RNA-containing antitumor agent and its use

Abstract

This antitumor agent comprises, as an active ingredient, an RNA which has a base sequence selected from the group consisting of the following (1) and (2) and has at least one nucleotide unit represented by formula (1) in a 3'-terminal region within 20 bases from the 3'-terminal thereof and / or in a 5'-terminal region within 25 bases from the 5'-terminal thereof. (1) A base sequence represented by SEQ ID NO: 2 or a base sequence having at least 90% identity with the sequence, and (2) a base sequence having 20 to 200 consecutive bases among the base sequences of (1) above (in formula (1), R1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, or a halogen atom; R2 represents NHR3 having a linking group, and the linking group is a divalent hydrocarbon group having at least one carbon atom; R3 represents a hydrogen atom, an alkyl group, or an alkenyl group; n represents 0 or 1; X1 represents an oxygen atom or a sulfur atom; X2 represents OH (or O-) or SH (or S-); and B represents a purine base or a pyrimidine base.)

Description

[Technical field]

[0001] This application claims priority based on Japanese patent application No. 2022-81120, filed with the Japan Patent Office on May 17, 2022, the contents of which are incorporated herein by reference as part of this specification. The present specification relates to the use of an antitumor agent containing RNA for suppressing tumor metastasis, a method for activating immune cells, and the like. [Background technology]

[0002] Recently, we have discovered potential anti-metastatic immune cells. Specifically, B220 + CD11c + NK1.1 + We found that natural killer (NK) cells migrated from the liver to the lungs of mice with cancer (Non-Patent Document 1). These NK cells reacted with fibrinogen + It accumulates in hyperpermeable sites, removes concentrated fibrinogen, and has the ability to destroy tumors by producing interferon gamma (IFNγ) (Non-Patent Document 2).

[0003] Liver B220 + CD11c + NK1.1 + NK cells and B220 in the lungs + CD11c + NK1.1 + Comparison of gene expression analysis between cells showed that ZC3H12D, which belongs to the RNA-binding zinc finger family, showed the highest fold change in lung cells. ZC3H12D has been reported as a putative tumor suppressor in lymphoma and lung cancer patients (Non-Patent Documents 3, 4). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] EMBOMolMed10,doi:10.15252 / emmm.201708643(2018) [Non-Patent Document 2] NatCommun4,1853,doi:10.1038 / ncomms2856(2013) [Non-Patent Document 3] CancerRes67,93-99,doi:10.1158 / 0008-5472.CAN-06-2723(2007) [Non-Patent Document 4] BrJHaematol139,161-163,doi:10.1111 / j.1365-2141.2007.06752.x(2007) Summary of the Invention

[0005] According to the present inventors, previous studies have suggested that ZC3H12D protein interacts with extracellular RNA (exRNA) in antimetastatic function, leading to the activation of NK cells.

[0006] The present specification provides an antitumor agent comprising, as an active ingredient, RNA capable of effectively interacting with ZC3H12D protein and activating NK cells, and uses thereof.

[0007] [1] An antitumor agent comprising as an active ingredient an RNA having a base sequence selected from the group consisting of (1) and (2) below, the RNA having at least one first nucleotide unit represented by the following formula (1) or at least one second nucleotide unit represented by the following formula (2) in its 3'-terminal region within 20 bases from the 3'-terminus and / or in its 5'-terminal region within 25 bases from the 5'-terminus. (1) The base sequence represented by SEQ ID NO:2 or a base sequence having 90% or more identity thereto (2) A base sequence of 20 to 200 consecutive bases from the base sequence of (1) above. [ka] (In formula (1), R 1represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, or a halogen atom; R 2 is an NHR having a linking group 3 The linking group is a divalent hydrocarbon group having one or more carbon atoms, and R 3 represents a hydrogen atom, an alkyl group, or an alkenyl group; n represents 0 or 1; X 1 represents an oxygen atom or a sulfur atom, X 2 is OH (or O - ) or SH(or S - ) and B represents a purine base or a pyrimidine base. [ka] (In formula (2), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, or a halogen atom; R 4 is an NHR having a linking group 5 The linking group is a divalent hydrocarbon group having one or more carbon atoms, and R 5 represents a hydrogen atom, an alkyl group, or an alkenyl group; n represents 0 or 1; X 1 represents an oxygen atom or a sulfur atom, X 2 is OH (or O - ) or SH(or S - ) and B represents a purine base or a pyrimidine base. [2] The antitumor agent according to [1], wherein the RNA has 1 to 20 of the nucleotide units in the 3'-terminal region and / or the 5'-terminal region. [3] The first nucleotide unit is represented by the formula (1), R 1 is a methoxy group or a fluorine atom, and R 2 The antitumor agent according to [1] or [2], wherein the nucleotide unit is an aminoethyl group. [4] In the first nucleotide unit, n is 1 and R 1 is a fluorine atom, and R 2 is a nucleotide unit which is an aminoethyl group, The antitumor agent according to [1] or [2], having at least one first nucleotide unit within 2 bases from the 3' end of the 3'-terminal region and at least one first nucleotide unit within 2 bases from the 5' end of the 5'-terminal region. [5] In the second nucleotide unit, n is 1 and R 1 is a methoxy group, and R 4 is a nucleotide unit which is an aminopropyl group, The antitumor agent according to [1] or [2], having at least one second nucleotide unit within 2 bases from the 3' end of the 3'-terminal region and at least one second nucleotide unit within 2 bases from the 5' end of the 5'-terminal region. [6] The antitumor agent described in [4] or [5], wherein the base sequence has 90% or more identity to a base sequence of 30 to 70 consecutive bases of the base sequence represented by SEQ ID NO: 4. [7] The antitumor agent described in [4] or [5], wherein the base sequence has 90% or more identity to a base sequence of 40 to 60 consecutive bases of the base sequence represented by SEQ ID NO: 4. [8] The antitumor agent according to [6] or [7], wherein the base sequence has 90% or more identity to the base sequence represented by SEQ ID NO: 6 or 10. [9] The antitumor agent described in [8], wherein the base of the first or second nucleotide unit is cytosine or uracil.

