JP2014217311A - Dna aptamer bound to cancer cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel DNA aptamer useful to diagnosis, treatment, metastasis prevention and the like of lung cancer.SOLUTION: A DNA aptamer specifically bound to non-small-cell lung cancer cells has a nucleotide sequence represented by 5'-P1-X-P2-3', wherein X is 1) a nucleotide sequence selected from a specific sequence, or 2) in the nucleotide sequence selected from a specific sequence, a sequence comprising a substitution, deletion, or addition of 1-3 nucleotides; P1 and P2 are a first and a second primer recognition sequences introduced for a PCR amplification. The DNA aptamer comprises at least one chemical modification selected from a group consisting of a chemical replacement at a sugar chain moiety, a chemical replacement at a phosphoric ester moiety and a chemical replacement at a nucleic-acid-base moiety, having a fluorescent labeling at 5' end or 3' end.

Description

  The present invention relates to a DNA aptamer that can specifically bind to cancer cells, particularly non-small cell lung cancer cells, and a composition comprising the DNA aptamer.

  DNA is a biopolymer composed of four nucleobases: guanine (G), cytosine (C), adenine (A), and thymine (T), and plays a role mainly in the preservation, expression and propagation of genes in vivo. Yes. However, in recent years, it has been clarified that there is DNA that specifically binds to a target substance, and research using DNA itself as a functional polymer has been activated. Nucleotide sequences that have high binding affinity for low molecular weight compounds such as drugs, DNA, RNA, peptides, and proteins have been discovered and selected so far, and they have selective recognition ability for specific molecules. DNA is called “DNA aptamer”.

  The selection of nucleic acid aptamers such as DNA aptamers is generally based on an in vitro selection method, in particular, a method based on a combinatorial chemistry called the evolutionary method of in vitro in vitro (the SELEX method). (For example, Non-Patent Documents 1 and 2; Patent Document 1). The SELEX method is a nucleic acid molecule (single-stranded DNA, RNA) having affinity for a target substance by selecting a nucleic acid ligand (aptamer) that binds to the target substance and repeating exponential amplification by PCR multiple times. Is to get 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 3) has been reported.

  Such a DNA aptamer is similar to an antibody in that it is a biopolymer with molecular recognition ability, but is easier to synthesize and modify than an antibody; it has excellent stability against environmental changes such as heat and pH. Theoretically, aptamers can be obtained for any substance, and the target substance to be targeted is not limited; it can be amplified quickly and inexpensively by PCR and other techniques, and the cost is excellent. It has the following advantages.

  On the other hand, the mortality rate of lung cancer in Japan has consistently increased since 1950, and since 1993, the number of lung cancer deaths has become the highest number of cancer deaths. Lung cancer is roughly classified into two types, non-small cell lung cancer and small cell lung cancer, and the former accounts for 80 to 85% of all lung cancers. Non-small cell lung cancer is classified into squamous cell carcinoma, adenocarcinoma, large cell carcinoma, etc. according to its histological type, 2/3 of which are already unresectable at the time of discovery, and drug therapy is the center of treatment. Increasing the therapeutic results of advanced lung cancer, that is, improving the results of chemotherapy, is essential for improving the therapeutic results of non-small cell lung cancer. For this purpose, a biomolecule that distinguishes normal cells from cancer cells and specifically binds to cancer cells is required. Therefore, there is a need for the development of new lung cancer biomarkers useful for earlier diagnosis and development of effective therapeutic agents.

Lee, et al. Curr Opin Chem Biol., 10 (3): 282,2006. Gilbert, et al., Circulation., 116 (23): 2678, 2007. Guo, et al., Int. J. Mol. Sci., 9 (4): 668, 2008. International Publication WO / 9119813

  Under the background as described above, an object of the present invention is to provide a novel DNA aptamer useful for diagnosis, treatment, prevention of metastasis, etc. of lung cancer.

  As a result of intensive studies to solve the above problems, the present inventors have identified a nucleotide sequence having a specific binding ability to non-small cell lung cancer 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 non-small cell lung cancer cells, and have completed the present invention.

