WO2011024955A1

WO2011024955A1 - Nucleic acid capable of binding to tgf-βii-type receptor, and use thereof

Info

Publication number: WO2011024955A1
Application number: PCT/JP2010/064612
Authority: WO
Inventors: 義一中村; 康子山村
Original assignee: 国立大学法人東京大学
Priority date: 2009-08-28
Filing date: 2010-08-27
Publication date: 2011-03-03
Also published as: JP2011045339A

Abstract

Disclosed is a substance which can be used for the elucidation of the physiological functions of TGF-ß and a TGF-ßII-type receptor and the analysis of mechanism of development of TGF-ß-induced diseases and the diagnosis and treatment of TGF-ß-induced diseases. Specifically disclosed are: an aptamer which is characterized by being capable of binding to a TGF-ßII-type receptor and being capable of inhibiting the binding between TGF-ß and a TGF-ßII-type receptor; a method for detecting a TGF-ßII-type receptor, which is characterized by using the aptamer; a medicinal agent comprising the aptamer; and others.

Description

Nucleic acids that bind to TGF-β type II receptors and uses thereof

The present invention relates to a nucleic acid that binds to a TGF-β type II receptor and a method for using the same.

Transforming Growth Factor-β (hereinafter referred to as “TGF-β”) has a strong growth-inhibiting effect on many cells such as epithelial cells, vascular endothelial cells, blood cells, and the like. It suppresses and functions as a canceration inhibitor. However, TGF-β is a multifunctional cytokine, and TGF-β produced by cancer cells in the late stage of canceration acts on normal tissue cells around the cancer tissue to cause angiogenesis, epithelial-mesenchymal transition, immunosuppression, It has been clarified that it induces extracellular matrix production and causes cancer invasion and metastasis. Furthermore, TGF-β was found to be an important factor in promoting extracellular matrix production and strongly causing tissue fibrosis and inducing chronic fibrotic diseases represented by pulmonary fibrosis, chronic renal failure, and cirrhosis. Has been. Therefore, TGF-β and TGF-β receptor are considered to be important targets for cancer invasion / metastasis, elucidation of molecular mechanisms of tissue fibrosis, and drug discovery.

The TGF-β receptor includes a TGF-β type I receptor (hereinafter sometimes referred to as “TβRI”) and a TGF-β type II receptor (hereinafter referred to as “TβRII”), both of which have a serine / threonine kinase region in the cell. In some cases). In recent years, the Smad transcription factor family (Smad2, Smad3, and Smad4), which are signaling molecules that function downstream of the TGF-β receptor complex, has been identified, and the major signaling pathway of TGF-β is being elucidated. When TGF-β binds to TβRII, TβRI is phosphorylated and activated by the serine / threonine kinase of TβRII. Smad2 or Smad3 present in the cytoplasm binds to the intracellular domain of activated TβRI, and is phosphorylated and activated by the serine / threonine kinase of TβRI. Thereafter, Smad2 and Smad3 are released from TβRI, form a heterocomplex with Smad4 in the cytoplasm, move to the nucleus, and function as a transcription factor.

Thus, TβRII is an essential cell surface molecule for binding to TGF-β, and TβRI alone cannot bind to TGF-β. Therefore, if the binding of TGF-β to TβRII can be inhibited, it is possible to suppress cancer invasion / metastasis and tissue fibrosis induced by TGF-β. So far, as an inhibitor of breast cancer metastasis targeting TβRII, solubilized TβRII (hereinafter sometimes referred to as “sTβRII”), solubilized TβRII and immunoglobulin Fc region fusion protein (sTβRII-Fc) Development is underway. The inhibitory effect was confirmed in an in vitro experimental system, and it was also reported that breast cancer metastasis to the lung, liver and spleen was suppressed in an in vivo experimental system using MMTV-neu transgenic mice. However, sTβRII and sTβRII-Fc have a large molecular weight, and because they require a sugar chain modification after protein translation, mass purification is not easy.

Incidentally, a technique called SELEX method has been developed as a technique for selecting and concentrating RNA that specifically binds to a target molecule in vitro (see Non-Patent Document 1). In the SELEX method, RNA having a random sequence having an appropriate length including a PCR primer sequence (including a T7 polymerase sequence on one end) is synthesized, and this is solid-phased and bound to a target molecule. After washing the unbound RNA, the RNA bound to the target molecule is recovered, amplified by RT-PCR, and used as an RNA template for the next round. By repeating this, an RNA aptamer that specifically binds to the target molecule is obtained. In this method, it is possible not only to clarify an RNA sequence that specifically binds to a target molecule, but also to obtain RNA having a physiological activity that promotes or inhibits the function of the target molecule. This suggests that the obtained aptamer can be applied as a pharmaceutical by targeting the pathogenic protein. Advantages of drug discovery using this SELEX method compared to conventional drug discovery are as follows: (1) Screening can be performed from a larger population than conventional chemical screening. (2) A large amount can be easily synthesized in a test tube. (3) Not immunogenic. (4) Chemical improvement can be easily performed. (5) It can target proteins that are highly conserved and difficult to produce antibodies. Etc. For example, Patent Document 1 discloses an RNA aptamer that specifically binds to a TGF-β type III receptor obtained by using the SELEX method.

JP 2007-43917 A

It is a figure which shows the binding analysis of a part of converged RNA and a human solubilization type TGF-beta type II receptor. 2RA5, 2RA1, 2RA6, and 2RA8 each represent a part of 20 types of convergent RNA aptamers. FIG. 3 is a view showing a binding analysis between the aptamer of the present invention and a human solubilized TGF-β type II receptor or a complex of TGF-β / human solubilized TGF-β type II receptor. “STβRII” represents a human solubilized TGF-βII type receptor, and “Tβ-sTβRII” represents a complex of TGF-β and a human solubilized TGF-βII type receptor. It is a figure which shows the predicted secondary structure of the deletion mutant which shortened the aptamer of this invention. The aptamer represented by SEQ ID NO: 1 (73 base length: 2RA5), the aptamer represented by SEQ ID NO: 2 (2RA5S1 from which 2RA5 has been deleted by 9 bases), and the aptamer represented by SEQ ID NO: 3 (14 bases of 2RA5) The deleted 2RA5S2) each secondary structure and the Kd value of binding to TβRII are shown. FIG. 3 shows that the aptamer of the present invention inhibits the cell growth inhibitory action of TGF-β.

The present invention elucidates the physiological function of TGF-β by obtaining an aptamer characterized by binding to TGF-β type II receptor and inhibiting the binding of TGF-β and TGF-β type II receptor. In addition, an object is to provide a substance that can be used for analysis, diagnosis, and treatment of the onset mechanism of diseases caused by excess TGF-β.

The present inventors prepared an RNA aptamer for a TGF-β type II receptor (TβRII), which is one of TGF-β receptors, as a candidate for such a substance. That is, SELEX was performed using TβRII as a target, and the binding activity between the obtained aptamer and the target was examined, and its usefulness was shown. The present invention has been completed based on these findings.