[10] In the first or second nucleotide unit, n is 1, and the RNA has, in each of the 3'-terminal region and / or the 5'-terminal region, X 1 Or X 2 Either S or SH(S - The antitumor agent according to any one of [1] to [9], wherein the first or second nucleotide unit is:

[11] The antitumor agent according to any one of [1] to

[10] , wherein 80% or more of the total number of uridine nucleosides in the RNA comprises a uridine analog selected from the group consisting of pseudouridine, 1-methylpseudouridine, 1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine.

[12] An antitumor agent described in any of [1] to

[11] , wherein the RNA is a mutant type of RNA having a base sequence represented by SEQ ID NO: 1, and the coding sequence from base 88 to base 897 of the base sequence represented by SEQ ID NO: 1 has bases substituted to have 1 to 5 additional stop codons compared to the natural coding sequence.

[13] The antitumor agent according to any one of [1] to

[12] , wherein the length of the RNA is 20 to 100 bases.

[14] An immunostimulant comprising, as an active ingredient, RNA having any one of a base sequence selected from the group consisting of (1) and (2) below, and having at least one first nucleotide unit represented by the following formula (1) or at least one second nucleotide unit represented by the following formula (2) in its 3'-terminal region within 20 bases from the 3'-terminal and / or its 5'-terminal region within 25 bases from the 5'-terminal: (1) The base sequence represented by SEQ ID NO:2 or a base sequence having 90% or more identity thereto (2) A base sequence of 20 to 200 consecutive bases from the base sequence of (1) above. [ka] (In formula (1), R 1represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, or a halogen atom; R 2 is an NHR having a linking group 3 The linking group is a divalent hydrocarbon group having one or more carbon atoms, and R 3 represents a hydrogen atom, an alkyl group, or an alkenyl group; n represents 0 or 1; X 1 represents an oxygen atom or a sulfur atom, X 2 is OH (or O - ) or SH(or S - ) and B represents a purine base or a pyrimidine base. [ka] (In formula (2), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, or a halogen atom; R 4 is an NHR having a linking group 5 The linking group is a divalent hydrocarbon group having one or more carbon atoms, and R 5 represents a hydrogen atom, an alkyl group, or an alkenyl group; n represents 0 or 1; X 1 represents an oxygen atom or a sulfur atom, X 2 is OH (or O - ) or SH(or S - ) and B represents a purine base or a pyrimidine base.

[15] An agent for suppressing tumor metastasis, comprising as an active ingredient RNA having a base sequence selected from the group consisting of (1) and (2) below, and having at least one first nucleotide unit represented by the following formula (1) or at least one second nucleotide unit represented by the following formula (2) in its 3'-terminal region within 20 bases from the 3'-terminus and / or its 5'-terminal region within 25 bases from the 5'-terminus. (1) The base sequence represented by SEQ ID NO:2 or a base sequence having 90% or more identity thereto (2) A base sequence of 20 to 200 consecutive bases from the base sequence of (1) above. [ka] (In formula (1), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, or a halogen atom; R 2 is an NHR having a linking group 3 The linking group is a divalent hydrocarbon group having one or more carbon atoms, and R 3 represents a hydrogen atom, an alkyl group, or an alkenyl group; n represents 0 or 1; X 1 represents an oxygen atom or a sulfur atom, X 2 is OH (or O - ) or SH(or S - ) and B represents a purine base or a pyrimidine base. [ka] (In formula (2), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, or a halogen atom; R 4 is an NHR having a linking group 5 The linking group is a divalent hydrocarbon group having one or more carbon atoms, and R 5 represents a hydrogen atom, an alkyl group, or an alkenyl group; n represents 0 or 1; X 1 represents an oxygen atom or a sulfur atom, X 2 is OH (or O - ) or SH(or S - ) and B represents a purine base or a pyrimidine base.

[16] An agent for enhancing the immunostimulating effect of an immunostimulant or the antitumor effect of an antitumor agent, comprising as an active ingredient RNA having a base sequence selected from the group consisting of (1) and (2) below, and having at least one first nucleotide unit represented by the following formula (1) or at least one second nucleotide unit represented by the following formula (2) in its 3'-terminal region within 20 bases from the 3'-terminus and / or its 5'-terminal region within 25 bases from the 5'-terminus. (1) The base sequence represented by SEQ ID NO:2 or a base sequence having 90% or more identity thereto (2) A base sequence of 20 to 200 consecutive bases from the base sequence of (1) above. [ka] (In formula (1), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, or a halogen atom; R 2 is an NHR having a linking group 3 The linking group is a divalent hydrocarbon group having one or more carbon atoms, and R 3 represents a hydrogen atom, an alkyl group, or an alkenyl group; n represents 0 or 1; X 1 represents an oxygen atom or a sulfur atom, X 2 is OH (or O - ) or SH(or S - ) and B represents a purine base or a pyrimidine base. [ka] (In formula (2), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, or a halogen atom; R 4 is an NHR having a linking group 5 The linking group is a divalent hydrocarbon group having one or more carbon atoms, and R 5 represents a hydrogen atom, an alkyl group, or an alkenyl group; n represents 0 or 1; X 1 represents an oxygen atom or a sulfur atom, X 2 is OH (or O - ) or SH(or S - ) and B represents a purine base or a pyrimidine base. Effect of the Invention