That is, the present invention, in one embodiment,
(1) A DNA aptamer having at least one nucleotide sequence selected from the sequences represented by SEQ ID NOs: 1 to 10, and specifically binding to non-small cell lung cancer cells;
(2) In at least one nucleotide sequence selected from the sequences represented by SEQ ID NOs: 1 to 10, it has a sequence containing 1 to 3 nucleotide substitutions, deletions or additions, and is used for non-small cell lung cancer cells. A DNA aptamer characterized in that it specifically binds to;
(3) a DNA aptamer that specifically binds to non-small cell lung cancer cells,
5′-P ₁ -XP ₂ -3 ′
Having a nucleotide sequence represented by
Here, X is 1) a nucleotide sequence selected from the sequences shown in SEQ ID NOs: 1 to 10, or 2) 1 to 3 nucleotides in the nucleotide sequence selected from the sequences shown in SEQ ID NOs: 1 to 10 A sequence containing substitutions, deletions or additions,
P ₁ and _{P 2} are the first and second primer recognition sequence introduced for PCR amplification,
The DNA aptamer;
(4) The DNA aptamer according to (3) above, wherein P ₁ is a first primer recognition sequence represented by SEQ ID NO: 11, and P ₂ is a second primer recognition sequence represented by SEQ ID NO: 12.
(5) The DNA aptamer according to any one of (1) to (4), wherein the non-small cell lung cancer cells are A549 cells;
(6) 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. The DNA aptamer according to any one of to (5);
(7) The DNA aptamer according to any one of (1) to (6) above, which has a fluorescent label at the 5 ′ end or the 3 ′ end; and (8) the fluorescent label is 6-carboxytetramethylrhodamine, The DNA aptamer according to (7) above, which is fluorescein isothiocyanate, 6-carboxyfluorocein-aminohexyl, or cyanine fluorescent dye.

In another aspect, the present invention relates to detection of lung cancer cells by the above DNA aptamer, and in particular,
(9) A composition for detecting lung cancer cells, comprising the DNA aptamer according to any one of (1) to (8) above;
(10) A kit for detecting lung cancer cells, comprising the DNA aptamer according to any one of (1) to (8) above;
(11) A method for detecting lung cancer cells, comprising using the DNA aptamer according to any one of (1) to (8) above; and (12) lung DNA, lung tissue, blood, Contacting with a sample collected from a living body selected from the group consisting of serum, plasma, saliva, and sputum, and detecting the presence of lung cancer cells by observing a response due to the binding between the sample and a DNA aptamer The detection method according to (11) above, comprising:
(13) The detection method according to (12), wherein the response is a fluorescence response.

In a further aspect, the present invention relates to the use of the above DNA aptamer in pharmaceutical compositions and drug delivery systems, in particular,
(14) A pharmaceutical composition for preventing or treating lung cancer metastasis, comprising the DA aptamer according to any one of (1) to (8) above;
(15) Use of the DNA aptamer according to any one of (1) to (8) above for producing a pharmaceutical composition for preventing or treating metastasis of lung cancer;
(16) A drug delivery system for preventing or treating lung cancer metastasis comprising the DNA aptamer according to any one of (1) to (8) above.

  According to the present invention, it is possible to efficiently detect such lung cancer cells by using a novel DNA aptamer that specifically binds to non-small cell lung cancer cells. In particular, by applying to a kit containing a DNA aptamer provided with a detection site such as a fluorescent label, simple and high-throughput detection or imaging using a lung cell or tissue collected from a living body as a measurement target can be performed. Detection of such lung cancer cells makes it possible to diagnose the onset of cancer, the presence or absence of metastasis, the prognosis of cancer, and the grade of malignancy.

  Further, since the DNA aptamer of the present invention has the property of being able to specifically bind to non-small cell lung cancer cells, the above-mentioned DNA aptamer can be used by conjugating a drug such as an anticancer agent to the DNA aptamer. Since the drug can act on the target site reliably, it can be expected to be useful as a pharmaceutical composition for preventing or treating lung cancer metastasis or a drug delivery system for such a pharmaceutical composition.

  Furthermore, since the consensus sequence in the DNA aptamer of the present invention is a relatively short region of only about 30 bases, it is possible to suppress the labor and cost for production, and to perform desired chemical modification or the like depending on various applications. There is also an advantage that it is easy to add a further function.

