JP2016021883A - Nucleic acids that specifically bind to human colon cancer cell colo205 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide novel DNA aptamers which are involved in human colon cancer (Colo205) and are useful such as for detection, diagnosis, treatment, and prevention of metastasis, of human colon cancer.SOLUTION: The invention provides a DNA aptamer which comprises a nucleotide sequence represented by a specific sequence or another specific sequence and specifically binds to human colon cancer cell. In a preferred embodiment, the DNA aptamer may be linked to a primer recognition sequence, a fluorescent label, biotin, avidin or streptavidin, or the other specific binding tag peptide, at the 5' terminal or 3' terminal thereof for facilitating detection.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a nucleic acid that can specifically bind to human colon cancer (Colo205) cells, a composition containing the nucleic acid, and the like.

  In recent years, research on diagnosis and treatment of colorectal cancer has been urgently studied as a cancer in which the increase in the onset tendency of Japanese people is remarkably observed. Colorectal cancer is expected to surpass lung cancer, which is currently the number one patient in 2015, and according to statistics in 2012, the third in mortality, especially in men, next to lung cancer and liver cancer. It ’s already ranked first among women. Since colorectal cancer is known to have a good prognosis by surgery if detected at an early stage, it has been the subject of early cancer screening, and numerous testing methods have been developed. In colorectal cancer, it is important to suppress cancer metastasis, and various studies have been conducted. It is known that the process of metastasis involves the interaction between metastatic cancer cells, various host cells (platelets, lymphocytes, endothelial cells), extracellular matrix and basement membrane.

  In order to analyze the characteristics of human colon cancer cells and the interaction with immune cells, several cell lines have been established from human colon adenocarcinoma patients (see, for example, Non-Patent Document 1). Colo205 is a cell line isolated from the ascites of human colon cancer patients, a malignant tumor cell line that has an epithelial cell morphology and produces large amounts of CEA. Colo205 cells are also known to have a mutation in the growth-driven oncogene BRAF, and that amplification contributes to the acquisition of resistance to MEK1 / 2 inhibitors.

  The tumor marker called CA50 is a sugar chain antigen recognized by monoclonal antibody C-50 prepared by immunizing mice with human colon cancer-derived cultured cell line Colo-205, and Lewis A belonging to type I sugar chain as an antigen. It has a determinant. Not only for colorectal cancer, but also suitable for screening because pancreatic cancer, hepatocellular carcinoma, biliary tract cancer, benign pancreatic / biliary system benign disease, dialysis patients, undialyzed renal failure patients, cirrhosis, and the positive rate is low. It is used as an effect judgment and monitoring of the detection of recurrent tumors. On the other hand, Patent Document 1 discloses a method for treating cancer in a subject containing a mutant KRAS or BRAF gene. The method includes administering to the subject an antibody or antigen-binding fragment thereof that competes with monoclonal antibody SC104 for binding to Colo205 of a human colon cancer cell line. However, the exact structure of this antigen binding site is not clear.

  All cells including Colo205 have their cell membrane surfaces covered with proteins produced by the cells. Numerous literatures have already introduced that nucleic acids having specific sequences adsorb to these expressed proteins or sugar chains bound to them. (For example, Patent Documents 2 and 3) Nucleic acid sequences that can specifically adsorb to these specific targets are called aptamers. These aptamers are characterized in that they can be produced by chemical synthesis methods. Therefore, it is not necessary to synthesize in vivo like an antibody which has been a typical presence of what specifically adsorbs with a pathogenic protein. When these aptamers are used for diagnosis, fluorescent labeling is often performed and detection with a fluorescence microscope is used (for example, Non-Patent Document 3).

  The in vitro selection method (in vitro selection method) is used as the most effective means for searching for new aptamers that specifically adsorb to proteins and cells. Particularly, the SELEX (Systematic Evolution of Ligands by Exponential enrichment) method is roughly divided into two steps: selection of the target nucleic acid molecule and amplification of the selected aptamer. (For example, Patent Document 4) By repeating this selection and amplification while increasing the selection pressure, a nucleic acid fragment having high affinity can be obtained. In addition, various improvements have been made in recent years, and aptamers can be recovered with fewer cycles. The method is excellent in efficiency and selectivity, and not only small molecules and proteins but also cells and tissues (exactly on the surface) A technique for obtaining an aptamer that binds to (molecule) (Cell-SELEX method; for example, Non-Patent Document 4) has been reported. Compared to the conventional SELEX method, this method requires no analysis of membrane proteins, can simultaneously select aptamers of various membrane proteins present on the cell surface, and selects aptamers that bind more specifically to the target cell. There is a feature that you can do. These aptamers can be synthesized chemically in a short time, not to mention their high affinity and specificity for the target, which is also a characteristic of antibodies that have been used for diagnosis and treatment in the past. There are various advantages of antibodies that can be performed inexpensively, have a simple mechanism of action, and have little immunogenicity.