[0008] According to the antitumor agent disclosed in the present specification, the active ingredient is RNA having a nucleotide unit containing a specific chemical modification in the 3'-terminal region and / or the 5'-terminal region of the RNA, and therefore the agent has excellent stability in the living body. In addition, the RNA disclosed in the present specification can efficiently interact with immune cells and activate the immune cells. This can effectively suppress tumors. According to such RNA, by the same action, it is possible to effectively activate immune cells, effectively suppress metastasis of tumor cells, and effectively activate immune cells, thereby enhancing the antitumor effect of, for example, an antitumor agent used in combination. The RNA disclosed in the present specification is useful for preventing and / or treating tumors. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows an outline of synthetic RNA used in the Examples. [Diagram 2] FIG. 1 shows the number of migrating cells when synthetic RNA was administered to 786-O cells overexpressing ZC3H12D. [Diagram 3] FIG. 1 shows the results of evaluating the effect of chemically modified RNA on IFNγ production in human CD56+CD3- NK cells. [Figure 4] FIG. 1 shows the results of evaluating the effect of chemically modified RNA on IFNγ production in human CD56+CD3- NK cells. [Diagram 5] FIG. 1 shows an outline of synthetic RNA used in the Examples. [Figure 6] FIG. 1 shows the results of evaluating the RNase resistance of chemically modified RNA in the serum of tumor-bearing mice. [Figure 7] FIG. 1 shows the results of evaluation of RNase resistance of chemically modified RNA in bovine serum. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present disclosure relates to synthetic RNA related to exILβ-mRNA, which is a sequence near the untranslated region on the RNA 3'-terminal side of IL-1β, and its use. The inventors have found that even when specific nucleotide units are introduced as chemical modifications into the 3'- and 5'-terminals of synthetic RNA corresponding to exILβ-mRNA, it can exert an NK cell activation effect equivalent to that of exIL1β-mRNA. Such synthetic RNA is considered to exert a role as an inherent intercellular neurotransmitter with anti-tumor or anti-metastasis function in vivo.

[0011] In this specification, the "chemical modification" of RNA may be any chemical modification applied to RNA, and may include, for example, modifications of the ribose, phosphodiester bond, and base of a nucleotide. With respect to bases, it includes not only replacement with a base sequence different from the natural base sequence, but also chemical modification of such bases even if the type of base is the same as that of the natural base sequence.

[0012] In the following, representative and non-limiting specific examples of the present invention will be described in detail with reference to the drawings as appropriate. This detailed description is intended simply to provide those skilled in the art with details for carrying out preferred examples of the present invention, and is not intended to limit the scope of the present invention. In addition, the additional features and inventions disclosed below can be used separately or together with other features and inventions to provide further improved antitumor agents and their uses.

[0013] Furthermore, the combinations of features and steps disclosed in the following detailed description are not essential to the practice of the invention in its broadest sense, but are specifically described only to illustrate representative embodiments of the invention. Furthermore, the various features of the representative embodiments described above and below, and the various features of those described in the independent and dependent claims, do not have to be combined in the exact embodiments described herein, or in the exact order listed, to provide additional and useful embodiments of the invention.

[0014] All features described in the specification and / or claims are intended to be disclosed individually and independently of one another as limitations to the original disclosure and claimed particulars, apart from any configuration of features described in the examples and / or claims. Furthermore, all numerical ranges and group or aggregate descriptions are intended to disclose their intermediate configurations as limitations to the original disclosure and claimed particulars.

[0015] In addition, hereinafter, the meaning of "lower" in a substituent in a compound described in the present specification means that the number of carbon atoms constituting the substituent is up to 10. For example, usually, the number of carbon atoms is 1 to 6, or 1 to 5, for example, the number of carbon atoms is 1 to 4, or for example, the number of carbon atoms is 1 to 3.

[0016] Various embodiments of the disclosure of this specification will be described in detail below. First, RNA used in this specification will be described, and then embodiments using RNA will be described.

[0017] (RNA) The RNA of the present disclosure has a base sequence of either (1) or (2) below, and can have at least one first nucleotide unit represented by formula (1) or at least one second nucleotide unit represented by formula (2) below in its 3'-terminal region within 20 bases from the 3'-terminus and / or in its 5'-terminal region within 25 bases from the 5'-terminus.

[0018] The RNA of the present disclosure has the following sequence: (1) The base sequence represented by SEQ ID NO:2 or a base sequence having 90% or more identity thereto (2) A base sequence of 20 to 200 consecutive bases from the base sequence of (1) above.

[0019] The RNA consisting of the base sequence represented by SEQ ID NO:2 corresponds to the 3' untranslated region (3'UTR) (exIL1β-mRNA) of IL1β-mRNA (the full-length base sequence of which is represented by SEQ ID NO:1), which is the mRNA of human interleukin-1β (IL1β, NM_000576).

[0020] The RNA of the present disclosure may have the full length of the base sequence represented by SEQ ID NO: 2, or may have a base sequence having 90% or more, for example, 95% or more, for example, 96% or more, for example, 97% or more, for example, 98% or more, or for example, 99% or more identity with the base sequence. Here, the identity of the base sequence can be determined, for example, by aligning two or more base sequences by blastn in NCBI's BLAST (BLAST: Basic Local Alignment Search Tool (nih.gov) page) (https: / / blast.ncbi.nlm.nih.gov / Blast.cg) under optimized conditions with highly similar sequence (megablast).

[0021] From the viewpoint of the present disclosure, a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 2 has the ability to activate NK cells. The ability to activate immune cells such as NK cells can be confirmed by the method disclosed in the Examples described below.

[0022] The RNA of the present disclosure may be a part of the base sequence shown in SEQ ID NO: 2 or a base sequence having 90% or more identity to said base sequence (hereinafter, such sequences are also collectively referred to as the base sequence number shown in SEQ ID NO: 2, etc.). The base sequence shown in SEQ ID NO: 2 has a total length of 605 bases, and a part of the base sequence shown in SEQ ID NO: 2, etc. may be, for example, a continuous portion of the base sequence shown in SEQ ID NO: 2, etc., that is 20 to 200 bases long. The base length is, for example, 25 bases or more, for example, 30 bases or more, for example, 40 bases or more, for example, 45 bases or more, and the base length is 180 bases or less, for example, 160 bases or less, for example, 150 bases or less, for example, 140 bases or less, for example, 130 bases or less, for example, 120 bases or less, for example, 110 bases or less, for example, 100 bases or less, for example, 90 bases or less, for example, 80 bases or less, for example, 75 bases or less, for example, 70 bases or less, for example, 60 bases or less. The range of the RNA of the present disclosure can be set by appropriately combining such lower and upper limits. For example, it can be 20 to 100 bases in length, 30 to 100 bases in length, 30 to 80 bases in length, or 40 to 60 bases in length.

