that specifically binds to CD166 expressed in cancer stem cells

[0137] Aptamers that specifically bind to CD166 expressed in cancer stem cells were constructed. CD166 aptamers (CD166 AT) were synthesized by Aptamer Sciences Inc. (Pohang, Korea). The sequences of the constructed aptamers that specifically bind to CD166 are shown in Table 1.

[0138] [Table 1]

[0139]

[0140]

[0141] Wherein, A, T(U), G and C are defined as 2'-deoxyadenosine, 2'-deoxythymidine (or 2'-deoxyuridine), 2'-deoxyguanosine and 2'-deoxycytidine, respectively. 'N' is defined as 5-(N-naphthylcarboxamide)-2'-deoxyuridine (NapdU), which is represented by the following formula 1.

[0142] [Formula 1]

[0143]

[0144] The constructed aptamer was dissolved in sterile triple distilled water and then heated to 95°C for 5 minutes. The heated aptamer was cooled to 25°C and used in the following experiments.

[0145] Example 8. Confirmation of the affinity of CD166 aptamers in ovarian cancer cells and ovarian cancer stem cells

[0146] 8-1. Confirmation of the affinity of CD166 aptamers for ovarian cancer cells and ovarian cancer stem cells

[0147] Confirm whether the CD166 aptamer constructed in Example 7 recognizes ovarian cancer cells and ovarian cancer stem cells. The following experiment was performed to analyze the interaction between the CD166 aptamer and the ovarian cancer cell line A2780 cells. First, each CD166 aptamer (AT-1, AT-2, AT-3, AT-4, AT-5 and AT-6) was dissolved in sterile tertiary water and then heated at 95°C for 5 minutes. After heating, cool to 25°C. The aptamer was added to the ovarian cancer cell line A2780 cells at a concentration of 500nM, and the cells were then cultured and stained at room temperature. Thereafter, the cells reacting with the CD166 aptamer were analyzed using a flow cytometer (fluorescence activated cell sorter, FACS), and the results were as shown in FIG. Fig.18 shown.

[0148] like Fig.18 As shown, it was confirmed that the CD166 aptamer showed interaction with A2780 cells, but the signal intensity was low. This is because the expression of CD166 in the ovarian cancer cell line A2780 is low.

[0149] Furthermore, in the same manner as above, ovarian cancer stem cell line A2780-SP cells were reacted with CD166 aptamer and then subjected to FACS. Fig.19 shown.

[0150] like Fig.19 As shown, it was confirmed that the CD166 aptamer interacted with the ovarian cancer stem cell line A2780-SP cells, and the signal intensity was high. In particular, it was confirmed that AT-5 among the CD166 aptamers had the most specific binding to the ovarian cancer stem cells.

[0151] 8-2. Comparison of affinity between CD166 aptamer and CD166 antibody in cancer stem cells

[0152] The affinity of CD166 aptamer AT-5 and CD166 FACS antibody (PE mouse anti-human CD166, Cat. No.: 559263, BD Pharminggen) as a control for ovarian cancer stem cell line A2780-SP cells was compared. The affinity comparison was carried out in the same manner as in Example 8-1. The results are shown in FIG. Fig. 20 shown.

[0153] like Fig. 20 As shown, it was confirmed that the binding of the CD166 aptamer and the CD166 antibody to A2780-SP cells exhibited similar binding specificity and affinity. The results showed that the CD166 aptamer specifically binds to CD166-expressing cells and that the aptamer has similar binding affinity to the CD166 antibody.

[0154] Example 9. Confirmation of the effect of CD166 aptamers on cell adhesion ability, cell proliferation, drug resistance and cell migration of ovarian cancer stem cells

[0155] The CD166 aptamers AT3, AT4, and AT5 were used to confirm the effect of the CD166 aptamer on cell adhesion. Specifically, CD166 expressing cells were isolated from ovarian cancer stem cells using CD166 FACS antibody. The isolated cells were used to perform a cell adhesion experiment with the CD166 aptamer. First, 1x10 5 Cells were seeded in 96-well plates and then blocked with 20 μg / ml collagen for 1 hour. After blocking, 0.1% bovine serum albumin (BSA) was added and incubated for 1 hour to limit nonspecific binding. The separated cells were each exposed to CD166 aptamer (500 nM) for 1 hour and then fixed with 4% formaldehyde. The fixed cells were stained with hoest33342. The stained cells were observed and counted using a microscope. The results are shown in Figure 2. Fig.21 shown.

[0156] like Fig.21 As shown, it was confirmed that cell adhesion was reduced in the experimental groups treated with the CD166 aptamers AT4 and AT5 having high affinity.