Special table 2013-509451 gazette JP 2006-149302 A JP 2011-92138 A International Publication No. WO91 / 19813

Semple, TU et al., Cancer Research. 38, 1345-1355, 1978 Little, AS et al., Sci. Signal., 4, ra17, 2011 Yen-An Shieh, Shu-Jyuan Yang, Ming-Feng Wei and Ming-JiumShieh., ACS NANO., Vol.4, No.3, 1433-1442, 2010. Guo, et al., Int. J. Mol. Sci., 9 (4): 668,2008

  Under the background as described above, an object of the present invention is to provide a novel DNA aptamer related to human colon cancer (Colo205) useful for detection, diagnosis, treatment, metastasis prevention, etc. of colon cancer. is there.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have identified a nucleotide sequence having specific binding ability to human colon cancer Colo205 cells by using the Cell-SELEX method. The inventors have newly found that a DNA having the sequence can function as a specific aptamer for human colon cancer Colo205 cells, and have completed the present invention.

  That is, the present invention provides, in one embodiment, a DNA aptamer comprising the nucleotide sequence represented by SEQ ID NO: 1 or 2 and specifically binding to human colon cancer cells. In a preferred embodiment of the present invention, the DNA aptamer has a primer recognition sequence, a fluorescent label, biotin, avidin or streptavidin, or other specific binding tag peptide at its 5 ′ end or 3 ′ end to facilitate detection. It may be connected with.

  In a different aspect of the present invention, there is provided a composition, a detection kit or a detection method for detecting human colorectal cancer cells comprising the DNA primer of the present invention. In a preferred embodiment, the step of bringing the DNA aptamer into contact with a sample collected from a living body selected from the group consisting of human colon cells, colon tissue, blood, serum and plasma, and a response by binding of the sample to the DNA aptamer Detecting the presence of human colon cancer cells by observing.

  In still another embodiment of the present invention, the pharmaceutical composition for treating human colon cancer cells, the use of the DNA primer of the present invention for the production of a pharmaceutical composition for treating human colon cancer, or the DNA primer of the present invention is included. A drug delivery system for treating human colorectal cancer is provided.

  ADVANTAGE OF THE INVENTION According to this invention, the DNA aptamer as a novel material useful in order to enable the onset of a human colon cancer, detection of a metastasis, diagnosis of cancer, and a diagnosis of a malignancy can be provided. That is, by providing a novel DNA aptamer that can specifically bind to human colorectal cancer, it is possible to search for a new antigen-binding site in human colorectal cancer or select a DNA aptamer having a different affinity from conventional antibodies. It is considered possible.

  In addition, since DNA aptamers can be easily chemically modified in accordance with detection means such as fluorescent labels, they are more advantageous than using antibodies in preparing cancer detection kits. Furthermore, drugs such as anticancer agents are conjugated. By making the gate, it is possible to reliably cause the drug to act on the target site. In addition, since chemical synthesis is easy, there is an advantage that a large-scale culture apparatus such as an antibody is not required and labor and cost for production can be suppressed.

It is a schematic diagram which shows single strand conversion of double stranded DNA using a magnetic microparticle. It is a real image figure of Colo205 cell dye | stained with the DNA aptamer which has a base sequence shown by sequence number 1. It is a fluorescence imaging figure of Colo205 cell dye | stained with the DNA aptamer which has a base sequence shown by sequence number 1. It is a real image figure of Colo205 cell dye | stained with the DNA aptamer which has a base sequence shown by sequence number 2. It is a fluorescence imaging figure of Colo205 cell dye | stained with the DNA aptamer which has a base sequence shown by sequence number 2.

  Hereinafter, embodiments of the present invention will be described. The scope of the present invention is not limited to these descriptions, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention.

1. DNA aptamer In the present application, “DNA aptamer” means a single-stranded oligo DNA capable of specifically recognizing a target molecule or substance. The DNA aptamer according to the present invention is specific to human colon cancer cells. It is a single-stranded oligo DNA having a function of binding.
The binding target of the DNA aptamer according to the present invention is preferably colon cancer cells, more preferably human colon cancer cells, and more preferably Colo205 cells. Colo205 cells are available from, for example, biological resource banks such as ATCC (American Type Culture Collection), or are commercially available as research human cell lines. In a typical embodiment, the DNA aptamer according to the present invention has a nucleotide sequence represented by SEQ ID NO: 1 or 2 shown below. In the present specification, nucleotide sequences are described from left to right in the direction from the 5 ′ end to the 3 ′ end.

As used herein, the term “specific” refers to the selective binding of a DNA aptamer according to the present invention to Colo205 cells. Aptamers can be tested for binding specificity by comparing binding to the appropriate antigen on Colo205 cells or their cell membranes to binding to unrelated cells or antigens or antigen mixtures under predetermined conditions. The DNA aptamer according to the present invention is at least 2-fold, at least 5-fold, at least 7-fold, preferably at least 2-fold relative to an appropriate antigen on Colo205 cells or their cell membranes relative to unrelated cells or antigens or antigen mixtures. An aptamer is considered specific when it binds 10-fold. The “irrelevant cell” may be a normal cell or a cancer cell other than human colorectal cancer, and only needs to be different from the human colorectal cancer cell in at least one of its origin, nature and form. The antigen on the cell membrane does not necessarily need to be present only in human colon cancer cells, but when present on the human colon cancer cell membrane due to some factor such as the expression level or three-dimensional structure difference on the cell membrane, the present invention is concerned. Any substance that specifically binds to a DNA aptamer may be used.
Since the DNA aptamer of the present invention can form a specific three-dimensional structure based on a specific base sequence, it is not nonspecific adsorption by a single-stranded DNA, but is specific to human colon cancer cells, particularly Colo205 cells. It is thought to combine.