[0023] The range of the part in the base sequence shown in SEQ ID NO: 2 is not particularly limited, and a region having NK cell activation ability can be appropriately selected. In the base sequence shown in SEQ ID NO: 2, the part can be selected, for example, from the range of 200 to 500 bases from the 5' end, or, for example, from 220 to 480 bases, or, for example, from 260 to 460 bases, or, for example, from 300 to 450 bases.

[0024] Examples of such base sequences include the base sequences represented by SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, as well as base sequences having 90% or more, for example, 95% or more, for example, 96% or more, for example, 97% or more, for example, 98% or more, for example, 99% or more identity with these sequences. From the perspective of the present disclosure, some sequences such as the base sequence represented by SEQ ID NO: 2 have the ability to activate NK cells. The mRNA consisting of the base sequence represented by SEQ ID NO: 5 is one of the fragments when human IL1β-mRNA is divided into 4 fragments containing 150 nucleotides. Also included are the base sequence represented by SEQ ID NO: 10 or a base sequence having 90% or more, for example, 95% or more, for example, 96% or more, for example, 97% or more, for example, 98% or more, for example, 99% or more identity with this base sequence.

[0025] <Chemical Modifications in RNA> (First Nucleotide Unit: Nucleotide Unit Represented by Formula (1)) In the nucleotide unit represented by the following formula (1), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, or a halogen atom.

[0026] [Chemical Formula]

[0027] Examples of the alkyl group include saturated hydrocarbon groups that are linear, branched, cyclic, or a combination thereof. In general, lower alkyl groups are preferred, and more preferred examples include lower alkyl groups having 1 to 6 carbon atoms or lower alkyl groups having 1 to 5 carbon atoms, and particularly preferred examples include lower alkyl groups having 1 to 4 carbon atoms or lower alkyl groups having 1 to 3 carbon atoms. Suitable examples of linear alkyl groups having 1 to 4 carbon atoms include methyl, ethyl, n-propyl, and n-butyl groups, and among these, methyl, ethyl, and n-propyl groups are preferred, and also, for example, methyl groups are preferred. Examples of branched alkyl groups having 1 to 4 carbon atoms include isopropyl, isobutyl, s-butyl, and t-butyl groups, and among these, isopropyl is a particularly preferred example. Examples of cyclic alkyl groups having 1 to 4 carbon atoms include cyclopropyl, cyclobutyl, and cyclopropylmethyl groups.

[0028] Alkenyl groups include saturated hydrocarbon groups which are linear, branched, cyclic, or a combination thereof. In general, lower alkenyl groups are preferred, and examples of lower alkenyl groups include ethenyl, 1-propenyl, 2-propenyl, 1-methyl-2-propenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-butenyl, and 2-butenyl groups.

[0029] The halogen atom is not particularly limited, but is, for example, a fluorine atom.

[0030] R 2 is an NHR having a linking group 3 That is, NHR 3 is attached to the 4' carbon atom via a linking group attached to the nitrogen atom.

[0031] The linking group can be, for example, a divalent hydrocarbon group having one or more carbon atoms. That is, examples of the divalent hydrocarbon group include an alkylene group having 1 to 8 carbon atoms and an alkenylene group having 2 to 8 carbon atoms.

[0032] The alkylene group as the linking group may be linear or branched, but is preferably linear. For example, a lower alkylene group is preferred, for example a lower alkylene group having 1 to 6 carbon atoms, or for example a lower alkylene group having 2 to 6 carbon atoms, or for example a lower alkylene group having 2 to 4 carbon atoms or 2 to 3 carbon atoms is preferred. Examples of linear alkylene groups having 1 to 4 carbon atoms include methylene, ethylene, propane-1,3-diyl, n-butane-1,1-diyl, n-pentyl-1,5-diyl, and n-hexyl-1,6-diyl. Examples of butane-1,2-diyl are also preferred. For example, ethylene, propane-1,3-diyl, and n-butane-1,1-diyl are particularly preferred.

[0033] The alkenylene group as the linking group may be linear or branched, and is preferably linear. For example, a lower alkenylene group is preferred, and examples of the lower alkenylene group include an ethene-1,2-diyl group, a propene-1,3-diyl group, and a butene-1,4-diyl group.

[0034] In the nucleotide unit represented by formula (1), a divalent hydrocarbon group such as an alkylene group having 1 to 3 carbon atoms, e.g., a methylene group, an ethylene group, or a propylene group, is preferred from the viewpoint of the NK cell activation ability of the RNA of the present disclosure.

[0035] R 3 Examples of the alkyl group include a hydrogen atom, an alkyl group, or an alkenyl group. In addition to the alkyl groups already described, preferred examples of the alkyl group include lower alkyl groups, such as an alkyl group having 1 to 3 carbon atoms, an alkyl group having 1 or 2 carbon atoms, and a methyl group. In addition to the alkenyl groups already described, preferred examples of the alkenyl group include lower alkenyl groups. R3 When is such a group, such as a hydrogen atom, the linking group is suitably an alkylene group having 2 or more, such as 3 or more, such as 4 or more, and such as 6 or less, such as 5 or less, such as 4 or less carbon atoms.

[0036] Also, R 3 When is a hydrogen atom, R 2 is an NH2 (amino group) having a linking group, that is, when the linking group is an alkylene group, it becomes an aminoalkyl group. 2 is an aminoalkyl group such as an aminoethyl group or an aminopropyl group, it is considered to be suitable as the RNA of the present disclosure.

[0037] n represents 0 or 1. When n is 0, the 3' position of the nucleotide unit is O - For example, it may be OH. When n is 1, X 1 represents an oxygen atom or a sulfur atom, X 2 is OH (or O - ) or SH(or S - ) stands for X. 1 Or X 2 is a sulfur atom or SH(S - ), the nucleotide unit has the next nucleotide unit at the 3' side via a phosphorothioate bond. When n is 1, the nucleotide unit can have a phosphate group or a phosphorothioate group at the 3' position. When it is a phosphate group, it may be in the form of a salt with an appropriate base.