[0157] The CD166 aptamer AT5 was used to confirm the effect of the CD166 aptamer on cell proliferation. Specifically, 1×10 4 Each cell was attached to a 24-well plate and then divided into a CD166 aptamer AT-5 treated group and an untreated group, and treated. After aptamer treatment for 0, 24, 48, and 72 hours, MTT solution (Sigma-Aldrich, Inc. St. Louis, MO) was added to the cells and reacted for 4 hours. The cells reacted with the MTT solution were treated with DMSO (sigma-Aldrich), and then the absorbance at 570 nm was measured. The confirmation results of the effect of the CD166 aptamer on cell proliferation are shown in Figure 2. Fig. 22 shown.

[0158] like Fig. 22 As shown, it was confirmed that the cell proliferation of cancer stem cells treated with the CD166 aptamer was inhibited.

[0159] The CD166 aptamer AT5 was used to confirm the effect of the CD166 aptamer on drug sensitivity. Specifically, 1×10 4Each cell was seeded into a 24-well plate and then treated with 0.1 μM of the anticancer agent paclitaxel and the CD166 aptamer AT-5. The control was treated with 0.1 μM paclitaxel alone. After aptamer treatment for 0, 24, 48, and 72 hours, MTT solution was added to the cells and reacted for 4 hours. The cells reacted with the MTT solution were treated with DMSO and then the absorbance at 570 nm was measured. The confirmation results of the effect of the CD166 aptamer on drug sensitivity are shown in Figure 2. Fig.23 shown.

[0160] like Fig.23 As shown, it was confirmed that the drug resistance, which is a characteristic of cancer stem cells, was reduced in cells treated with the CD166 aptamer.

[0161] The CD166 aptamer AT5 was used to confirm the effect of the CD166 aptamer on cell migration. Specifically, 1×10 4 Each cell was seeded into a 24-well plate and then divided into a CD166 aptamer AT-5 treated group and an untreated group. The migration degree of cells treated with the aptamer was confirmed in vitro. The results are shown in Fig.24 shown.

[0162] like Fig.24 As shown, it was confirmed that the cell migration of cancer stem cells treated with the CD166 aptamer was reduced.

[0163] The above results indicate that when CD166-expressing cells were treated with an aptamer that specifically binds to CD166, cell adhesion, cell proliferation, drug resistance, and cell migration, which are the main characteristics of ovarian cancer stem cells, were reduced, thereby inhibiting ovarian cancer stem cells.

[0164] Example 10. Confirmation of the inhibition of focal adhesion signaling pathway in the process of CD166 aptamer inhibiting the adhesion ability of ovarian cancer stem cells

[0165] Isolation of CD166 from the ovarian cancer stem cell line A2780-SP 高 Cancer cells. The separated cells were reacted with the CD166 aptamer AT-5 for 1 hour, and then the focal adhesion signaling pathway in the cells in which CD166 expression was suppressed was confirmed by Western blotting. Western blotting was performed in the same manner as in Example 5. The confirmation results of the focal adhesion signaling pathway in the cells in which CD166 expression was suppressed are shown in FIG. Fig.25 shown.

[0166] like Fig.25 As shown, in A2780-SP cells in which CD166 expression was suppressed, the expression of p-PAXILLIN and p-SRC involved in cell adhesion was confirmed to be suppressed. In particular, it was confirmed that the expression of p-FAK Y925 site, which is known to be involved in adhesion, among FAX phosphorylation sites, was reduced in the cells.

[0167] Example 11. Confirmation of the effect of CD166 aptamer on tumor formation

[0168] Confirm the effect of CD166 aptamer AT-5 on tumor formation. Specifically, cells with high expression of CD166 were isolated from the ovarian cancer stem cell line A2780-SP using flow cytometry (ARIA3). The isolated cells were reacted with CD166 aptamer AT-5 for 1 hour. The cells (1×10 5 The nude mice were subcutaneously inoculated with 50% aptamer-treated cells (100% aptamer-treated cells) in the nude mice. At this time, the left side of the nude mice were inoculated with cells that were not treated with the aptamer, and the right side of the nude mice were inoculated with cells that were treated with the CD166 aptamer AT-5. After inoculation, the size of the tumor formation was measured from the 18th day. The nude mice were euthanized on the 38th day of inoculation. The tumors were isolated from the nude mice, and finally the weight and size of the tumors were measured. The observation results of the isolated tumors are shown in Fig.26 The results of measuring tumor size are shown in Fig. 27 The results of tumor weight measurement are shown in Fig.28 .