<SEQ ID NO: 1>
5'-CCGTGGTGTGGGGGTTGGGGGGTTGTCGTCCGCG-3 '
<SEQ ID NO: 2>
5′-CGAGTGGGGGTATGCTTGGGGGTGTCGTTCGCCG-3 ′

  As long as the DNA aptamer according to the present invention has a function of specifically binding to human colon cancer cells, one or more nucleotides in the sequence shown in SEQ ID NO: 1 or 2 are substituted, deleted, or added. It may be an array. Preferably, the number of nucleotides to be substituted, deleted or added is 1 to 3, more preferably 1 or 2, and still more preferably 1.

  When such nucleotide substitution, deletion, or addition is present, the sequence of the DNA aptamer according to the present invention is 90% or more, preferably 93% or more, more preferably 96, with each of the above SEQ ID NOs: 1 or 2. % Or more homologous sequences (hereinafter sometimes referred to as “homologues”). Here, as used herein, the term “homology” refers to the generally accepted meaning in the art. The term is typically referred to when examined by sequence analysis programs (eg, Karlin and Altschul, 1990, PNAS 87: 2264-2268; Karlin and Altschul, 1993, PNAS 90: 5873-5877) or by visual inspection. The number of nucleotides of the subject nucleic acid sequence that match the same nucleotides of the nucleic acid sequence.

  When one or more nucleotides are substituted, the substitution can be made with a universal base. The term “universal base” indicates its commonly accepted meaning in the art. That is, the term generally refers to nucleotide base analogs that form base pairs with each base of standard DNA / RNA almost indistinguishably and are recognized by intracellular enzymes (see, eg, Loakes et al., 1997, J. MoI. Mol.Bio.270: 426-435). Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carbozamide, and nitroazole derivatives (3′-nitropyrrole, 4-nitro Indole, 5-nitroindole, and 6-nitroindole) (Loakes, 2001, Nucleic Acids Res. 29: 2437).

  In addition, the DNA aptamer according to the present invention has no upper limit as long as it has a function of specifically binding to human colon cancer cells. However, in consideration of easiness of synthesis, antigenicity problems, etc., the upper limit of the length of the DNA aptamer in the present embodiment is, for example, 200 bases or less, preferably 150 bases or less, more preferably 100 bases or less. .

When the total number of bases is small, chemical synthesis and mass production are easier, and the cost advantage is great. Moreover, chemical modification is easy, safety in the living body is high, and toxicity is low. The lower limit is the number of bases in SEQ ID NO: 1 or 2 or more, that is, 34 bases or more. The DNA aptamer is preferably single-stranded (ssDNA), but even when a double-stranded structure is partially formed by taking a hairpin loop structure, the length of the DNA aptamer is one. Calculate as the length of the chain.

In a preferred embodiment, the DNA aptamer according to the present invention may be a nucleotide sequence comprising either the sequence of SEQ ID NO: 1 or 2 and a primer recognition sequence on the 5 ′ and 3 ′ terminal sides thereof. That is, in this case, the DNA aptamer is
5′-P ₁ -XP ₂ -3 ′
It has the nucleotide sequence represented by these. Here, X is a nucleotide sequence selected from the sequences shown in SEQ ID NO: 1 or 2, or a sequence comprising 1 to 3 nucleotide substitutions, deletions or additions in those sequences. P ₁ and _{P 2} are the first and second primer recognition sequence introduced for the PCR amplification. Preferably, P ₁ is GCCTGTTGGAGCCCTCCT (SEQ ID NO: 3) and P ₂ is CGCTTATTTCTTGCTCCC (SEQ ID NO: 4).

  The DNA aptamer according to the present invention may be chemically modified in order to increase stability in vivo. Non-limiting examples of such chemical modifications include chemical substitution at the sugar chain moiety (eg 2′-0 methylation), chemical substitution at the phosphate ester moiety (eg phosphorothioation, amino group) , Lower alkylamino groups, acetyl groups, and the like), and chemical substitution at the base moiety. Similarly, it can have additional bases at the 5 'or 3' end. The length of the additional base is usually 5 bases or less. The additional base may be DNA or RNA, but if DNA is used, the stability of the aptamer may be improved in some cases. Examples of such an additional base sequence include ug-3 ′, uu-3 ′, tg-3 ′, tt-3 ′, ggg-3 ′, guuu-3 ′, gttt-3 ′, and tttt-3. Examples of such sequences include, but are not limited to, ', uuuu-3'.

  In addition, the DNA aptamer according to the present invention can have a detection label linked to, for example, the 5 'end or the 3' end for use in the human colorectal cancer cell detection method described later or the detection kit. As the detection label, a fluorescent label is preferable, but a Raman scattering label, an enzyme label, and an infrared label may be used.