[0038] B represents a purine base or a pyrimidine base. The purine base is not particularly limited, but includes, for example, a purine-9-yl group, as well as various substituted purine-9-yl groups such as 2,6-dimethoxypurine-9-yl and 2,6-dichloropurine-9-yl. The pyrimidine base includes, for example, a 2-oxo-pyrimidin-1-yl group and a 2-oxo-pyrimidin-1-yl group. Furthermore, substituted 2-oxo-pyrimidin-1-yl groups such as 2-oxo-4-methoxy-pyrimidin-1-yl and 4-(1H-1,2,4-triazol-1-yl)-pyrimidin-1-yl are included.

[0039] Additionally, B may be a modified base as described below.

[0040] (Second nucleotide unit: a nucleotide unit represented by formula (2)) More specifically, the second nucleotide unit represented by the following formula (2) is a nucleotide unit represented by either or both of the following formulas (2a) and 2(b). The nucleotide unit represented by formula (2a) is an R-form with respect to the asymmetric carbon atom at the 5' position, and the nucleotide unit represented by formula (2b) is an S-form with respect to the asymmetric carbon atom at the 5' position. In addition, in the RNA disclosed herein, these nucleotide units may be used in combination of the R-form and the S-form, or only one of them may be used. For example, in the RNA disclosed herein, it may be preferable to use only the S-form as the nucleotide unit represented by formula (2) from the viewpoints of nuclease resistance, stability in serum, and thermal stability of the RNA / RNA duplex. In addition, in this specification, the nucleotide unit represented by formula (2) and the second nucleotide unit refer to the S-form and / or the R-form, unless otherwise specified.

[0041] [ka]

[0042] [ka]

[0043] In the second nucleotide unit, R 1 has the same meaning as in formula (1). R 1 may preferably represent a hydroxyl group or a halogen atom in which a hydrogen atom is substituted with an alkyl group or an alkenyl group. 1 is preferably a hydroxyl group in which a hydrogen atom is substituted with an alkyl group having 1 to 3 carbon atoms. For example, a methyl group, an ethyl group, or an n-propyl group is preferable, and for example, a methyl group or an ethyl group is preferable, and for example, a methyl group is preferable. The halogen atom is not particularly limited, but may be preferably a fluorine atom, for example.

[0044] In the second nucleotide unit, R 4 is an NHR having a linking group 5 That is, NHR 5 is attached to the 5' carbon atom via a linking group attached to the nitrogen atom.

[0045] In the second nucleotide unit, a linking group and R 5 is R in formula (1). 2 The linking group and R 3 The linking group is, for example, a lower alkylene group, and may be, for example, a lower alkylene group having 1 to 6 carbon atoms, or may be, for example, a lower alkylene group having 2 to 6 carbon atoms, or may be, for example, a lower alkylene group having 2 to 4 carbon atoms or 2 to 3 carbon atoms. Examples of the linking group include a methylene group, an ethylene group, a propane-1,3-diyl group, an n-butane-1,1-diyl group, an n-pentyl-1,5-diyl group, an n-hexyl-1,6-diyl group, and the like. Examples of the linking group include a butane-1,2-diyl group, and the like. Examples of the linking group include an ethylene group, a propane-1,3-diyl group, and an n-butane-1,1-diyl group, and the like are particularly preferred in some cases, and a propane-1,3-diyl group is preferred in other cases.

[0046] The alkenylene group as a linking group may be linear or branched, and is preferably linear. For example, a lower alkenylene group may be preferred, and examples of the lower alkenylene group include an ethene-1,2-diyl group, a propene-1,3-diyl group, and a butene-1,4-diyl group.

[0047] In the second nucleotide unit, a divalent hydrocarbon group such as an alkylene group having 1 to 3 carbon atoms, for example, a methylene group, an ethylene group, or a propane-1,3-diyl group, is preferred in terms of the NK cell activation ability of the RNA of the present disclosure.

[0048] In the second nucleotide unit, R 5 In some cases, R is preferably a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, an alkyl group having 1 or 2 carbon atoms, or a methyl group. 5 is a hydrogen atom or any of these groups, the linking group may preferably be an alkylene group having 2 or more, for example 3 or more, for example 4 or more, and for example 6 or less, for example 5 or less, for example 4 or less carbon atoms, and preferably an ethylene group or a propane-1,3-diyl group.

[0049] Also, R 5 When is a hydrogen atom, R 4 is an NH having a linking group 2 (amino group), that is, when the linking group is an alkylene group, it becomes an aminoalkyl group. 4 is an aminoalkyl group such as an aminoethyl group or an aminopropyl group, it is considered to be suitable as the RNA of the present disclosure.

[0050] In the second nucleotide unit, X 1 and X 2has the same meaning as in the first nucleotide unit, and may take the same embodiment. In the second nucleotide unit, n represents 0 or 1. When n is 0 or 1, the second nucleotide unit may take the same embodiment as in the first nucleotide unit. In addition, B has the same meaning as in the first nucleotide unit, and may take the same embodiment.

[0051] The RNA of the present disclosure has at least one nucleotide unit represented by formula (1) and / or at least one nucleotide unit represented by formula (2) in its 5'-terminal region within 25 bases from the 5'-terminal and / or in its 3'-terminal region within 25 bases from the 3'-terminal. The RNA of the present disclosure may have any of these nucleotide units only in the 5'-terminal region, may have any of these nucleotide units only in the 3'-terminal region, or may have any of these nucleotide units in both the 5'-terminal region and the 3'-terminal region.