FIG. 1 is a schematic diagram showing the formation of single-stranded double-stranded DNA using magnetic particles. FIG. 2 shows the sequence of the DNA aptamer of the present invention selected by sequencer analysis. FIG. 3 is a fluorescence imaging diagram of A549 cells by a DNA aptamer having SEQ ID NO: 1. FIG. 4 is a fluorescence imaging diagram of A549 cells by a DNA aptamer having SEQ ID NO: 2. FIG. 5 is a fluorescence imaging diagram of A549 cells by a DNA aptamer having SEQ ID NO: 3. FIG. 6 is a fluorescence imaging diagram of A549 cells with a DNA aptamer having SEQ ID NO: 4. FIG. 7 is a fluorescence imaging diagram of A549 cells with a DNA aptamer having SEQ ID NO: 5. FIG. 8 is a fluorescence imaging diagram of A549 cells with a DNA aptamer having SEQ ID NO: 8. FIG. 9 is a fluorescence imaging diagram of A549 cells by a DNA aptamer having SEQ ID NO: 9.

  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 that can specifically recognize a target molecule or substance, and the DNA aptamer according to the present invention is specific to non-small cell lung cancer cells. It is a single-stranded oligo DNA having a function of binding to.

  In general, non-small cell lung cancer is classified into squamous cell carcinoma (ASC), adenocarcinoma (ADC), and large cell carcinoma (LCC) mainly depending on the tissue type. Thus, non-limiting examples of “non-small cell lung cancer cells” herein include squamous cell carcinoma cells NCI-H226, NCI-H647; lung adenocarcinoma cells A549, LC319, PC-3. , PC-9, PC-14, A427, NCI-H1373, and LX1 which is a large cell lung cancer cell. The binding target of the DNA aptamer according to the present invention is preferably lung adenocarcinoma cells, more preferably A549 cells (for details of A549 cells, see Giard, DJ, Aarson, SA, Todaro, G.J., Arstain, P., Kersey, J.H., Dosik, H., and Parks, W.P. In vitro culture of human der smer slid Natl. Cancer Inst., 51: 1417-1423, 1973, etc.).

In a typical embodiment, the DNA aptamer according to the present invention has a nucleotide sequence represented by any of SEQ ID NOs: 1 to 10 shown below. The nucleotide sequence is written from left to right in the direction from the 5 ′ end to the 3 ′ end.
<SEQ ID NO: 1>
ATA TTG GTG TTC GGT ATT TGG TGG TGT TTG GTC
<SEQ ID NO: 2>
GGA CAC CAG ACC ACG TAA TAA ACA CAT AGC CCT T
<SEQ ID NO: 3>
GCA TTG GTG GGT TTG TTC GGT TAC TGT TTG TGT C
<SEQ ID NO: 4>
ACG TTG GGT GTG TTC GGC TTG GTG CAG TTC GCC G
<SEQ ID NO: 5>
ATC GGT CGG TGG TGC CGG GTG GTG CAG TTC GCC A
<SEQ ID NO: 6>
AGA CCC ACC GCA AAC ACC AGT AAC CAC CTT GGT T
<SEQ ID NO: 7>
TGG GTT GGA AGA GTG GAC ATG GCT GCC GTC CGC C
<SEQ ID NO: 8>
TTA AAG TGG GAC CGT CTT TTG TGT GGT TCC GCC C
<SEQ ID NO: 9>
GGA CAC ACC AAC CCA GTA CTA CAC GTA ACT TAA C
<SEQ ID NO: 10>
ACT GGT GAT ATC GCG GTG GGT TGT AGT GTC ACC T

  As long as the DNA aptamer according to the present invention has a function of specifically binding to non-small cell lung cancer cells, one or a plurality of nucleotides in the above SEQ ID NOs: 1 to 10 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. In addition, 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 to 10. % 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. 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. 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).

  Moreover, the DNA aptamer according to the present invention has no upper limit as long as it has a function of specifically binding to non-small cell lung cancer cells. However, in consideration of easiness of synthesis and antigenicity problems, 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 NOS: 1 to 10 or more, that is, 33 bases or 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 any one of the sequences of SEQ ID NOs: 1 to 10 and a primer / 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 NOs: 1 to 10, or a sequence containing 1 to 3 nucleotide substitutions, deletions or additions in these sequences. P ₁ and _{P 2} are the first and second primer recognition sequence introduced for the PCR amplification. Preferably, P ₁ is GCC TGT TGT GAG CCT CCT (SEQ ID NO: 11) and P ₂ is CGC TTA TTC TTG TCT CCC (SEQ ID NO: 12).