As the fluorescent label, a fluorescent labeling agent commonly used in the art can be used. For example, green fluorescent protein (GFP), fluorescent protein, fluorescein isothiocyanate (FITC), 6-carboxyfluorocein-amino Hexyl, fluorescein derivative, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 647, Alexa Fluor 750, rhodamine, 6-carboxytetramethylrhodamine, TAMRA®, phycoerythrin (PE), phycocyanin (PC) ), PC5, PC7, Cy dye, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, TexasRed, allophycocyanin (APC), amino Methylcoumarin acetate (AMCA), Marina Blue, Cascade Blue, Cascade Yellow, Pacific Blue, SPRD, tetramethylrhodamine isothiocyanate (TRITC), R110, mC1B, CellTracker dye, CFSE, JC-1, HKD, FKD, HKD , FDA, Calcein AM, nitrobenzooxadiazole (NBD) group, dimethylaminosulfonylbenzooxadiazole group, acidine (Acd), dansyl (Dns), 7-dimethylaminocoumarin-4-acetic acid (DMACA), 5-((2-Aminoethyl) amino) naphthalene-1-sulfonic acid (EDANS), naphthalene, anthracene Fluorophore can be mentioned can be introduced in commercially available oligonucleotide solid phase synthesis services such fine protoporphyrin 9.

  Furthermore, a quencher (quenching substance) that absorbs fluorescence energy emitted from the fluorescent substance may be further bound in the vicinity of the fluorescent substance. In such an embodiment, the fluorescence is detected by separating the fluorescent substance and the quencher during the detection reaction.

  Examples of enzyme labels include β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like. Further, as a luminescent substrate, luminol, luminol derivatives, luciferin, lucigenin, or the like may be used as a labeling agent.

  The Raman scattering label is not particularly limited as a substituent having the ability to bind to the metal surface, for example, at the 5 ′ end or 3 ′ end of the fluorescently labeled aptamer, but a thiol (SH) group, an alkylamino group Then, by introducing an aromatic amino group and a carboxyl group and adsorbing them on the surface of, for example, a gold nanoparticle, cell recognition is performed by detecting enhanced Raman scattered light emitted from the aptamer adsorbing metal particle. In this case, although not particularly limited, gold, silver, copper, iron, silicon, quantum dots, or the like can be used as the metal nanoparticles.

2. Selection of DNA aptamer The DNA aptamer of the present invention can be selected and obtained using in vitro selection methods well known in the art. As a preferable example of such a technique, an in vitro evolution method (System Evolution of Ligands by EXPERIMENTAL ENRICHEMENT: SELEX method) is used.

  In the SELEX method, nucleic acid ligands (aptamers) that bind to a target substance are selected multiple times and exponential amplification by PCR (Polymerase Chain Reaction) is repeated a plurality of times. Strand DNA, RNA).

  Further, as an improved technique, for example, Guo, et al. , Int. J. Mol. Sci. 9 (4): 668, 2008 (Non-Patent Document 4), it is preferable to use the Cell-SELEX method. Since this method can use the cell itself as a target, it does not require analysis of membrane proteins on the cell surface and can simultaneously select multiple aptamers that can bind to the cell surface, compared to the conventional SELEX method. And an aptamer that more specifically binds to the target cell can be selected.

  In addition to these, the DNA aptamer of the present invention can be selected and obtained using a method well known in the art.

  As described above, the “in vitro selection method” selects an aptamer molecule having affinity for a target molecule or cell from a pool of nucleic acid molecules (so-called DNA pool) containing a random nucleotide sequence, and determines the affinity. It is a method of eliminating molecules that do not have. By repeating the cycle of amplifying only the selected aptamer molecule by PCR or the like, and further selecting by affinity, aptamer molecules having strong binding ability can be concentrated.

  Specifically, first, a single-stranded nucleic acid molecule containing a random nucleotide sequence (base sequence) region of about 20 to 300 bases, preferably 30 to 150 bases, more preferably about 30 to 100 bases, for example, oligo DNA is prepared. To do. In order to enable PCR amplification, it is preferable to use an oligo DNA having a base sequence to serve as a primer at both ends.

The primer recognition sequence portion may have an appropriate restriction enzyme site so that the primer portion can be excised with a restriction enzyme after PCR amplification. The length of the primer recognition sequence portion to be used is not particularly limited, but is about 20 to 50, preferably about 20 to 30 bases. Further, in order to make it possible to separate the single-stranded DNA after PCR amplification by electrophoresis or the like, the 5 ′ end may be labeled with a radiolabel, a fluorescent label, or the like.

  Next, the nucleic acid molecule (library pool) having the random nucleotide sequence obtained above and the target cell are mixed at an appropriate concentration ratio and incubated under an appropriate condition. After incubation, the mixture is centrifuged to separate the nucleic acid molecule-target cell complex and free nucleic acid molecule. The supernatant portion of the separation solution is removed, and a cell-bound nucleic acid sequence is amplified by performing a PCR reaction using the obtained nucleic acid molecule-target cell complex. Thereafter, the nucleic acid molecule forming a complex with the target cell is made into a single strand according to a technique well known in the art. As such a technique, for example, separation utilizing the binding of streptavidin-immobilized magnetic particles and biotin can be mentioned. Thereby, ssDNA having cell binding ability can be separated from the amplified nucleic acid duplex, and unnecessary coexisting substances contained in the PCR reaction solution such as DNA polymerase can be removed. Thereafter, the same operation is performed using the recovered ssDNA as a library pool.