[0052] The RNA of the present disclosure may also have a combination of the first nucleotide unit and the second nucleotide unit in the 5'-terminal region. The RNA of the present disclosure may also have a combination of the first nucleotide unit and the second nucleotide unit in the 3'-terminal region. The RNA of the present disclosure may also have an appropriate combination of the first nucleotide unit and the second nucleotide unit in the 5'-terminal region and the 3'-terminal region. For example, the RNA may have the first nucleotide unit and the second nucleotide unit in the 5'-terminal region and the 3'-terminal region, respectively, or vice versa.

[0053] The 5'-terminal region is, for example, within 20 bases, for example, within 15 bases, or for example, within 10 bases from the 5'-terminal. For example, it is within 8 bases, for example, within 6 bases, for example, within 4 bases, for example, within 3 bases, for example, within 2 bases, or for example, within 1 base (5'-terminal). The 3'-terminal region is, for example, within 20 bases, for example, within 15 bases, for example, within 10 bases, for example, within 8 bases, for example, within 6 bases, for example, within 5 bases, or for example, within 4 bases from the 3'-terminal. For example, it is within 3 bases, for example, within 2 bases, or for example, within 1 base (3'-terminal).

[0054] The RNA of the present disclosure has at least one nucleotide unit represented by formula (1) and / or nucleotide unit represented by formula (2) in the 5'-terminal region. The number of nucleotide units represented by the nucleotide units represented by formula (1) and formula (2) in the region is not particularly limited, and may be, for example, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2. The nucleotide units represented by formula (1) and formula (2) are provided, for example, in a region within 6 bases from the 5'-terminal. This is believed to effectively protect the 5'-terminal side. It may be effective to provide the nucleotide units represented by formula (1) and formula (2) at least at the base position of the 5'-terminal or two bases from the 5'-terminal.

[0055] The RNA of the present disclosure has at least one nucleotide unit represented by formula (1) and / or one nucleotide unit represented by formula (2) in the 3'-terminal region. The number of the nucleotide units represented by formula (1) and the nucleotide units represented by formula (2) in the region is not particularly limited, but may be, for example, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2. The nucleotide units represented by formula (1) and the nucleotide units represented by formula (2) are provided, for example, in a region within 6 bases from the 3'-terminal. This is believed to effectively protect the 3'-terminal side. It may be effective to provide the nucleotide units represented by formula (1) and the nucleotide units represented by formula (2) at least at the base position of the 3'-terminal or 2 bases from the 5'-terminal.

[0056] The RNA of the present disclosure can have at least one nucleotide unit represented by formula (1) and / or one nucleotide unit represented by formula (2) in both the 3'-terminal region and the 5'-terminal region. Since such an RNA has these nucleotide units in these terminal regions, it has excellent nuclease resistance, and as a result, it exhibits excellent antitumor and metastasis suppression effects. The nucleotide units represented by formula (1) and the nucleotide units represented by formula (2) provided in each of these terminal regions are, for example, 1 to 5, or, for example, 1 to 4, or, for example, 1 to 3, or, for example, 1 to 2. In each of these terminal regions, it may be preferable to have one or two nucleotide units represented by formula (1) and / or formula (2) within 3 bases or 2 bases from each end. Furthermore, at least one or all of the nucleotide units represented by formula (1) in each of these terminal regions may preferably have a fluorine atom at the 2' position of ribose and an aminoethyl group at the 4' position, and at least one or all of the nucleotide units represented by formula (2) in each of these terminal regions may preferably have a methoxy group at the 2' position of ribose and an aminopropyl group at the 5' position. Furthermore, it may be preferable that the 5'-terminal region of RNA has a nucleotide unit represented by formula (2) having an aminopropyl group or the like at the 5' position in one or two regions from the 5' end, and the 3'-terminal region of RNA has a nucleotide unit represented by formula (1) having an aminoethyl group or the like at the 4' position in one or two regions from the 3' end. This is because in this form, the aminoalkyl group is closer to the 5' end and 3' end exposed to nuclease. In addition, the type of base in these nucleotide units is not particularly limited, but may be, for example, a base having a pyrimidine ring, or may be, for example, cytosine or uracil.

[0057] In view of the above, for example, the following aspects of the RNA of the present disclosure may be preferable. [1] The nucleotide unit represented by formula (1) is: n is 1; R 1 is a fluorine atom, and R 2 is a nucleotide unit which is an aminoethyl group, The nucleotide unit represented by formula (2) is R 1 is a methoxy group, and R 4 is a nucleotide unit which is an aminopropyl group, The 3'-terminal region has at least one nucleotide unit represented by formula (1) and / or one nucleotide unit represented by formula (2) within 2 bases from the 3'-terminal, and the 5'-terminal region has at least one nucleotide unit within 2 bases from the 5'-terminal, particularly, the 5'-terminal region has a nucleotide unit represented by formula (2) and the 3'-terminal region has a nucleotide unit represented by formula (1). [2] In [1], the base sequence has an identity of 90% or more with a base sequence of 30 to 70 consecutive bases of the base sequence represented by SEQ ID NO: 4. [3] In the above [1], the base sequence has an identity of 90% or more with a base sequence of 40 to 60 consecutive bases in the base sequence represented by SEQ ID NO: 4. [4] In the above [1] to [3], the base sequence has 90% or more identity with the base sequence represented by SEQ ID NO: 6 or 10. [5] In the above [1] to [4], the base of the nucleotide unit is cytosine or uracil.

[0058] The 5'-terminal region and 3'-terminal region of the RNA of the present disclosure may have a structure in which riboses are linked by phosphorothioate bonds, in addition to having a nucleotide unit represented by formula (1) and / or a nucleotide unit represented by formula (2). Linkages by phosphorothioate bonds may also be provided in the nucleotide units represented by formula (1) and the nucleotide units represented by formula (2), but it may be advantageous for nucleotides corresponding to other bases not substituted by the nucleotide units to also be linked by phosphorothioate bonds.

[0059] The introduction site and number of phosphorothioate bonds in the 5'-terminal region and the 3'-terminal region are not particularly limited. For example, the 5'-terminal and 3'-terminal regions may have 1 to 8, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 consecutive phosphorothioate bonds, respectively, including the initial linkage from the 5'-terminal and 3'-terminal, or may have 1 to 4, for example, 1 to 2 bases apart from the initial linkage from the terminal.