That is, the gist of the present invention is as follows.
[1] An aptamer that binds to a TGF-β type II receptor and inhibits the binding between TGF-β and a TGF-β type II receptor;
[2] The aptamer according to [1], comprising all or part of a nucleotide sequence selected from any one of SEQ ID NOs: 1 to 3;
[3] The aptamer according to [2], wherein at least one of the pyrimidine nucleotides is a modified nucleotide;
[4] The aptamer according to [1], which is either (a) or (b) below:
(A) an aptamer comprising all or part of a nucleotide sequence selected from any one of SEQ ID NOs: 1 to 3, wherein the 2 ′ position of the ribose of the pyrimidine nucleotides contained in the aptamer is the same or different and is a fluorine atom Or an aptamer substituted with an atom or group selected from the group consisting of a hydrogen atom, a hydroxy group and a methoxy group;
(B) an aptamer comprising all or part of a nucleotide sequence in which 1 to 5 nucleotides are substituted, deleted, inserted or added in a nucleotide sequence selected from any of SEQ ID NOs: 1 to 3, The 2 ′ position of the ribose of the pyrimidine nucleotide contained in the aptamer is the same or different and is a fluorine atom, or is substituted with an atom or group selected from the group consisting of a hydrogen atom, a hydroxy group and a methoxy group, Aptamer;
[5] A complex comprising the aptamer according to any one of [1] to [4] and a functional substance;
[6] The complex according to [5], wherein the functional substance is an affinity substance, a labeling substance, an enzyme, a drug delivery vehicle or a drug;
[7] A TGF-β type II receptor is detected and / or characterized by using the aptamer according to any one of [1] to [4] or the complex according to [5] or [6]. A method of quantification;
[8] A prophylactic or therapeutic agent for a disease caused by the action of TGF-β, comprising the aptamer according to any one of [1] to [4] or the complex according to [5] or [6];
[9] A disease caused by the action of TGF-β, comprising administering the aptamer according to any one of [1] to [4] or the complex according to [5] or [6] Prevention or treatment methods;
[10] The aptamer according to any one of [1] to [4] or the complex according to [5] or [6] for the prevention or treatment of a disease caused by the action of TGF-β.

The present invention provides an aptamer that has the ability to bind to a TGF-β type II receptor and inhibits the binding between TGF-β and a TGF-β type II receptor. Such aptamers are aptamers that inhibit the function of TGF-β. The aptamer of the present invention is useful for treating diseases caused by excessive TGF-β expression.

The present invention provides an aptamer that has the ability to bind to a TGF-β type II receptor and inhibits the binding between TGF-β and a TGF-β type II receptor.

TGF-β is a multifunctional cytokine that induces cell growth inhibition, angiogenesis, epithelial-mesenchymal transition, immunosuppression, extracellular matrix production, and the like. When TGF-β binds to a TGF-β type II receptor on the cell surface, the TGF-β type II receptor phosphorylates and activates the TGF-β type I receptor. When the intracellular signaling molecule is phosphorylated by the activated TGF-β type I receptor and activated, a TGF-β signal is transmitted into the cell.

The TGF-β, TGF-β type I receptor and TGF-β type II receptor shown in the present invention are produced in an animal body, and are produced using mammalian cells such as mice, insect cells, cells such as E. coli. Can also be produced by chemical synthesis. Moreover, if it is produced by cultured cells or chemical synthesis, a mutant can be produced easily. Here, “mutant” means an amino acid sequence in which one to several amino acids are substituted or a part of various proteins, and originally TGF-β, TGF-β type I receptor and TGF-β type II receptor are It means a protein or peptide having at least one activity. When an amino acid is substituted, the substituted amino acid can be either a natural or non-natural amino acid. The TGF-β, TGF-β type I receptor and TGF-β type II receptor referred to in the present invention include these variants.

Aptamer refers to a nucleic acid molecule having binding activity to a predetermined target molecule. Aptamers can inhibit the activity of a given target molecule by binding to the given target molecule. The aptamer of the present invention may preferably be an RNA aptamer, a modified RNA aptamer or a mixture thereof. The aptamer of the present invention may also be in a linear or cyclic form.

The aptamer of the present invention has a binding ability to a TGF-β type II receptor and is characterized by inhibiting the binding between TGF-β and a TGF-β type II receptor. Specifically, the aptamer of the present invention competitively inhibits the binding between TGF-β and the TGF-β type II receptor by binding to the TGF-β binding region of the TGF-β type II receptor. Therefore, the aptamer of the present invention can inhibit the binding between TGF-β and TGF-β type II receptor. Examples of such aptamers include aptamers including a nucleotide sequence selected from any one of SEQ ID NOs: 1 to 3. Here, “having the ability to bind to a TGF-β type II receptor” means having a higher binding activity to a TGF-β type II receptor than the RNA pool of a random sequence.

The aptamer of the present invention is not particularly limited as long as it can bind to the TGF-β type II receptor and inhibit the binding between TGF-β and the TGF-β type II receptor, and the existing aptamer can be easily used. For this purpose, such aptamer may be, for example, “an aptamer containing a part of a nucleotide sequence selected from any one of SEQ ID NOS: 1 to 3, which comprises TGF-βII An aptamer that binds to a type I receptor and inhibits the binding of TGF-β to a type TGF-β receptor ”.

The aptamer of the present invention has nucleotide substitutions, deletions, insertions or additions as long as it binds to the TGF-β type II receptor and can inhibit the binding of TGF-β to the TGF-β type II receptor. It also includes aptamers. The number of nucleotides that may be substituted, deleted, inserted or added is not particularly limited, but is usually 1 to 5, preferably 1 to 3.

In the present specification, “an aptamer comprising a part of a nucleotide sequence selected from any one of SEQ ID NOs: 1 to 3, which binds to a TGF-β type II receptor and receives TGF-β and TGF-β type II receptor” Examples of aptamers that inhibit binding to the body include

(Where
N1 and N2 represent nucleobases capable of complementary base pairing with each other by 3 base pairs,
N3 and N4 represent nucleobases capable of 4 base pair complementary base pairing with each other;
N5 represents one nucleobase,
N6 and N7 represent nucleobases capable of 5 base pair complementary base pairing with each other;
N8 represents at least 4 nucleobases,
The 3 ′ terminal base of N2 and the 5 ′ terminal base of N3 are continuous,
N4 3 ′ terminal base and N5 are continuous,
The 5 ′ terminal bases of N5 and N6 are continuous,
N7 3 ′ terminal base and N8 5 ′ terminal base are continuous,
The straight line connecting each of N1 to N8 indicates a hydrogen bond between nucleobases, and the curve connecting each of N1 to N8 indicates a loop portion. And aptamers that bind to the TGF-β type II receptor and inhibit the binding of TGF-β to the TGF-β type II receptor.