  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 alkylamine 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 a method for detecting lung cancer cells described later or the detection kit. As such a detection label, a fluorescent label is preferable, but an enzyme label or an infrared label may be used. As the fluorescent label, a fluorescent labeling agent commonly used in the art can be used. For example, 6-carboxytetramethylrhodamine (TAMRA (trademark)), fluorescein isothiocyanate (FITC), 6-carboxyfluorothein- Examples thereof include fluorophores that can be introduced by commercially available oligonucleotide solid phase synthesis services such as aminohexyl (FAM) and cyanine fluorescent dyes (Cy3, Cy5). 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, etc.) may be used as a labeling agent.

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. The SELEX method is a nucleic acid molecule (single-stranded DNA, RNA) having affinity for a target substance by selecting a nucleic acid ligand (aptamer) that binds to the target substance and repeating exponential amplification by PCR multiple times. Is to get As an improved technique, it is preferable to use a Cell-SELEX method as described in, for example, Guo, et al., Int. J. Mol. Sci., 9 (4): 668, 2008. 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. Sensing composition cancer cells, detection methods, and kits as described above, DNA aptamers according to the present invention has a function to specifically bind to non-small cell lung cancer cells, of the lung cancer cells The composition for detection that can be suitably used for detection and contains the DNA aptamer of the present invention can also be used as a tumor marker for non-small cell lung cancer.

  Specifically, the detection composition comprising 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 lung cells, lung tissue, blood, serum, plasma, saliva, and sputum, Thereafter, the presence of lung cancer cells is detected by observing a response (the presence or absence of a signal) due to the binding between the sample and the DNA aptamer. The “sample collected from a living body” is a sample collected from an animal, preferably a human, and is in the form of a sample that can be secured with minimal invasiveness or a secretory fluid, in vitro cell culture fluid component sample, etc. It is not limited. The “response” for detecting the presence of lung cancer cells is preferably a fluorescence response. Therefore, as described above, the fluorescence of TAMRA ™ FITC or the like is present at the 5 ′ end or 3 ′ end of the DNA aptamer. It is preferable to link a labeling agent.

  Moreover, the composition for detecting lung cancer cells of the present invention can also be provided as a kit containing a DNA aptamer in order to improve its simplicity 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 preventing or treating lung cancer metastasis comprising the above DNA aptamer. Preferably, in addition to the DNA aptamer, the pharmaceutical composition can contain an effective amount of a pharmaceutical compound (active ingredient) for preventing or treating metastasis of lung cancer, and a pharmaceutically acceptable carrier. For example, there is a possibility that this aptamer bound to gold nanoparticles can be used as a reagent for cancer thermotherapy.

  The “lung cancer” is a non-small cell lung cancer including squamous cell carcinoma (ASC), adenocarcinoma (ADC), and large cell carcinoma (LCC), preferably an adenocarcinomas associated with A549 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 a different site from the primary lesion and prevent secondary cancer from occurring at the site.

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 lung cancer metastasis, but is preferably an anticancer agent. Examples of such anticancer agents include alkylating agents, antimetabolites, antitumor antibiotics, chemotherapeutic agents, and other anticancer agents. Examples of alkylating agents include nitrogen mustard, chlorambutyl, dibromodalcitol, thiotepa, carmustine, and busulfan. Antibiotics such as mitomycin C, bleomycin, peplomycin, doxorubicin, THP-adriamycin, actinomycin D, and other anticancer agents include
Amrubicin hydrochloride, irinotecan hydrochloride, ifosfamide, etoposidrastat, gefitinib, cyclophosphamide, cisplatin, trastuzumab, fluorouracil, imanitive mesylate, methotrexate, rituxan, adriamycin, carboplatin, tamoxifen, camptothecin, melphalan cyclatonase, melphalan Etc.