[0060] The RNA of the present disclosure comprises at least one nucleotide unit represented by formula (1) and / or one represented by formula (2) in the 5'-terminal region and / or 3'-terminal region, and the other nucleotide units may be natural nucleotides, i.e., nucleotide units comprising natural ribose, or may be nucleotide units in which the OH group of ribose has been modified, for example, by introducing a methoxy group at the 2'-position of ribose, or may be nucleotide units in which other known chemical modifications have been made to the ribose. For example, the RNA of the present disclosure may be RNA in which all nucleotide units other than the nucleotide unit represented by formula (1) in the 5'-terminal region and / or 3'-terminal region comprise chemically modified ribose having a methoxy group at the 2'-position of ribose.

[0061] In view of the above, for example, in any one of the above aspects (1) to (5) of the RNA disclosed herein, it may be preferable that all nucleotide units other than the nucleotide units have chemically modified ribose having a methoxy group at the 2' position of the ribose.

[0062] The RNA of the present disclosure can further comprise a chemical modification that changes the uridine in RNA to a uridine analogue. The chemical modification that changes uridine to a uridine analogue, for example, is not particularly limited in the percentage of the total number of uridines in the RNA of the present disclosure. For example, 80% or more of the total number of uridines can be uridine analogues, for example, 90% or more, or for example, 100%.

[0063] Nucleobases and nucleosides containing uridine analogues include, but are not limited to, uridine analogues selected from the group consisting of uridine pseudouridine, 1-methyl pseudouridine, 1-ethyl pseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine. For example, the uridine analogue is 1-methyl-pseudouridine.

[0064] The RNA of the present disclosure can further comprise chemical modification of the cytidine in RNA to cytidine analogue.The chemical modification of cytidine to cytidine analogue, for example, is not particularly limited to the ratio of the total number of cytidines in the RNA of the present disclosure.For example, 80% or more of the total number of cytidines can be cytidine analogue, for example, 90% or more, or for example, 100%.

[0065] Nucleobases and nucleosides, including cytidine analogs, include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, 4-acetyl-cytidine, 5-formylcytidine, 4-methylcytidine, 5-methyl-cytidine, 5-halo-cytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-pseudoisocytidine, 5-amino-1-methyl-1-de ... -zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine, α-thio-cytidine, 2'-O-methyl-cytidine, 5,2'-O-dimethyl-cytidine, 4-acetyl-2'-O-methyl-cytidine, 4,2'-O-dimethyl-cytidine, 5-formyl-2'-O-methyl-cytidine (f5Cm), 4,4,2'-O-trimethyl-cytidine, 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0066] The RNA of the present disclosure can further comprise a chemical modification of the adenine in RNA to an adenine analogue. The chemical modification of adenine to an adenine analogue is not particularly limited, for example, in the total number of adenines in the RNA of the present disclosure. For example, 80% or more of the total number of adenines can be adenine analogues, for example, 90% or more, or for example, 100%.

[0067] Nucleobases and nucleosides containing adenine analogs include 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-de ... ,6-Diaminopurine, 7-Deaza-8-Aza-2,6-Diaminopurine, 1-Methyladenosine, 2-Methyl-adenine, 6-Methyladenosine, 2-Methylthio-6-Methyl-adenosine, 6-Isopentenyladenosine, 2-Methylthio-6-Isopentenyl-adenosine, 6-(cis-Hydroxyisopentenyl)adenosine, 2-Methylthio-6-(cis-Hydroxyisopentenyl)adenosine, 6-Glycinylcarbamoyladenosine, N6- Threonylcarbamoyl adenosine, 6-methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-6-threonylcarbamoyl-adenosine, 6,6-dimethyl-adenosine, 6-hydroxynorvalylcarbamoyl-adenosine, 2-methylthio-6-hydroxynorvalylcarbamoyl-adenosine, 6-acetyl-adenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2'-O -methyl-adenosine, 6,2'-O-dimethyl-adenosine, 6,6,2'-O-trimethyl-adenosine, 1,2'-O-dimethyl-adenosine, 2'-O-ribosyladenosine (phosphate), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and 6-(19-amino-pentaoxanonadecyl)-adenosine.

[0068] The RNA of the present disclosure can further comprise a chemical modification that makes guanine in RNA into a guanine analogue. The chemical modification that makes guanine into a guanine analogue instead of guanine is not particularly limited, for example, in the total number of guanines in the RNA of the present disclosure. For example, 80% or more of the total number of guanines can be guanine analogues, for example, 90% or more, or for example, 100%.

[0069] Nucleobases and nucleosides containing guanine analogues include inosine, 1-methyl-inosine, wyosine, methylwyosine, 4-demethyl-wyosine, isowyosine, wyosine, peroxywyosine, hydroxywyosine, unmodified hydroxywyosine, 7-deaza-guanosine, queosine, epoxyqueosine, galactosyl-queosine, mannosyl-queosine, 7-cyano-que ... Ano-7-deaza-guanosine, 7-aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methylguanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methylguanosine, 2-methyl-guanosine Guanosine, 2,2-dimethyl-guanosine, 2,7-dimethyl-guanosine, 2,2,7-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, 2-methyl-6-thio-guanosine, 2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine, 2-methyl-2'-O-methyl-guanosine , 2,2-dimethyl-2'-O-methyl-guanosine, 1-methyl-2'-O-methyl-guanosine, 2,7-dimethyl-2'-O-methyl-guanosine, 2'-O-methyl-inosine, 1,2'-O-dimethyl-inosine, 2'-O-ribosylguanosine (phosphate), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.

[0070] When the base sequence defined in (1) and (2) is defined as the first region, the RNA of the present disclosure may have a 5' untranslated region (UTR) on the 5' end side of the first region. The 5'UTR may be the natural 5'UTR of the target polypeptide encoded by the first region, or may be a 5'UTR different from the encoded target polypeptide. For example, the 5'UTR may contain at least one translation initiation sequence, such as a Kozak sequence, an internal ribosome entry site (IRES), and / or a fragment thereof. The sequences and designs of these 5'UTRs in RNA medicines are well known.