Here, “nucleic acid bases capable of complementary base pairing of 3 base pairs to each other” represented by N1 and N2 means “three nucleic acids capable of forming complementary base pairs with each other across the loop portion” “Base”, for example, when N1 is GGG, N2 is CCC. “Nucleobase capable of complementary base pairing of 4 base pairs to each other” represented by N3 and N4 means “four nucleobases capable of forming complementary base pairs with each other across the loop portion” For example, when N3 is CGGG, N4 is CCCG. "Nucleobase capable of complementary base pairing of 5 base pairs to each other" represented by N6 and N7 means "5 nucleobases capable of forming complementary base pairs with each other across a loop part" For example, when N6 is GGCCG, N7 is CGGCC.
The “one nucleobase” represented by N5 may be any nucleobase, for example, selected from the group consisting of A (adenine), C (cytosine), G (guanine) and U (uracil). Nucleobase. A is preferred.
The “at least 4 nucleobases” represented by N8 may be any nucleobase, for example, selected from the group consisting of A (adenine), C (cytosine), G (guanine) and U (uracil). 4 or more nucleobases are combined. Of the four nucleobases, the first four bases are preferably GACA.
N2 3 ′ terminal base and N3 5 ′ terminal base, N4 3 ′ terminal base and N5, N5 and N6 5 ′ terminal base and N7 3 ′ terminal base and N8 5 The bonding mode of each of the 'terminal bases is not particularly limited. For example, a phosphodiester bond such as a 3'-5' phosphodiester bond or a 2'-5 'phosphodiester bond, or a part of oxygen constituting the phosphodiester bond Or the phosphorothioate bond etc. which substituted all by the sulfur atom are mentioned.

The aptamer of the present invention may have an activity of inhibiting the binding between any mammalian TGF-β and TGF-β type II receptor. Such mammals include, for example, primates (eg, humans, monkeys), rodents (eg, mice, rats, guinea pigs), and pets, livestock and working animals (eg, dogs, cats, horses, cows). , Goats, sheep, pigs).

The length of the aptamer of the present invention is not particularly limited, and can usually be about 10 to about 200 nucleotides, for example, about 100 nucleotides or less, preferably about 75 nucleotides or less. If the total number of nucleotides is small, chemical synthesis and mass production are easier, and the merit in cost is also great. In addition, chemical modification is easy, the in vivo stability is high, and the toxicity is considered low.

Each nucleotide contained in the aptamer of the present invention is a nucleotide containing a hydroxy group at the 2 ′ position of ribose (eg, ribose of pyrimidine nucleotide, ribose of purine nucleotide) (ie, unsubstituted nucleotide), or Same or different, a nucleotide in which the hydroxy group is substituted (modified) with an arbitrary atom or group at the 2 ′ position of ribose (in the present invention, sometimes referred to as “substituted nucleotide” or “modified nucleotide”) ).

Examples of such an arbitrary atom or group include a hydrogen atom, a fluorine atom, —O-alkyl group (eg, —O—Me group), —O-acyl group (eg, —O—CHO group) or amino And nucleotides substituted with groups (eg, —NH ₂ groups). The aptamer of the present invention can also be an aptamer in which at least one of the pyrimidine nucleotides is a modified nucleotide.

The aptamer of the present invention is also a nucleotide in which at least one of pyrimidine nucleotides, preferably all pyrimidine nucleotides are substituted with a fluorine atom at the 2′-position of ribose, or the same or different, and any atom described above. Or a nucleotide, preferably a nucleotide substituted with an atom or group selected from the group consisting of a hydrogen atom, a hydroxy group and a methoxy group.

The aptamer of the present invention is also
(A) an aptamer that binds to a TGF-β type II receptor and inhibits the binding between TGF-β and a TGF-β type II receptor, and is selected from any one of SEQ ID NOs: 1 to 3 From the group consisting of a hydrogen atom, a hydroxy group and a methoxy group, wherein the 2 ′ position of the ribose of the pyrimidine nucleotides contained in the aptamer is the same or different and is a fluorine atom. An aptamer substituted with a selected atom or group;
(B) An aptamer that binds to a TGF-β type II receptor and inhibits the binding between TGF-β and a TGF-β type II receptor, and is selected from any one of SEQ ID NOs: 1 to 3 1 to 5 (preferably 1 to 3, more preferably 1 or 2) nucleotides comprising all or part of the nucleotide sequence substituted, deleted, inserted or added, An aptamer in which the ribose 2 ′ position of pyrimidine nucleotides contained in the aptamer is the same or different and is a fluorine atom or substituted with an atom or group selected from the group consisting of a hydrogen atom, a hydroxy group and a methoxy group ;
(C) a connected product selected from the group consisting of a plurality of connected products of (a), a plurality of connected products of (b), and a plurality of connected products of (a) and (b);
It can be.

Among the aptamers described above, the aptamer characterized in that it binds to the TGF-β type II receptor and inhibits the binding between TGF-β and the TGF-β type II receptor in (a), comprising: Including a part of a nucleotide sequence selected from any one of 3 to 3, wherein the 2 ′ position of the ribose of the pyrimidine nucleotide contained in the aptamer is the same or different and is a fluorine atom, or a hydrogen atom, a hydroxy group and a methoxy group Examples of an aptamer substituted with an atom or group selected from the group consisting of groups are aptamers comprising a part of the nucleotide sequence selected from any of the above-mentioned “SEQ ID NOs: 1 to 3” As an example of an aptamer that binds to a TGF-β type II receptor and inhibits the binding of TGF-β to a TGF-β type II receptor ” Among the aptamers, the ribose 2 ′ position of the pyrimidine nucleotides contained in the aptamer is the same or different and is a fluorine atom, or an atom or group selected from the group consisting of a hydrogen atom, a hydroxy group and a methoxy group. Aptamers that are substituted are mentioned.

Among the aptamers, the aptamer characterized in that it binds to the TGF-β type II receptor and inhibits the binding between TGF-β and TGF-β type II receptor in (b), In a nucleotide sequence selected from any one of 1 to 3, a nucleotide sequence in which 1 to 5 (preferably 1 to 3, more preferably 1 or 2) nucleotides are substituted, deleted, inserted or added The ribose 2 ′ position of the pyrimidine nucleotides contained in the aptamer is the same or different and is a fluorine atom or an atom or group selected from the group consisting of a hydrogen atom, a hydroxy group and a methoxy group Examples of the substituted aptamer include a part of the nucleotide sequence selected from any of the aforementioned “SEQ ID NOs: 1 to 3”. 1-5 nucleotides from the aptamer described as an example of an aptamer that binds to TGF-β type II receptor and inhibits binding of TGF-β to TGF-β type II receptor ” From the group consisting of a hydrogen atom, a hydroxy group, and a methoxy group, wherein the 2′-position of the ribose of the pyrimidine nucleotide that is deleted, inserted or added and is the same or different is a fluorine atom. Aptamers that are substituted with a selected atom or group are included.