  A series of operations from mixing the above-described nucleic acid molecule and target cell, separation of the nucleic acid molecule bound to the target cell, PCR amplification, and use of the amplified nucleic acid molecule again for binding to the target cell are performed in several rounds. By repeating rounds, nucleic acid molecules that bind to target cells more specifically can be selected. The obtained nucleic acid molecule can be subjected to sequence analysis by a technique well known in the art.

3. Composition for detecting cancer cell, detection method and kit As described above, the DNA aptamer according to the present invention has a function of specifically binding to human colon cancer cells. The detection composition containing the DNA aptamer of the present invention can be suitably used for detection, and can also be used as a tumor marker for human colorectal cancer.

  Specifically, the detection composition containing the DNA aptamer of the present invention is brought into contact with a sample collected from a living body selected from the group consisting of human colon cells, colon tissue, blood, serum and plasma, and then the sample The presence of human colorectal cancer cells is detected by observing the response (the presence or absence of a signal) due to the binding between the DNA and the aptamer.

The sample collected from the living body is collected from an animal, and the form thereof is not particularly limited as long as it is a sample that can be secured with minimal invasiveness, a secretory fluid, an in vitro cell culture fluid component sample, or the like.

In addition, the “response” for detecting the presence of colon cells is preferably a fluorescence response or a Raman scattering response, and as described above, in the fluorescence response, at the 5 ′ end or 3 ′ end of the DNA aptamer. It is preferable to link a fluorescent labeling agent such as TAMRA (registered trademark) or FITC. In the Raman scattering response, a fluorescent labeling agent such as Cy3.5 (registered trademark), TAMRA (registered trademark), FITC or the like is linked to the 5 ′ end or 3 ′ end of the DNA aptamer, and the thiol (SH) group, It is preferable to introduce an alkylamino group, an aromatic amino group, or a carboxyl group and to adsorb it on, for example, the gold nanoparticle surface.

  In addition, the composition for detecting human colon cancer cells of the present invention can be provided as a kit containing a DNA aptamer in order to improve its convenience and portability. In the kit, the DNA aptamer can be provided usually in the form of an aqueous solution dissolved at an appropriate concentration, or in the form of a DNA array in which the DNA aptamer is immobilized on a solid phase carrier.

  For example, biotin is bound to the end of a DNA aptamer to form a complex, streptavidin is immobilized on the surface of the solid support, and the DNA aptamer is immobilized on the solid support by the interaction of biotin and streptavidin. can do. The kit may appropriately contain other reagents as necessary. For example, additives such as a solubilizing agent, a pH adjusting agent, a buffering agent, and a tonicity agent may be used as an additive. These blending amounts can be appropriately selected by those skilled in the art.

4). Pharmaceutical composition and DDS
In another aspect, the present invention provides a pharmaceutical composition for detecting, diagnosing, preventing or treating metastasis of human colorectal cancer, comprising the above DNA aptamer. Preferably, the pharmaceutical composition contains, in addition to a DNA aptamer, an effective amount of a pharmaceutical compound (active ingredient) for detection, diagnosis, metastasis prevention or treatment of human colorectal cancer, and a pharmaceutically acceptable carrier. Can do. For example, there is a possibility that this aptamer bound to gold nanoparticles can be used as a reagent for cancer thermotherapy.

  The “human colorectal cancer” is preferably human colorectal cancer associated with Colo205 cells. “Metastasis prevention or treatment” can include cancer cell release, migration, metastasis, suppression of invasion or proliferation, induction of apoptosis. Suppression of metastasis means that cancer cells reach different sites from the primary lesion and prevent secondary cancer from occurring at the sites.

  The active ingredient contained in the pharmaceutical composition of the present invention is not particularly limited as long as it is effective for preventing or treating metastasis of human colon cancer, but is preferably an anticancer agent. Examples of such anticancer agents include alkylating agents, antimetabolites, antitumor antibiotics, chemotherapeutic agents, molecular targeted therapeutic agents, other anticancer agents other than these.

Examples of the alkylating agent include nitrogen mustard, chlorambutyl, dibromodalcitol, thiotepa, carmustine, busulfan and the like.

  Examples of the antimetabolite include 6-mercaptopurine, fluorouracil, tegafur, doxyfluridine, cytarabine, enocitabine, methotrexate and the like.

  Antibiotics include mitomycin C, bleomycin, pepromycin, doxorubicin, THP-adriamycin, actinomycin D, and other anticancer agents include amrubicin hydrochloride, irinotecan hydrochloride, ifosfamide, etoposidetate, gefinitib, cyclophosphamide, cisplatin, trastuzumab , Fluorouracil, imatinib mesylate, methotrexate, rituxan, adriamycin, carboplatin, tamoxifen, camptothecin, melphalan, L-asparaginase, acecraton, schizophyllan and the like.