  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 introducing a nucleic acid. 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 the DNA aptamer of the present invention to the surface of a transport material such as a liposome or a nanoparticle, the pharmaceutical component contained in the transport material can be selectively transported to lung cancer cells. Therefore, in a further aspect, the present invention provides a drug delivery system for preventing or treating lung cancer metastasis comprising the above DNA aptamer. The pharmaceutical ingredients that can be transported by the drug delivery system are typically the above-mentioned anticancer agents, but as long as they are substances useful for the prevention or treatment of lung cancer metastasis, there are other toxins and cancer growth inhibitors. It can also be a siRNA (small interfering RNA) that inhibits the expression of a gene, a suicide gene, or a gene that plays an important role in the growth and metastasis of lung cancer.

  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 A549 cells, which are non-small cell lung cancer cells, were selected from a DNA pool having a random sequence using Cell-SELEX method. Each step in the Cell-SELEX method is as follows.
1) Preparation of DNA pool (solution preparation of DNA aptamer candidate group)
2) Mixing with target substance-A549 cells 3) Separation of target-binding DNA and non-binding DNA 4) Replication of target-binding DNA (step of amplifying DNA aptamer bound to target substance)
5) Purification of target-binding DNA (step of purifying amplified DNA aptamer into single-stranded DNA)
6) Cloning of target-binding nucleic acid (pretreatment for sequence analysis of the obtained DNA aptamer)
7) Perform 8 rounds of steps 1) -6) 8) Sequence analysis of target-binding nucleic acid (Analysis of nucleotide sequence of DNA aptamer by sequencer)

  A more detailed experimental procedure is as follows.

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: Random34 (manufactured by Japan Genetic Research Institute)
- sequence: 5'-GCC TGT TGT GAG CCT CCT (N 34) CGC TTA TTC TTG TCT CCC-3 '
・ Length: 70 bases (random sequence is the center 34 bases)
・ Molecular weight: 21391.3 g / mol
Molar extinction coefficient: 630475 L / mol · cm

  Both sides of the random sequence are sequences that are recognized by primers in later PCR and allow amplification. A 1 μM DNA pool was prepared from the random DNA using a cell culture medium (Wako Pure Chemical Industries, Ltd .: RPM1-1640) buffer as a solvent.

Thereafter, A549 cells were cultured in a culture dish and cultured until the number of cells reached 10 ⁶ to 10 ⁷ . After the culture, the medium was removed, and 2 mL of phosphate buffered saline (hereinafter referred to as PBS) was added to the petri dish to wash the cells. 1 mL of 0.05% trypsin solution containing EDTA was added to the petri dish, and left in a 37 ° C. incubator for 2 minutes. After confirming that the cells were detached from the petri dish, 4 mL of PBS was added, and this was collected in a 15 mL centrifuge tube and centrifuged at 200 g for 3 minutes. Thereafter, the supernatant was removed with an aspirator. PBS (3 mL) was added thereto, the cells were suspended by pipetting, centrifuged at 200 g for 3 minutes, and the washing step for removing the supernatant was performed twice. Were prepared medium to 10 ⁶ cells in 330UL. To the prepared suspension, 370 μL of a 1 μM random DNA (DNA pool) solution prepared in advance was mixed and mixed thoroughly by vortexing.

  The obtained mixed solution was allowed to stand in a 37 ° C. incubator for 1 hour. Thereafter, centrifugation was performed at 400 g for 4 minutes using the same centrifuge tube. After removing the supernatant, 500 μL of PBS was added and centrifuged at 400 g for 4 minutes. This washing operation was performed three times. After removing the supernatant, 200 μL of PBS was added, and the mixture was heated at 95 ° C. for 10 minutes. Centrifugation was performed at 13000 g for 5 minutes, and the supernatant was collected.

  The separated A549 cell-binding DNA was amplified by PCR. The apparatus used was a Thermal Cycler (TAKARA-TP600). As the primer, an 18-base primer corresponding to the common sequence of the random DNA was used (manufactured by Nippon Genetic Institute). The 5 'terminal side of the primer is modified with biotin to enable separation of single-stranded DNA as described later.

  Streptavidin is added to the purified DNA and adsorbed on the magnetic particles. After collecting the magnetic particles with a magnet, the supernatant is removed, and then single-stranded DNA not bound to the magnetic particles is recovered in the supernatant by denaturation with alkaline buffer ( Figure 2). Thereafter, the alkaline buffer was replaced with a PBS buffer, and the single-stranded DNA, which was the target compound bound to the magnetic particles, was recovered. This was defined as one round, and this operation was performed 8 times.