The aptamer (b) can also be obtained by introducing a mutation into the aptamer (a) and performing SELEX described later again. The aptamer thus obtained may have a higher binding activity and physiological activity than the RNA of (a). Thus, aptamers suitable for various purposes can be produced by introducing mutations into RNA.

In the above (c), the connection can be performed by tandem coupling. In addition, a linker may be used for the connection. Linkers include nucleotide chains (eg, 1 to about 20 nucleotides), non-nucleotide chains (eg, — (CH ₂ ) _n -linker, — (CH ₂ CH ₂ O) _n -linker, hexaethylene glycol linker, TEG linker , A linker containing a peptide, a linker containing a —S—S— bond, a linker containing a —CONH— bond, and a linker containing a —OPO ₃ — bond). The number of the plurality of connected objects is not particularly limited as long as it is 2 or more, but may be 2, 3, or 4, for example.

The aptamer of the present invention may be one in which the sugar residue (eg, ribose) of each nucleotide is modified in order to enhance the binding property, stability, drug delivery property, etc. to the TGF-β type II receptor. Examples of the site modified in the sugar residue include those in which the oxygen atom at the 2′-position, 3′-position and / or 4′-position of the sugar residue is replaced with another atom. Examples of modifications include fluorination, O-alkylation (eg, O-methylation, O-ethylation), O-allylation, S-alkylation (eg, S-methylation, S-ethylation). ), S-allylation, amination (eg, —NH ₂ ). Such modification of sugar residues can be performed by a method known per se (for example, Sproat et al., (1991) Nucl. Acid. Res. 19, 733-738; Cotton et al., (1991)). Nucl. Acid. Res. 19, 2629-2635; Hobbs et al., (1973) Biochemistry 12, 5138-5145).

The aptamer of the present invention may be one in which a nucleobase (eg, purine or pyrimidine) is modified (eg, chemically substituted) in order to enhance the binding property to the TGF-β type II receptor. Examples of such modifications include 5-position pyrimidine modification, 6- and / or 8-position purine modification, modification with exocyclic amine, substitution with 4-thiouridine, substitution with 5-bromo or 5-iodo-uracil. Can be mentioned. Moreover, the phosphate group contained in the aptamer of the present invention may be modified so as to be resistant to nuclease and hydrolysis. For example, the P (O) O group may be P (O) S (thioate), P (S) S (dithioate), P (O) NR ₂ (amidate), P (O) R, R (O) OR ′. , CO or CH ₂ (formacetal) or 3′-amine (—NH—CH ₂ —CH ₂ —) optionally substituted [wherein each R or R ′ is independently H Or substituted or unsubstituted alkyl (eg, methyl, ethyl)].
Examples of the linking group include —O—, —N—, and —S—, which can be bonded to adjacent nucleotides through these linking groups.
Modifications may also include 3 ′ and 5 ′ modifications such as capping.

Modifications are further made of polyethylene glycol, amino acids, peptides, inverted dT, nucleic acids, nucleosides, Myristoy, Lithocholic-oleyl, Docosanyl, Lauroyl, Stearoyl, Palmitoyl, Oleoyl, Linoleoyl, other lipids, vitamins, steroids, cholesterol, steroids It can be performed by adding a fluorescent substance, an anticancer agent, a toxin, an enzyme, a radioactive substance, biotin, a column carrier, or the like to the end. When adding a nucleic acid, the length of the added nucleic acid is not particularly limited, but is preferably 100 mer or less, and more preferably 30 mer or less. Examples of the column carrier include agarose and sepharose. See, for example, US Pat. Nos. 5,660,985 and 5,756,703 for such modifications.

The aptamer of the present invention can be chemically synthesized by the disclosure in the present specification and a method known per se in the technical field. Aptamers bind to a target substance by various binding modes such as ionic bonds using the negative charge of the phosphate group, hydrophobic bonds and hydrogen bonds using ribose, hydrogen bonds using nucleobases and stacking bonds. In particular, the ionic bond utilizing the negative charge of the phosphate group that exists in the number of constituent nucleotides is strong and binds to the positive charge of lysine or arginine present on the surface of the protein. For this reason, nucleobases that are not involved in direct binding to the target substance can be substituted. In particular, the stem structure portion is already base-paired and faces the inside of the double helix structure, so that the nucleobase is difficult to bind directly to the target substance. Therefore, the activity of the aptamer is often not reduced even if the base pair is replaced with another base pair. Even in a structure that does not form a base pair, such as a loop structure, base substitution is possible when the nucleobase is not involved in direct binding to the target molecule. Regarding the modification of the 2 'position of ribose, in rare cases, the functional group at the 2' position of ribose may directly interact with the target molecule, but in many cases it is irrelevant and can be replaced with other modified molecules. Is possible. Thus, aptamers often retain their activity unless functional groups involved in direct binding to the target molecule are replaced or deleted. It is also important that the overall three-dimensional structure does not change significantly.

Aptamers are produced by the SELEX method and its improved methods (for example, AD Ellington et al., (1990) Nature, 346, 818-822; C. Tuerk et al., (1990) Science, 249, 505-510). It can be produced by using. In the SELEX method, by increasing the number of rounds or using a competitive substance, aptamers having a stronger binding power to the target substance are concentrated and selected. Therefore, by adjusting the number of rounds of SELEX and / or changing the competition state, aptamers with different binding strengths, aptamers with different binding modes, aptamers with the same binding strength or binding mode but different base sequences You may be able to get it. In addition, the SELEX method includes an amplification process by PCR. By introducing a mutation by using manganese ions in the process, it becomes possible to perform SELEX with more diversity.

An aptamer obtained by SELEX is a nucleic acid having a high affinity for a target substance, which does not mean binding to the active site of the target substance. Therefore, it should be noted that the aptamer obtained by SELEX does not necessarily affect the function of the target substance. The active aptamer thus selected can be further improved in performance by performing optimized SELEX. In the optimized SELEX, a template in which a part of an aptamer having a predetermined sequence is made into a random sequence or a template in which about 10 to 30% of a random sequence is doped is prepared, and SELEX is performed again.

The aptamer obtained by SELEX has a length of about 70 nucleotides, and it is difficult to make it as a medicine as it is. Therefore, it may be shortened to a length of about 50 nucleotides or less which can be easily chemically synthesized. The aptamer of the present invention is not particularly limited as long as it can bind to the TGF-β type II receptor and inhibit the binding between TGF-β and the TGF-β type II receptor. For example, an aptamer that inhibits the binding between TGF-β and a TGF-β type II receptor, which includes a part of a nucleotide sequence selected from any one of SEQ ID NOs: 1 to 3. That is, the aptamer of the present invention is an aptamer that binds to the TGF-β type II receptor and inhibits the binding between TGF-β and the TGF-β type II receptor, and is selected from any one of SEQ ID NOs: 1 to 3. Also included are aptamers that contain all or part of the nucleotide sequence to be processed.
The aptamer obtained by SELEX may vary in ease of subsequent minimization depending on the primer design.