  Targets of molecular targeted therapeutic agents are classified into cell surface antigens, growth factors / receptors / signal transduction systems, cell cycle, apoptosis, telomere / telomerase, metastasis / angiogenesis.

  Molecular targeted therapeutics that target cell surface antigens are often antibodies. Representative examples include Rituximb (Rituxan), Trastuzumab, Cetuximab, Oncophage, and Provenge.

  The molecular targeted therapeutic agent related to the growth factor / receptor / signal transduction system is not particularly limited, and examples thereof include Trastumab, c-kit and BCR-ABL tyrosine kinase inhibitors, particularly EGFR tyrosine kinase.

  Examples of molecular targeted therapeutic agents for colorectal cancer include Bevacizumab, Thalidomide, Lenalimide, which is an antibody against VEGF itself.

  Currently studied as molecular target therapeutics for colorectal cancer include 1) anti-VEGF antibody Bevacizumab, 2) anti-EGFR antibody Cetuximab, and 3) anti-EGFR antibody Panitumab.

  Examples of the pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present invention include excipients such as sucrose and starch, binders such as cellulose and methylcellulose, disintegrants such as starch and carboxymethylcellulose, and stearic acid. Lubricants such as magnesium and aerosil, fragrances such as citric acid and menthol, preservatives such as sodium benzoate and sodium bisulfite, stabilizers such as citric acid and sodium citrate, suspending agents such as methylcellulose and polyvinylpyrrolidone, and interfaces Examples include, but are not limited to, dispersants such as active agents, diluents such as water and physiological saline, and base waxes.

  In order to promote introduction of the pharmaceutical composition of the present invention into cancer cells, the composition may further contain a reagent for nucleic acid introduction. As the reagent for introducing nucleic acid, cationic lipids such as atelocollagen, liposome, nanoparticle, lipofectin, lipfectamine, DOGS (transfectam), DOPE, DOTAP, DDAB, DHDAB, HDAB, polybrene, or polyethyleneimine are used. I can do it.

  The pharmaceutical composition of the present invention can be administered to, for example, mammals (eg, humans, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.).

  The pharmaceutical composition of the present invention can take various dosage forms, for example, capsules, tablets, liquids and the like, but is not limited, but more generally, it is liquefied and made into injections. Or an oral agent or a sustained-release agent.

  The injection can be prepared by a well-known method in the technical field. For example, it can be prepared by dissolving in an appropriate solvent such as sterilized water, buffer solution, physiological saline, etc., sterilizing by filtration with a filter or the like, and then filling in an aseptic container.

  Oral preparations are formulated into dosage forms such as tablets, granules, fine granules, powders, soft or hard capsules, solutions, emulsions, suspensions, syrups and the like.

  As a sustained release agent, it is formulated into dosage forms such as tablets, granules, fine granules, powders, soft or hard capsules, microcapsules and the like. In the case of formulating, a stabilizer such as albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol or the like can be preferably added.

  Furthermore, in the formulation of the pharmaceutical composition of the present invention, necessary auxiliary additives such as excipients, solubilizers, antioxidants, soothing agents, tonicity agents and the like may be included.

  In the case of a liquid preparation, it is desirable to store it after removing moisture by freeze storage or freeze drying. The freeze-dried agent is used by re-dissolving it by adding distilled water for injection at the time of use.

  In the case of a sustained release agent, examples of sustained release carriers include soluble collagen or soluble collagen derivatives, proteins such as gelatin, ceramic porous bodies, polyamino acids, polylactic acid, chitin or chitin derivatives, water-swellable polymer gels, etc. Can be used.

  The pharmaceutical composition of the present invention can be administered by an appropriate administration route depending on the form. It can be administered orally or parenterally, but is preferably administered parenterally. For example, it can be administered into a vein, artery, subcutaneous, intramuscular, etc. in the form of an injection. Moreover, it can administer by implanting in the living body, for example, an affected part, subcutaneous, intramuscular, etc. in the form of a sustained release agent.

  The dose and frequency of administration vary depending on the purpose of administration, administration method, type and size of cancer, and the situation (gender, age, body weight, etc.) of the subject of administration, but basically a desirable dosage form for the above active ingredients Follow.

  In addition, by attaching or encapsulating the DNA aptamer of the present invention to the surface of a transport material such as a liposome or a nanoparticle, a pharmaceutical component contained in the transport material can be selectively transported to human colon cancer cells. Accordingly, in a further aspect, the present invention provides a drug delivery system for preventing or treating metastasis of human colorectal cancer, comprising the above DNA aptamer.

  The pharmaceutical ingredient that can be transported by the drug delivery system is typically the above-described anticancer agent, but as long as it is a substance useful for preventing or treating metastasis of human colon cancer, in addition to these, a toxin, a cancer growth inhibitory gene, It can also be a siRNA (small interfering RNA) that inhibits the expression of a suicide gene or a gene that plays an important role in the growth and metastasis of human colon cancer.