[0169] like Figures 26 to 28 As shown, it was confirmed that tumor formation was inhibited at the site where cells treated with the CD166 aptamer AT-5 were inoculated, and the tumor size was smaller than that of the aptamer-untreated group. In addition, it was confirmed that the tumor weight at the site where cells treated with the CD166 aptamer AT-5 were inoculated was significantly reduced compared to the control.

[0170] Example 12. Confirmation of CD166 expression in patient-derived ovarian cancer stem cells

[0171] Flow cytometry (FACS) was performed to confirm the expression of CD166 in cancer stem cells isolated from ovarian cancer patients. The isolation of patient-derived ovarian cancer cells was performed by a previously described method (STEM CELLS 2016; 34: 551-564). More specifically, for flow cytometry, a CD166 FACS antibody (PE mouse anti-human CD166, Cat. No.: 559263, BD Pharminggen) was used, and patient-derived ovarian cancer stem cells were stained. Thereafter, flow cytometry was performed using FACS Canto II (BD Bioscience).

[0172] The results of confirming the expression of CD166 in patient-derived ovarian cancer stem cells by flow cytometry are as follows Fig.29 shown.

[0173] like Fig.29 As shown, it was confirmed that the expression level of CD166 was high in patient-derived ovarian cancer stem cells.

[0174] Example 13. Confirmation of the effect of CD166 expression inhibition on focal adhesion signaling pathway, cell proliferation, drug resistance, sphere formation ability and cell migration of patient-derived ovarian cancer stem cells

[0175] siRNA was used to inhibit CD166 expression in patient-derived ovarian cancer stem cells, and the focal adhesion signaling pathway in cells in which CD166 expression was inhibited was confirmed by Western blotting. The inhibition of CD166 expression and Western blotting were performed in the same manner as in Examples 4 and 5. The results of confirming the focal adhesion signaling pathway in cells in which CD166 expression was inhibited are shown in FIG. Fig.30 shown.

[0176] like Fig.30 As shown, it was confirmed that the expression of p-PAXILLIN and p-SRC involved in cell adhesion was suppressed in ovarian cancer stem cells derived from patients whose CD166 expression was suppressed. In particular, it was confirmed that the expression of p-FAK Y925 site, which is known to be involved in adhesion, among the FAX phosphorylation sites, was reduced in cells. The expression of cancer stem cell-related genes was confirmed.

[0177] Next, the proliferation, drug resistance, spheroid formation ability, cell migration and cell adhesion of cells with suppressed CD166 expression were measured in the same manner as in Example 2. The results were as follows: Figures 31 to 35 shown.

[0178] like Figures 31 to 35 As shown, it was confirmed that the cell proliferation ability of patient-derived ovarian cancer stem cells with suppressed CD166 expression was reduced, drug sensitivity was increased, and sphere formation ability, cell migration and cell adhesion were reduced. The results show that in patient-derived ovarian cancer stem cells, the expression of CD166 plays an important role in stem cell function.

[0179] Example 14. Confirmation of the affinity of CD166 aptamers for patient-derived ovarian cancer stem cells

[0180] It was confirmed whether the CD166 aptamer AT5 showing high affinity in Example 8 recognizes patient-derived ovarian cancer stem cells. The affinity confirmation was carried out in the same manner as in Example 8. The results are as follows: Fig.36 As shown in A.

[0181] like Fig.36 As shown in A, it was confirmed that the CD166 aptamer AT5 interacted with the patient-derived ovarian cancer stem cells, and the signal intensity was high.

[0182] Example 15. Confirmation of the effect of CD166 aptamers on cell adhesion ability, drug resistance and cell migration of patient-derived ovarian cancer stem cells

[0183] The CD166 aptamers AT3 and AT5 were used to confirm the effects of the CD166 aptamers on cell adhesion, drug resistance and cell migration. Specifically, the cell adhesion, drug resistance and cell migration were performed in the same manner as in Example 9, and the results were as follows: Fig.36 As shown in B, 37 and 38.

[0184] like Fig.36 As shown in Figures B, 37 and 38, the results indicate that when patient-derived ovarian cancer stem cells are treated with an aptamer that specifically binds to CD166, cell adhesion, drug resistance, and cell migration, which are the main characteristics of cancer stem cells, are reduced, thereby inhibiting the characteristics of patient-derived ovarian cancer stem cells.