Aptamers can be easily modified because they can be chemically synthesized. Aptamers can predict their secondary structure using the MFOLD program. It is also possible to predict the three-dimensional structure by X-ray analysis or NMR analysis. With these structure predictions, it is possible to predict which nucleotides can be substituted or deleted, and where new nucleotides can be inserted. Aptamers of the predicted new sequence can be easily chemically synthesized, and whether or not the aptamer retains activity can be confirmed by an existing assay system. Further, the modification can be designed or altered to a high degree as well as the length of the sequence.

As described above, aptamers can be highly designed or modified. The present invention also includes a predetermined sequence (eg, a sequence corresponding to a portion selected from a stem portion, an internal loop portion, a hairpin loop portion, and a single-stranded portion: hereinafter, abbreviated as a fixed sequence if necessary). Provided is a method for producing an aptamer in which the aptamer can be highly designed or modified.

For example, the method for producing such aptamer is as follows:

[In the above, (N) a represents a nucleotide chain consisting of a N, (N) b represents a nucleotide chain consisting of b N, and N is the same or different, respectively, A, G, It is a nucleotide selected from the group consisting of C, U and T (preferably A, G, C and U). a and b are the same or different and may be any number, for example, 1 to about 100, preferably 1 to about 50, more preferably 1 to about 30, even more preferably 1 to about 20 or 1 Can be from about 10 to about 10. ] Each of a single type of nucleic acid molecule or a plurality of types of nucleic acid molecules (eg, a library of nucleic acid molecules having different numbers of a, b, etc.) and primer sequences (i), (ii) Producing an aptamer comprising a fixed sequence using the corresponding primer pair.

The present invention also provides a complex comprising the aptamer of the present invention and a functional substance bound thereto. The bond between the aptamer and the functional substance in the complex of the present invention can be a covalent bond or a non-covalent bond. The complex of the present invention may be a conjugate of the aptamer of the present invention and one or more (eg, 2 or 3) of the same or different functional substances. The functional substance is not particularly limited as long as it newly adds some function to the aptamer of the present invention or can change (eg, improve) some property that can be retained by the aptamer of the present invention. Examples of the functional substance include proteins, peptides, amino acids, lipids, carbohydrates, monosaccharides, polynucleotides, and nucleotides. Examples of functional substances include, for example, affinity substances (eg, biotin, streptavidin, polynucleotides having affinity for target complementary sequences, antibodies, glutathione sepharose, histidine), labeling substances (eg, fluorescent substances, Luminescent substances, radioisotopes), enzymes (eg, horseradish peroxidase, alkaline phosphatase), drug delivery vehicles (eg, liposomes, microspheres, peptides, polyethylene glycols), drugs (eg, calicheamicin and duocarmycin) Used in missile therapy, nitrogen mustard analogs such as cyclophosphamide, melphalan, ifosfamide or trophosphamide, ethyleneimines such as thiotepa, nitrosourea such as carmustine, temozolomide or dacarbazine Lyostat, antifolate-like antimetabolites such as methotrexate or raltitrexed, purine analogues such as thioguanine, cladribine or fludarabine, pyrimidine analogues such as fluorouracil, tegafur or gemcitabine, vinca alkaloids such as vinblastine, vincristine or vinolerubin and their analogues Podophyllotoxin derivatives such as etoposide, taxane, docetaxel or paclitaxel, anthracyclines and analogs such as doxorubicin, epirubicin, idarubicin and mitoxantrone, other cytotoxic antibiotics such as bleomycin and mitomycin, cisplatin, carboplatin and Platinum compounds such as oxaliplatin, pentostatin, miltefosine, estramustine, Tecan, irinotecan and bicalutamide), toxins (e.g., ricin toxin, rear toxins and Vero toxins) can be mentioned. These functional molecules may eventually be removed. Furthermore, it may be a peptide that can be recognized and cleaved by an enzyme such as thrombin, matrix metal protease (MMP), Factor X, or a polynucleotide that can be cleaved by a nuclease or a restriction enzyme.

The aptamer or complex of the present invention can be used as a medicine or a diagnostic agent, a test agent, or a reagent. In particular, the aptamer or complex of the present invention inhibits intracellular signal transduction of TGF-β through the TGF-β type II receptor, and thus, for example, various diseases caused by the action of TGF-β, such as the action of TGF-β. It is useful as a medicament, diagnostic agent, test agent, reagent, etc. for various fibrosis caused by tissue fibrosis or various diseases associated with tissue fibrosis.

Here, “various diseases resulting from the action of TGF-β” include renal diseases (eg, renal fibrosis, nephritis, renal failure, nephrosclerosis, etc.), lung diseases (eg, pulmonary fibrosis, pneumonia, etc.) ), Liver disease (eg, liver tissue fibrosis, cirrhosis, hepatitis, etc.), skin disease (eg, wound, scleroderma, psoriasis, keloid, etc.), arthritis (eg, rheumatoid arthritis, osteoarthritis, etc.), Examples include vascular diseases (eg, vascular restenosis, rheumatic vasculitis, etc.). Examples of “various diseases associated with tissue fibrosis” include cancer in various organs (particularly cancer invasion and metastasis), arteriosclerosis, and the like.

The medicament of the present invention may be formulated with a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include excipients such as sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone , Gelatin, gum arabic, polyethylene glycol, sucrose, starch and other binders, starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate , Aerosil, Talc, Lubricant such as sodium lauryl sulfate, Citric acid, Menthol, Glycyllysine / Ammonium salt, Fragrance, such as Glycine, Orange powder, Sodium benzoate Preservatives such as sodium, sodium bisulfite, methylparaben, propylparaben, stabilizers such as citric acid, sodium citrate, acetic acid, suspensions such as methylcellulose, polyvinylpyrrolidone, aluminum stearate, dispersants such as surfactants, Examples include, but are not limited to, water, physiological saline, diluents such as orange juice, base waxes such as cacao butter, polyethylene glycol, and white kerosene.

Preparations suitable for oral administration include a solution in which an effective amount of a ligand is dissolved in a diluent such as water, physiological saline, orange juice, a capsule containing an effective amount of the ligand as a solid or a granule, a sachet or Examples thereof include tablets, suspensions in which an effective amount of an active ingredient is suspended in a suitable dispersion medium, and emulsions in which a solution in which an effective amount of an active ingredient is dissolved is dispersed in an appropriate dispersion medium and emulsified.