  After 8 rounds, PCR amplification was performed using an 18-base primer not modified with biotin, and the PCR product was subjected to sequencer analysis. The analysis was performed by a sequencer analyzer Ion PGMTM (registered trademark) manufactured by Life Technologies Japan, and the sequences shown in FIG. 3 were selected (corresponding to SEQ ID NOs: 1 to 10 in order from the top).

Example 2: Staining of non-small cell lung cancer cells with fluorescently labeled DNA aptamer A549 cells are cultured in a culture dish and cultured until the number of cells reaches 10 ⁶ to 10 ⁷ . An aptamer solution in which the 5 ′ end of a DNA aptamer consisting of a nucleotide sequence having affinity for A549 cells found by sequencer analysis was modified with TAMRA ™, and the DNA was adjusted to 100 μM with ultrapure water. Was added while being dispersed in a culture dish. This was left still in an incubator at 37 ° C. for 1 hour. Thereafter, the cells were washed three times with PBS, and further observed with an inverted fluorescence microscope IX51 manufactured by Olympus with 2 mL of PBS added. The results with DNA aptamers having SEQ ID NOs: 1 to 5, 8, and 9 are shown in FIGS. 3 to 9, respectively. It was revealed that these fluorescently labeled DNA aptamers specifically bind to A549 cells, thereby obtaining a good fluorescence imaging image for A549 cells.

Claims (16)

A DNA aptamer having at least one nucleotide sequence selected from the sequences represented by SEQ ID NOs: 1 to 10 and specifically binding to non-small cell lung cancer cells. At least one nucleotide sequence selected from the sequences shown in SEQ ID NOs: 1 to 10 and having a sequence containing 1 to 3 nucleotide substitutions, deletions or additions, and specific for non-small cell lung cancer cells DNA aptamer characterized by binding to the target. A DNA aptamer that specifically binds to non-small cell lung cancer cells,
5′-P ₁ -XP ₂ -3 ′
Having a nucleotide sequence represented by:
X is 1) a nucleotide sequence selected from the sequences shown in SEQ ID NOs: 1 to 10; or 2) substitution of 1 to 3 nucleotides in the nucleotide sequence selected from the sequences shown in SEQ ID NOs: 1 to 10 A sequence containing a loss or addition,
P ₁ and P ₂ are DNA aptamers that are first and second primer recognition sequences introduced for PCR amplification. P ₁ is a first primer recognition sequence shown in SEQ ID NO: 11, and P ₂ is a second primer recognition sequences as shown in SEQ ID NO: 12, DNA aptamer according to claim 3. The DNA aptamer according to any one of claims 1 to 4, wherein the non-small cell lung cancer cells are A549 cells. 6. The method of claim 1, comprising at least one chemical modification selected from the group consisting of chemical substitution with a sugar chain moiety, chemical substitution with a phosphate ester moiety, and chemical substitution with a 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 DNA aptamer according to claim 7, wherein the fluorescent label is 6-carboxytetramethylrhodamine, fluorescein isothiocyanate, 6-carboxyfluorocein-aminohexyl, or a cyanine fluorescent dye. A composition for detecting lung cancer cells, comprising the DNA aptamer according to any one of claims 1 to 8. A kit for detecting lung cancer cells, comprising the DNA aptamer according to any one of claims 1 to 8. A method for detecting lung cancer cells, wherein the DNA aptamer according to any one of claims 1 to 8 is used. Contacting the DNA aptamer with a sample collected from a living body selected from the group consisting of lung cells, lung tissue, blood, serum, plasma, saliva, and sputum, and a response due to the binding of the sample and the DNA aptamer. The detection method according to claim 11, comprising a step of detecting the presence of lung cancer cells by observing. The detection method according to claim 12, wherein the response is a fluorescence response. A pharmaceutical composition for preventing or treating lung cancer metastasis, comprising the DNA aptamer according to any one of claims 1 to 8. Use of the DNA aptamer according to any one of claims 1 to 8 for producing a pharmaceutical composition for preventing or treating lung cancer metastasis. A drug delivery system for preventing or treating lung cancer metastasis, comprising the DNA aptamer according to any one of claims 1 to 8.

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