[0185] Example 16. Confirmation of the inhibition of focal adhesion signaling pathway in the process of CD166 aptamer inhibiting the cell adhesion ability of patient-derived ovarian cancer stem cells

[0186] Patient-derived ovarian cancer stem cells were reacted with CD166 aptamers AT-3 and AT-5 for 1 hour, respectively, and then the focal adhesion signaling pathway in cells in which CD166 expression was suppressed was confirmed by Western blotting. Western blotting was performed in the same manner as in Example 5. The results of confirming the focal adhesion signaling pathway in cells in which CD166 expression was suppressed are shown in FIG. Fig.39 shown.

[0187] like Fig.39 As shown, it was confirmed that the expression of p-PAXILLIN and p-SRC involved in cell adhesion was inhibited by the CD166 aptamer AT-5. In particular, it was confirmed that the expression of the p-FAK Y925 site known to be involved in adhesion among the FAX phosphorylation sites was reduced in the cells.

[0188] Example 17. Comparison of the affinity of CD166 aptamers and CD166 antibodies for mesenchymal stem cells

[0189] The affinity of CD166 aptamer AT-5 and CD166 FACS antibody (PE mouse anti-human CD166, Cat. No.: 559263, BD Pharminggen) as a control for mesenchymal stem cells (normal stem cells) was compared. The affinity comparison was carried out in the same manner as in Example 8-1. The results are shown in FIG. Fig.40 shown.

[0190] like Fig.40 As shown, it was confirmed that the binding of the CD166 aptamer and the CD166 antibody to mesenchymal stem cells exhibited similar binding specificity and affinity. The results showed that the CD166 aptamer specifically binds to CD166-expressing cells and that the aptamer has similar binding affinity to the CD166 antibody.

[0191] Example 18. Confirmation of the effect of CD166 aptamer on cell proliferation ability, cell migration and cell adhesion of mesenchymal stem cells

[0192] The CD166 aptamer AT5 was used to confirm the effects of the CD166 aptamer on cell proliferation ability, cell migration and cell adhesion. Specifically, the cell proliferation ability, cell migration and adhesion were performed in the same manner as in Example 9.

[0193] like Figures 41 to 43 As shown, it was confirmed that when mesenchymal stem cells, which are normal stem cells, were treated with an aptamer that specifically binds to CD166, there was no change in cell proliferation ability, cell migration, and cell adhesion. The results indicate that it does not affect the characteristics of normal stem cells.

[0194] Example 19. Confirmation of focal adhesion signaling pathway generated by CD166 aptamer and CD166 expression inhibition in mesenchymal stem cells

[0195] Mesenchymal stem cells were reacted with the CD166 aptamer AT-5 for 1 hour, and then the focal adhesion signaling pathway in cells in which CD166 expression was suppressed was confirmed by Western blotting. Western blotting was performed in the same manner as in Example 5. In addition, the focal adhesion signaling pathway caused by the suppression of CD166 expression in mesenchymal stem cells was confirmed by the method of Example 4. The results were as follows: Fig.44 shown.

[0196] like Fig.44 As shown, it was confirmed that the expression of p-PAXILLIN and p-SRC involved in cell adhesion in normal stem cells was not affected by the CD166 aptamer AT-5 treatment and the inhibition of CD166 expression. In addition, it was confirmed that the expression of the p-FAK Y925 site known to be involved in adhesion among the phosphorylation sites of FAX was not affected in the cells.

[0197] In general, the inventors have developed an aptamer that specifically binds to CD166 expressed in cancer stem cells, and confirmed that the aptamer reduces cell adhesion ability, cell proliferation, drug resistance and cell migration that are characteristics of cancer stem cells, thereby showing an anti-cancer effect. Therefore, the aptamer of the present invention can be used in cancer diagnosis, prognosis prediction and treatment fields in various ways.

[0198] Hereinafter, the present invention will be described in more detail by preparation examples. The preparation examples are only for illustrating the present invention, and the scope of the present invention should not be interpreted as being limited by the preparation examples.

[0199] Preparation Example 1. Preparation of a pharmaceutical composition for preventing or treating cancer

[0200] 1-1. Preparation of powder

[0201] 1mg of aptamer that specifically binds to cancer stem cells

[0202] 100mg lactose

[0203] 10mg talc

[0204] The above ingredients were mixed and filled in a sealed bag to make powder.

[0205] 1-2. Preparation of tablets

[0206] 1mg of aptamer that specifically binds to cancer stem cells

[0207] 100mg corn starch

[0208] 100mg lactose

[0209] 2mg magnesium stearate

[0210] The above ingredients are mixed and then compressed according to a conventional tablet manufacturing method to prepare tablets.

[0211] 1-3. Preparation of capsules

[0212] 1mg of aptamer that specifically binds to cancer stem cells

[0213] 3mg crystalline cellulose

[0214] 14.8mg lactose

[0215] 0.2mg magnesium stearate

[0216] According to a conventional capsule manufacturing method, the above ingredients are mixed and filled in a gelatin capsule to prepare a capsule.