In addition, the medicament of the present invention can be coated by a method known per se for the purpose of taste masking, enteric solubility or sustainability, if necessary. As a coating agent used for coating, for example, hydroxypropylmethylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, polyoxyethylene glycol, Tween 80, Pluronic F68, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxymethylcellulose acetate succinate, Eudragit (manufactured by Rohm, Germany, methacrylic acid / acrylic acid copolymer) and pigments (eg, Bengala, titanium dioxide, etc.) are used. The medicine may be either an immediate release preparation or a sustained release preparation. Examples of the sustained release substrate include liposomes, atelocollagen, gelatin, hydroxyapatite, and PLGA.

Suitable formulations for parenteral administration (eg, intravenous, subcutaneous, intramuscular, topical, intraperitoneal, nasal, pulmonary) are aqueous and non-aqueous isotonic. There are sterile injection solutions, which may contain antioxidants, buffers, antibacterial agents, isotonic agents and the like. Also, aqueous and non-aqueous sterile suspensions can be mentioned, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The preparation can be enclosed in a container in unit doses or multiple doses like ampoules and vials. In addition, the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile solvent immediately before use. In addition to injectable solutions, inhalants and ointments are also possible. In the case of an inhalant, the lyophilized active ingredient is refined and administered by inhalation using an appropriate inhalation device. In the inhalant, conventionally used surfactants, oils, seasonings, cyclodextrins or derivatives thereof can be appropriately blended as necessary.

The dose of the medicament of the present invention varies depending on the type / activity of the active ingredient, the severity of the disease, the animal species to be administered, the drug acceptability of the administration target, body weight, age, etc. The amount of active ingredient may be about 0.0001 to about 100 mg / kg, such as about 0.0001 to about 10 mg / kg, preferably about 0.005 to about 1 mg / kg.

The present invention also provides a solid phase carrier on which the aptamer or complex of the present invention is immobilized. Examples of the solid phase carrier include a substrate, a resin, a plate (eg, multiwell plate), a filter, a cartridge, a column, and a porous material. The substrate may be one used for DNA chips, protein chips, etc., for example, nickel-PTFE (polytetrafluoroethylene) substrate, glass substrate, apatite substrate, silicon substrate, alumina substrate, etc. And the like coated with a polymer or the like. Examples of the resin include agarose particles, silica particles, copolymers of acrylamide and N, N′-methylenebisacrylamide, polystyrene-crosslinked divinylbenzene particles, particles obtained by crosslinking dextran with epichlorohydrin, cellulose fibers, and allyldextran. Examples include N, N'-methylenebisacrylamide cross-linked polymers, monodisperse synthetic polymers, monodisperse hydrophilic polymers, sepharose, and Toyopearl. Also included are resins in which various functional groups are bonded to these resins. . The solid phase carrier of the present invention can be useful for, for example, purification of TGF-β type II receptor, detection of TGF-β type II receptor, quantification of TGF-β type II receptor, and the like.

The aptamer or complex of the present invention can be immobilized on a solid support by a method known per se. For example, an affinity substance (for example, one described above) or a predetermined functional group is introduced into the aptamer or complex of the present invention, and then immobilized on a solid phase carrier using the affinity substance or the predetermined functional group. A method is mentioned. The present invention also provides such a method. The predetermined functional group may be a functional group that can be subjected to a coupling reaction, and examples thereof include an amino group, a thiol group, a hydroxy group, and a carboxyl group. The present invention also provides an aptamer having such a functional group introduced therein.

The present invention also provides a method for detecting and quantifying the TGF-β type II receptor. The detection and quantification method of the present invention may comprise measuring the TGF-β type II receptor utilizing the aptamer of the present invention (eg, by using the complex of the present invention and a solid phase carrier). The method for detecting and quantifying the TGF-β type II receptor can be performed by the same method as the immunological method except that the aptamer of the present invention is used instead of the antibody. Therefore, by using the aptamer of the present invention in place of an antibody, an enzyme immunoassay (EIA) (eg, direct competition ELISA, indirect competition ELISA, sandwich ELISA), radioimmunoassay (RIA), fluorescence immunoassay ( FIA), Western blotting (eg, use as a substitute for secondary antibody in Western blotting), immunohistochemical staining, cell sorting, and similar methods can be used for detection and quantification. . Such a method may be useful, for example, for measuring the amount of TGF-β type II receptor in a biological or biological sample.

The contents of all publications, including patents and patent application specifications cited in this specification, are hereby incorporated by reference herein to the same extent as if all were explicitly stated. Is.

Hereinafter, the experimental technique of the present invention will be described in detail with reference to examples. In addition, these Examples illustrate the present invention and do not limit the scope of the present invention.

[Example 1] Purification of recombinant TGF-β type II receptor Human solubilized TGF-β type II receptor (sTβRII-8His) in which the intracellular domain and transmembrane domain are deleted and the C-terminus is labeled with histidine (His) ) Expression vector was constructed. This expression vector was then introduced into FreeStyle 293 cells using 293fectin-transfection reagent (Invitrogen). Subsequently, recombinant sTβRII-8His was purified from the culture supernatant using TALON Metal Affinity Resins (Clontech).

[Example 2] Cell culture The FreeStyle 293 cell line was cultured using a FreeStyle 293 expression medium (manufactured by Invitrogen). The 293T cell line is DMEM (Invitrogen), the Hep3B cell line is MEM (Invitrogen), the SNU-16 cell line is RPMI1640 (Invitrogen), 10% fetal bovine serum (JRH Biosciences), 100 units / ml penicillin. −100 μg / ml streptomycin (Invitrogen) was added and cultured.

[Example 3] Selection of RNA aptamer RNA aptamer by the SELEX method was obtained by the method of Ellington et al. (Ellington AD and Szostak JW, in vitro selection of RNA molecules that binds specific 46: -822, 1990), and the method of Tuerk et al. (Tuerk C. and Gold L., Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteria9 T4 DNA 9 Ie purified The T [beta] RII-8His, immobilized on TALON Metal Affinity Resins or Ni-NTA Agarose, was obtained by performing the SELEX method for RNA pool with a random sequence of 40 bases (N40 random RNA pool). Specifically, it is as follows.
First, the N40 random RNA pool used in the SELEX method was prepared from a chemically synthesized template having the following F1 primer and a random sequence of 40 bases (commissioned from Operon Biotechnology and Invitrogen). The random sequence template was amplified by PCR using Ex Taq Hot Start Version (manufactured by Takara Bio Inc.). Using this DNA fragment as a template for transcription, the promoter sequence of T7 RNA polymerase contained in the F1 primer was used to generate a DuraScribe T7 Transcript Kits ( (Epicentre Technologies, Inc.) was used for in vitro transcription, phenol extraction and gel filtration to obtain an N40 random RNA pool.