  The present invention uses a pretargeting strategy to bind the DNA aptamer of the present invention to a targetable conjugate and administer it to a subject, optionally clearing the DNA aptamer remaining at the non-targeted site, and then diagnosis. The substance or therapeutic substance can be bound to the targeting site. A pre-targeting strategy based on avidin or streptavidin and biotin can be used as described in Goldenberg US Pat. No. 5,525,338 entitled “Detection and Therapy of Lesions with Biotin / Avidin Conjugates.” . The avidin / streptavidin system is highly versatile and has been used in several configurations. The DNA aptamer of the present invention can be coupled with streptavidin or biotin. This is followed by administration of a diagnostic or therapeutic substance conjugated with biotin or avidin / streptavidin, respectively. Another configuration can use a three-step approach that initially targets biotin-conjugated DNA aptamers of the invention, followed by streptavidin / avidin cross-linking, followed by biotin-conjugated diagnostic agent or A therapeutic agent is given. Methods for synthesizing biotin and avidin / streptavidin conjugates for the DNA aptamers of the present invention are well known in the art.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by these.

[Example 1] Selection of aptamers Aptamers that specifically bind to COLO205 colon cancer cells were selected from a DNA pool having a random sequence of 34 bases using the Cell-SELEX method. Each step of the Cell-SELEX method is as follows.
The random DNA to be used is a single strand, which is hereinafter referred to as a sense strand.

The DNA pool used was an oligo DNA having the following sequence with a total length of 70 bases and a random sequence part (N) of 34 bases.
DNA pool: Random 34 (manufactured by Nippon Genetic Institute)
- _{sequence: 5'-GCC TGT TGT GAG CCT} CCT (N 34) CGC TTA TTC TTG TCT CCC-3 '( SEQ ID NO: 5)
・ Length: 70 bases (random base is 34 bases in the center)
Molecular weight: 21391.3 g / mol
Molar extinction coefficient: 630475 L / mol · cm

The base sequences at both ends of the random sequence are primer sequences used during PCR.
Prepare 100 μL of DNA pool prepared to a concentration of 1 μM. COLO205 cells (cell concentration 1 × 10 ⁶ cells / mL) cultured on a petri dish with a culture medium RPMI-1640 were removed with an EDTA-containing 0.5% trypsin solution and dispersed again in 200 μL of the same medium; The prepared 1 μM DNA pool (100 μL) was mixed. Thereafter, it was left in a 37 ° C. incubator for 1 hour to promote the interaction between cells and DNA. Thereafter, target-binding DNA and non-binding DNA were separated by centrifugation at 1500 rpm for 5 minutes, the supernatant was removed, and the precipitated cells were washed with 1 mL of phosphate buffer. After repeating this washing operation twice, 200 mL of phosphate buffer was added to the cells, and the cells were heated at 95 ° C. for 10 minutes to peel off the DNA adsorbed on the cells. After completion, the cells were not mixed and centrifuged at 13000 × g for 5 minutes, and the supernatant was collected.

Since the collected supernatant liquid contains sense strand DNA having cell adsorption ability, it was amplified using PCR. As a primer, an 18-base primer complementary to the common sequence of the random DNA was used. As will be described later, biotin modification is applied to the 5 ′ end side of the primer constituting the antisense strand to enable separation of single-stranded DNA (sense strand). Since the amplified DNA has a double-stranded structure, the target sense strand was recovered from this double strand by the method shown in FIG. 1 using magnetic particles. That is, streptavidin is added to the amplified DNA and adsorbed on the magnetic fine particles. After collecting the magnetic fine particles with a magnet, the supernatant is removed, and then the single-stranded DNA that is not bound to the magnetic fine particles is recovered in the supernatant by denaturation with an alkaline buffer. did.
The recovered single-stranded DNA was mixed with the target cells again, and the above procedure was repeated. After 6 cycles, sequence analysis was performed using the next-generation sequencer ionPGM ^™ , and a red fluorescent dye (CY3.5) was introduced into the 5 ′ end of only DNA having a sequence of 1000 or more homologous strands to perform a cell recognition test. It was.
The structure of the actually used DNA is as follows.

<SEQ ID NO: 1>
5'-CCGTGGTGTGGGGGTTGGGGGGTTGTCGTCCGCG-3 '
<SEQ ID NO: 2>
5′-CGAGTGGGGGTATGCTTGGGGGTGTCGTTCGCCG-3 ′

[Example 2] Staining of colon cancer cells COLO205 with fluorescently labeled DNA aptamer COLO205 cells were cultured in a culture dish and cultured until the number of cells reached 10 ⁶ to 10 ⁷ . A 100 μM solution was prepared by modifying the 5 ′ end of a DNA aptamer having affinity for COLO205 cells found by sequencer analysis with CY3.5 (trademark). This was added to a final concentration of 10 μM while being dispersed in the culture dish. This was left still in an incubator at 37 ° C. for 1 hour. Thereafter, the cells were washed three times with HBSS (Hank's Balanced Salt Solution), and further observed with an inverted fluorescence microscope IX51 made by Olympus in a state where 2 mL of HBSS was added.