[0217] 1-4. Preparation of injections

[0218] 1mg of aptamer that specifically binds to cancer stem cells

[0219] 180mg mannitol

[0220] 2974mg sterile distilled water for injection

[0221] 26mgNa2HPO4·2H2O

[0222] According to a conventional method for producing injections, the injections are prepared so that the above ingredients are contained in one ampoule (2 ml).

[0223] 1-5. Liquid preparation

[0224] 1mg of aptamer that specifically binds to cancer stem cells

[0225] 10g isomerized sugar

[0226] 5g mannitol

[0227] Appropriate amount of purified water

[0228] According to the conventional liquid preparation method, each component is added to purified water to dissolve, and an appropriate amount of lemon essence is added. Then the above components are mixed, and purified water is added to adjust the total amount to 100 ml. Then, it is filled into a brown bottle and sterilized to prepare a liquid.

[0229] Above, specific parts of the present invention are described in detail. It is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the protection scope of the present invention is not limited thereby. Therefore, the essential scope of the present invention is intended to be limited by the attached claims and their equivalents. Sequence Listing <110> Hessel Technology Co., Ltd. <120> Aptamer specifically binding to cancer stem cells and its use <130> 1-385P <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> AT1, clone 1 <400> 1 tcagccgcca gccagttccn ncagccnngn agagncanga ncgnnancgg ancngaggga 60 ccagagcacc acagag 76 <210> 2 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> AT2, clone 2 <400> 2 tcagccgcca gccagttcan nganaaggnn aggacncnnn cngnnggnan ccncacngga 60 ccagagcacc acagag 76 <210> 3 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> AT3, clone 3 <400> 3 tcagccgcca gccagttcgc gcncgnnncg gaggggangc agaccccncn cgcnnngcga 60 ccagagcacc acagag 76 <210> 4 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> AT4, clone 4 <400> 4 tcagccgcca gccagttccn ncnagagnca ngcacgnnan cggaccngnc ggcgaangga 60 ccanancacc acagag 76 <210> 5 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> AT5, clone 5 <400> 5 tcagccgcca gccagttcan nnganaaagc nccgnnancg ggccccnngn agagncanga 60 ccagagcacc acagag 76 <210> 6 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> AT6, clone 6 <400> 6 tcagccgcca gccagttcnc gcnnncggnc ancangaacg gcncgnnncg gaacngncga 60 ccagagcacc acagag 76

Claims

1. An aptamer that specifically binds to cancer stem cells, the aptamer consisting of a nucleic acid sequence represented by SEQ ID NO:

5. 2 . The aptamer that specifically binds to cancer stem cells according to claim 1 , wherein the aptamer specifically binds to CD166. 3 . The aptamer that specifically binds to a cancer stem cell according to claim 1 , wherein the cancer stem cell is at least one selected from the group consisting of breast cancer stem cells, lung cancer stem cells, ovarian cancer stem cells, and colon cancer stem cells.

4. A composition for diagnosing cancer, comprising the aptamer that specifically binds to cancer stem cells according to claim 1, The cancer is at least one selected from the group consisting of breast cancer, lung cancer, ovarian cancer and colon cancer.

5. A kit for diagnosing cancer, the kit comprising the composition for diagnosing cancer according to claim 4, The cancer is at least one selected from the group consisting of breast cancer, lung cancer, ovarian cancer and colon cancer. 6 . The kit for diagnosing cancer according to claim 5 , wherein the kit for diagnosing cancer is a PCR kit, a DNA chip kit, a protein chip kit or a microarray kit.

7. A pharmaceutical composition for preventing or treating cancer, comprising the aptamer that specifically binds to cancer stem cells according to claim 1, The cancer is at least one selected from the group consisting of breast cancer, lung cancer, ovarian cancer and colon cancer.

8. An anticancer adjuvant composition, comprising the aptamer that specifically binds to cancer stem cells according to claim 1, The cancer is at least one selected from the group consisting of breast cancer, lung cancer, ovarian cancer and colon cancer.

9. A composition for cancer-specific drug delivery, comprising the aptamer that specifically binds to cancer stem cells according to claim 1, The cancer is at least one selected from the group consisting of breast cancer, lung cancer, ovarian cancer and colon cancer.

10. Use of the aptamer according to claim 1 in preparing a drug for preventing or treating cancer, The cancer is at least one selected from the group consisting of breast cancer, lung cancer, ovarian cancer and colon cancer.

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