The binding of sTβRII-8His immobilized on TALON Metal Affinity Resins or Ni-NTA Agarose (Qiagen) to the RNA in the N40 random RNA pool was performed by buffer A [20 mM Tris-HCl, 80 mM potassium acetate, 2.5 mM magnesium acetate. acetate, 1 mM dithiothreitol].
After TALON Metal Affinity Resins or Ni-NTA Agarose was washed with buffer A, sTβRII-8His and RNA bound thereto were released using 300 mM imidazole. RT-PCR was performed based on this RNA to amplify the DNA template, and then transcription was performed in vitro to prepare the next round RNA pool.

After repeating this cycle up to 6 rounds, the cDNA corresponding to the concentrated RNA was cloned into pGEM T-EASY Vector (Promega) and transformed into E. coli strain DH5α (Toyobo). A clone was obtained. After extracting the plasmid DNA, the base sequence was determined with a DNA sequencer Model 3100 (manufactured by ABI) using the following sequence primers. As a result, 20 kinds of converged RNA were obtained.

(SEQ ID NO: 4) F1 primer:
5′- TAATACGACTCACTATA GGGACACAATGACG-3 ′ (T7 promoter sequence is underlined)
(SEQ ID NO: 5) Random sequence template:
5′-CTCTCATGTCGGGCCGTTA-N40-CGTCCATTGGTCCCTATATGGAGTCGTTTA-3 ′
(SEQ ID NO: 6) Sequence primer:
5'-GTTTTCCCAGTCACGAC-3 '

MFOLD program was applied to the obtained base sequence to predict RNA secondary structure.

[Example 4] Analysis of binding reaction rate by surface plasmon resonance The binding activity of converged RNA to TβRII was examined by surface plasmon resonance analysis. The 5 ′ end of poly (dT) ₁₆ oligonucleotide was biotinylated and immobilized on a streptavidin sensor chip (GE Healthcare Bioscience). Furthermore, convergent RNA having poly (A) ₁₆ added to the 3 ′ end was added and immobilized.
Subsequently, sTβRII (manufactured by R & D Systems) or Tβ-sTβRII was added, and surface plasmon resonance analysis was performed using BIACORE 2000 (manufactured by Biacore AB) to measure the binding reaction rate (FIG. 1). When RNA binds to sTβRII or Tβ-sTβRII, an optical change occurs, and an increase in Resonance Units is observed on the sensorgram. The measurement was performed using non-specific binding between the N40 random RNA pool and sTβRII or Tβ-sTβRII as a background. The obtained sensorgram was analyzed with BIAevaluation (manufactured by BIACORE) software, and the dissociation constant (Kd value) was calculated. Among the converged RNAs, 13 types were anti-TβRII RNA aptamers having binding ability to sTβRII. The calculated bond dissociation constant (Kd) was 2.9 × 10 ⁻⁹ M to 10.4 × 10 ⁻⁹ M.

[Example 5] Cell staining with fluorescently labeled RNA or fluorescently labeled antibody Next, it was examined whether the anti-TβRII RNA aptamer can bind to TβRII expressed on the cell membrane.
N40 random RNA pools and RNA aptamers were labeled with Alexa Fluor 488 using ULYSIS Nucleic Acid Labeling Kits (Invitrogen).
Subsequently, it was confirmed that the labeled aptamer bound to sTβRII in the same manner as the unlabeled RNA aptamer by surface plasmon resonance analysis.
A human fetal kidney cell line 293T cultured on Poly-D-Lycine BioCoat culture slides (BD Biosciences) was used with FuGENE 6 Transfection Reagent (Roche Applied Science II). Introduced into sex, the human TGF-β type II receptor was expressed. After 48 hours, cells were fixed with 4% paraformaldehyde. Further, after blocking with 10% BSA-PBS, the cells were stained with Alexa Fluor 488-labeled RNA or anti-human TβRII antibody (Santa Cruz) and Alexa Fluor 488-labeled anti-rabbit IgG antibody (Invitrogen). Cell nuclear DNA was stained with DAPI Nucleic Acid Stain (manufactured by Invitrogen). It was embedded using VECTASHIELD Mounting Medium (manufactured by Vector Laboratories), and fluorescence was observed with an inverted research microscope IX71 (Olympus).
When staining human TβRII-expressing 293T cells with Alexa Fluor 488-labeled anti-TβRII RNA aptamer, fluorescence was observed in the same manner as immunostaining with anti-human TβRII antibody. Not observed. On the other hand, Alexa Fluor 488-labeled N40 random RNA was used to stain human TβRII-expressing 293T cells and parental strain 293T cells, but no fluorescence was observed in either. From these results, it was shown that the obtained anti-TβRII RNA aptamer specifically recognizes and binds to TβRII expressed on the cell surface, similarly to the anti-human TβRII antibody.

[Example 6] Search for anti-TβRII RNA aptamer that recognizes TGF-β binding region Is there an RNA aptamer that recognizes the TGF-β binding region of TβRII among 13 types of anti-TβRII RNA aptamers capable of binding to sTβRII? Searched for no.
As described above, the RNA aptamer was immobilized on the streptavidin sensor chip, and sTβRII or sTβRII (Tβ-sTβRII) previously bound with TGF-β was added to perform surface plasmon resonance analysis (FIG. 2). Of the 13 aptamers, an RNA aptamer named 2RA5 bound to sTβRII, but not to Tβ-sTβRII. On the other hand, 12 other RNA aptamers, including an aptamer named 2RA1, bound to both sTβRII and Tβ-sTβRII.
As a result, it was found that only 2RA5 recognized the TGF-β binding region of TβRII and bound to this region. 2RA5 is an RNA aptamer comprising the nucleotide sequence represented by SEQ ID NO: 1 (hereinafter, the RNA aptamer comprising the nucleotide sequence represented by SEQ ID NO: 1 may be referred to as “2RA5”).

[Example 7] Precipitation of TβRII and Tβ-TβRII by RNA aptamer Furthermore, RNA aptamer 2RA5 obtained in Example 6 was transformed into TβRII or a complex of TGF-β and TβRII (Tβ-II) from a cell lysate. It was examined whether or not (TβRII) could be separated.
Polyester (A) ₁₆ was added to each 3 ′ end of N40 random RNA and RNA aptamer (2RA1, 2RA5) and Oligotex-dT30 Super (manufactured by Takara Bio Inc.) was immobilized on Oligo (dT) ₃₀ Immobilized via base pairing. Next, 293T cells expressing hemagglutinin (HA) -labeled human TβRII (TβRII-HA) were unstimulated or stimulated with TGF-β, and then treated with 2 mM DSS (Pierce) to bind TGF-β. And TβRII were cross-linked to obtain a cell lysate. Oligotex-dT30 Super immobilized with RNA and cell lysate were mixed and reacted, and then washed with PBS. RNA and the protein binding thereto were recovered from Oligotex-dT30 Super and analyzed by Western blotting using SDS-PAGE and peroxidase (HRP) -labeled anti-HA monoclonal antibody. In the Western blotting, Amersham ECL Plus Westing Bleaching Detection Reagents (manufactured by GE Healthcare) and HRP are reacted with each other, detected by LAS-1000 Plus (manufactured by Fuji Film), and TβRII and Tβ-TβRII are precipitated by RNA aptamer. I analyzed.
As a result, 2RA5 separated TβRII, but Tβ-TβRII could not be separated. On the other hand, 2RA1 separated not only TβRII but also Tβ-TβRII, and neither N40 random RNA was separated. The above results revealed that 2RA5 is bound to the TGF-β binding region of TβRII, suggesting that 2RA5 may inhibit the binding of TGF-β and TβRII and suppress the physiological action of TGF-β. It was.