The results are shown in FIGS.
FIG. 2 is a real image of Colo205 cells stained with a DNA aptamer having the base sequence shown in SEQ ID NO: 1, and FIG. 3 is a diagram showing the fluorescence imaging thereof.
4 is a real image of Colo205 cells stained with a DNA aptamer having the base sequence shown in SEQ ID NO: 2, and FIG. 5 is a diagram showing the fluorescence imaging thereof.
As is clear from these results, it can be seen that, in most cells, strong fluorescence is exhibited particularly in cells that adhere to the culture dish.

Claims (19)

  A DNA aptamer comprising the nucleotide sequence represented by SEQ ID NO: 1 or 2, and specifically binding to human colon cancer cells.   The nucleotide sequence represented by SEQ ID NO: 1 or 2 has a sequence containing 1 to 3 nucleotide substitutions, deletions or additions, and specifically binds to human colon cancer cells DNA aptamer. A DNA aptamer that specifically binds to human colon cancer cells,
5'-P1-X-P2-3 '
Having a nucleotide sequence represented by:
X has 1) a nucleotide sequence represented by SEQ ID NO: 1 or 2, or 2) a nucleotide sequence represented by SEQ ID NO: 1 or 2, which has a sequence containing 1 to 3 nucleotide substitutions, deletions, or additions. And a nucleotide sequence that specifically binds to human colon cancer cells,
P1 and P2 are DNA aptamers that are first and second primer recognition sequences introduced for PCR amplification. The DNA aptamer according to claim 3, wherein P1 is a first primer recognition sequence represented by SEQ ID NO: 3, and P2 is a second primer recognition sequence represented by SEQ ID NO: 4.   The DNA aptamer according to any one of claims 1 to 4, wherein the human colon cancer cells are Colo205 cells.   6. The method according to claim 1, comprising at least one chemical modification selected from the group consisting of chemical substitution at the sugar chain moiety, chemical substitution at the phosphate ester moiety, and chemical substitution at the nucleobase moiety. Or a DNA aptamer according to claim 1.   The DNA aptamer according to any one of claims 1 to 6, which has a fluorescent label at the 5 'end or 3' end.   The fluorescent label is green fluorescent protein (GFP), fluorescent protein, fluorescein isothiocyanate (FITC), 6-carboxyfluorocein-aminohexyl, fluorescein derivative, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor. 647, Alexa Fluor 750, rhodamine, 6-carboxytetramethylrhodamine, TAMRA (registered trademark), phycoerythrin (PE), phycocyanin (PC), PC5, PC7, Cy dye, Cy3, Cy3.5, Cy5, Cy5. 5, Cy7, Texas Red, allophycocyanin (APC), aminomethylcoumarin acetate (AMCA), Marina Blue, Casc de Blue, Cascade Yellow, Pacific Blue, SPRD, Tetramethylrhodamine isothiocyanate (TRITC), R110, mC1B, CellTracker dye, CFSE, JC-1, PKH, DCFH-DA, DHR, FDA, Calcein AM, Nitrobenzodia Azole (NBD) group, dimethylaminosulfonylbenzooxadiazole group, acidine (Acd), dansyl (Dns), 7-dimethylaminocoumarin-4-acetic acid (DMACA), 5-((2-aminoethyl) amino ) Emission wavelength including one or more selected from naphthalene-1-sulfonic acid (EDANS), naphthalene, anthracene and protoporphyrin 9 to 421 nm to 7 An organic fluorophore 2 nm, DNA aptamer according to claim 7.   A DNA aptamer, wherein the DNA aptamer according to any one of claims 1 to 8 is adsorbed on the surface of metal nanoparticles (quantum dots) that can emit fluorescence.   The one or more particles selected from gold, silver, copper, iron and silicon, wherein the DNA aptamer according to any one of claims 1 to 8 having a fluorescent label at the 5 'end or the 3' end is a metal having Raman scattering activity DNA aptamer adsorbed on the surface.   The DNA aptamer according to any one of claims 1 to 6, wherein the 5 'end or 3' end is linked to biotin, avidin or streptavidin or other specific binding tag peptide.   A composition for detecting human colon cancer cells, comprising the DNA aptamer according to any one of claims 1 to 11.   A kit for detecting human colon cancer cells, comprising the DNA aptamer according to any one of claims 1 to 11.   A method for detecting human colon cancer, comprising using the DNA aptamer according to any one of claims 1 to 11.   Contacting the DNA aptamer with a sample collected from a living body selected from the group consisting of human colon cells, colon tissue, blood, serum, and plasma, and observing a response due to the binding between the sample and the DNA aptamer. The detection method of Claim 14 including the process of detecting presence of a human colon cancer cell by.   The detection method according to claim 15, wherein the response is a fluorescence response.   A pharmaceutical composition for preventing or treating metastasis of human colorectal cancer, comprising the DNA aptamer according to any one of claims 1 to 11.   Use of the DNA aptamer according to any one of claims 1 to 11 for the manufacture of a pharmaceutical composition for preventing or treating metastasis of human colon cancer.   A drug delivery system for preventing or treating metastasis of human colon cancer, comprising the DNA aptamer according to any one of claims 1 to 11.