[Example 8] Inhibition of TGF-β signal transduction by RNA aptamer Next, examination was made as to whether the RNA aptamer of the present invention inhibits the binding of TGF-β and TβRII in vitro and suppresses TGF-β signal transduction. did.
When TGF-β binds to TβRII, Smad2 and Smad3, which are TGF-β signaling molecules present in the cytoplasm, are activated. Activated Smad2 and Smad3 form a heterocomplex with Smad4, and move from the cytoplasm to the nucleus to function as a transcription factor. When FLAG-tagged Smad3 (FLAG-Smad3), Smad4, TβRI, TβRII expression plasmids were introduced into 293T cells and FLAG-Smad3 expression plasmids were introduced into human hepatoma cell line Hep3B and expressed, and stimulated with TGF-β, FLAG-Smad3 Intranuclear translocation was observed. Therefore, cells were stimulated with TGF-β in the presence of sTβRII, N40 random RNA, and RNA aptamer (2RA1, 2RA5), and FLAG-Smad3 nuclear translocation was anti-FLAG monoclonal antibody and Alexa Fluor 568-labeled anti-mouse IgG antibody. Was observed.
As a result, like sTβRII, FLAG-Smad3 translocation into the nucleus was markedly inhibited in the presence of 2RA5. In contrast, FLAG-Smad3 translocation into the nucleus was not inhibited in the presence of N40 random RNA and 2RA1. From the above results, it was found that 2RA5 inhibits the binding of TGF-β to TβRII, thereby inhibiting the signal transduction pathway mediated by TGF-β and suppressing the physiological action derived from TGF-β.

[Example 9] The size of 2RA5 was attempted for the purpose of reducing the number of bases and reducing the molecular weight while maintaining the ability of the RNA aptamer to bind to a shortened target substance.
Based on the secondary structure predicted by the MFOLD program of 2RA5, a plurality of deletion mutants of 2RA5 were prepared, and the binding ability of each deletion mutant to sTβRII was examined by surface plasmon resonance analysis. The RNA aptamer named “2RA5S1” deleted by 9 bases from 2RA5 of 73 base length and the RNA aptamer named “2RA5S2” deleted by 14 bases are similar to the secondary structure similar to 2RA5. (Fig. 3).
The nucleotide sequences of 2RA5S1 and 2RA5S2 are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[Example 10] Inhibition test of TGF-β cell growth inhibitory action by RNA aptamer Human gastric cancer cell line SNU-16 maintains reactivity to TGF-β, and when stimulated with TGF-β, cell growth is strongly suppressed. The Thus, SNU-16 cells were stimulated with TGF-β in the presence of anti-human TβRII antibody, N40 random RNA, 2RA1, 2RA5, or 2RA5S1 to examine whether the cell growth inhibitory action of TGF-β was inhibited (Fig. 4).
Cells were seeded in a 96-well plate, medium, anti-human TβRII antibody (R & D Systems), N40 random RNA pool, or RNA aptamer was added, and allowed to stand at 37 ° C., 5% CO ₂ for 1 hour. Thereafter, medium or 40 pM TGF-β was added and cultured for 16 hours. Cell Proliferation Reagent WST-1 (Roche Applied Science) was added and allowed to stand at 37 ° C., 5% CO ₂ for 4 hours, and then the absorbance of the sample was measured at a wavelength of 450 nm using a microplate reader. The reference wavelength was 655 nm.
As a result, similar to the anti-human TβRII antibody, suppression of cell proliferation induced by TGF-β was markedly inhibited in the presence of 2RA5 or 2RA5S1. On the other hand, such inhibition of cell growth was not observed in the presence of N40 random RNA or 2RA1. From these results, it became clear that 2RA5 and 2RA5S1 inhibit the cell growth inhibitory action of TGF-β in vitro.

This application is based on Japanese Patent Application No. 2009-198813 (filing date: August 28, 2009) filed in Japan, the contents of which are incorporated in full herein.

SEQ ID NO: 1 is a nucleic acid capable of binding to the TGF-β type II receptor.
SEQ ID NO: 2 is a nucleic acid capable of binding to the TGF-β type II receptor.
SEQ ID NO: 3 is a nucleic acid capable of binding to the TGF-β type II receptor.

Claims

An aptamer that binds to a TGF-β type II receptor and inhibits the binding between TGF-β and a TGF-β type II receptor.
The aptamer according to claim 1, comprising all or part of a nucleotide sequence selected from any one of SEQ ID NOs: 1 to 3.
The aptamer according to claim 2, wherein at least one of the pyrimidine nucleotides is a modified nucleotide.
The aptamer according to claim 1, which is either (a) or (b) below:
(A) an aptamer comprising all or part of a nucleotide sequence selected from any one of SEQ ID NOs: 1 to 3, wherein the 2 ′ position of the ribose of the pyrimidine nucleotides contained in the aptamer is the same or different and is a fluorine atom Or an aptamer substituted with an atom or group selected from the group consisting of a hydrogen atom, a hydroxy group and a methoxy group;
(B) an aptamer comprising all or part of a nucleotide sequence in which 1 to 5 nucleotides are substituted, deleted, inserted or added in a nucleotide sequence selected from any of SEQ ID NOs: 1 to 3, The 2 ′ position of the ribose of the pyrimidine nucleotide contained in the aptamer is the same or different and is a fluorine atom, or is substituted with an atom or group selected from the group consisting of a hydrogen atom, a hydroxy group and a methoxy group, Aptamer.
A complex comprising the aptamer according to any one of claims 1 to 4 and a functional substance.
The complex according to claim 5, wherein the functional substance is an affinity substance, a labeling substance, an enzyme, a drug delivery vehicle or a drug.
A method for detecting and / or quantifying a TGF-β type II receptor, comprising using the aptamer according to any one of claims 1 to 4 or the complex according to claim 5 or 6.
A prophylactic or therapeutic drug for a disease caused by the action of TGF-β, comprising the aptamer according to any one of claims 1 to 4 or the complex according to claim 5 or 6.
A method for preventing or treating a disease caused by the action of TGF-β, comprising administering the aptamer according to any one of claims 1 to 4 or the complex according to claim 5 or 6.
The aptamer according to any one of claims 1 to 4 or the complex according to claim 5 or 6 for the prevention or treatment of a disease caused by the action of TGF-β.