[0071] The RNA of the present disclosure may further comprise a 5' cap and a polyA tail. The 5' cap is usually a modified nucleotide component and generally refers to a structure added to the 5' end of a mature mRNA.

[0072] The 5' cap may be formed by a modified nucleotide, in particular a derivative of a guanine nucleotide. Preferably, the 5' cap is attached to the 5' end via a 5'-5'-triphosphate bond. The 5' cap may be methylated, for example m7GpppN, where N is the terminal 5' nucleotide of the 5' capped nucleic acid.

[0073] The polyA tail is also called a polyA tail and refers to a polynucleotide sequence of consecutive adenines. The length of the polyA is not particularly limited, but is preferably 20 or more, more preferably 50 or more. The upper limit of the length is not particularly limited, but is, for example, 200 or less.

[0074] The RNA of the present disclosure may be mRNA or a part thereof. In the case of mRNA or a part thereof, it may contain 5'UTR and / or poly A tail. When the RNA of the present disclosure is mRNA and is a mutant type of RNA having a coding region, for example, a base sequence represented by SEQ ID NO: 1, and contains a coding sequence from base 88 to base 897 of the base sequence represented by SEQ ID NO: 1, it may contain a mutation that contains about 1 to 5 stop codons. For example, the base sequence represented by SEQ ID NO: 3 (containing 3 stop codons) may be mentioned. In addition, the RNA of the present disclosure may contain a G→A mutation to avoid a series of bases such as G, if necessary.

[0075] The RNA of the present disclosure may be an RNAi-inducing agent, an RNAi agent, an siRNA, an shRNA, an miRNA, an antisense RNA, an RNA, an aptamer, a vector, and the like.

[0076] The RNA of the present disclosure may have a labeling molecule, such as a fluorescent substance or a chromogenic substance, attached to a portion thereof.

[0077] The above various chemical modifications are known or well known to those skilled in the art, and based on the disclosure of this specification and the common general technical knowledge at the time of filing this application, a person skilled in the art can obtain RNA using a desired chemical modification. RNA having a nucleotide unit represented by formula (1) and / or a nucleotide unit represented by formula (2) can be produced by the methods described in WO 2018 / 110678 and WO 2019 / 088179.

[0078] Administration of the RNA of the present disclosure to cells overexpressing the ZC3H12D protein can result in activation of the cell's migratory ability, induction of the cell's anti-metastatic activity, and, in the case of the cells being natural killer (NK) cells, increased tumor-destructive activity.

[0079] The various embodiments of the RNA of the present disclosure are applicable, either alone or in combination, to each of the following aspects of the RNA of the present disclosure.

[0080] (Anti-tumor agent and method for inhibiting tumor proliferation) The antitumor agent of the present disclosure contains the RNA of the present disclosure as an active ingredient. The method of inhibiting tumor cell proliferation of the present disclosure is to administer the RNA of the present disclosure to humans or non-human animals in vitro. The RNA of the present disclosure has immune cell activation ability and the like by administering the RNA of the present disclosure to humans or non-human animals in vitro, and thus can exert an antitumor effect.

[0081] The RNA of the present disclosure as an active ingredient of an antitumor agent is an RNA that can activate immune cells or increase the antitumor activity of cells. The cells are cancer cells, tumor cells, or immune cells, and examples of immune cells include natural killer cells (NK cells), macrophages, eosinophils, neutrophils, basophils, dendritic cells, lymphocytes, etc. Preferably, the immune cells are natural killer cells (NK cells), macrophages, or both.

[0082] Tumors include head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, liver cancer, gallbladder cancer, bile duct cancer, biliary tract cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, kidney cancer, bladder cancer, prostate cancer, testicular tumors, osteosarcoma, multiple myeloma, skin cancer, brain tumors, and mesothelioma.

[0083] The activation of immune cells can be evaluated by IFNγ production in immune cells, improvement of the migration ability of immune cells or tumor cells expressing the ZC3H12D protein, etc. The antitumor activity can be evaluated by a known method for evaluating antitumor activity.

[0084] (Activator (stimulator) of immune cells and method for activating (stimulating) immune cells) The immune cell activator of the present disclosure contains the RNA of the present disclosure as an active ingredient. The immune cell activation method of the present disclosure can include administering the RNA of the present disclosure to humans or non-human animals in vitro. For the cells, immune cells, and activation of immune cells, the above-mentioned embodiments for the antitumor agent are applicable.

[0085] (Tumor cell metastasis inhibitor and method for inhibiting tumor cell metastasis) The tumor cell metastasis inhibitor of the present disclosure contains the RNA of the present disclosure as an active ingredient. The method for inhibiting tumor cell metastasis disclosed herein can include administering the RNA of the present disclosure to a human or non-human animal in vitro. The RNA of the present disclosure can activate immune cells to increase the antitumor activity of the cells and inhibit the metastasis of tumor cells. The cells, immune cells, activation of immune cells, and antitumor activity are the same as those described above for the antitumor agent.

[0086] Inhibition of metastasis of tumor cells can be evaluated by known methods such as counting the number of metastatic tumor cells.

[0087] The treatment intended to suppress the metastasis of primary cancer by the metastasis inhibitor is performed in combination with the treatment of primary cancer or independently. For example, when the metastasis inhibitor is used in combination, it can be used before, after, or simultaneously with one or more of the following treatments for primary cancer: surgical treatment, anticancer drug treatment, radiation therapy (X-ray, proton beam, heavy particle beam) and electromagnetic wave therapy (radio wave). Combination with surgical treatment is one of the preferred embodiments.

[0088] Typically, the following applications are mentioned: (a) When primary cancer remains after surgical treatment of the primary cancer, it is used in combination with one or more of anticancer drug therapy, radiation therapy, and electromagnetic therapy. (b) When the primary cancer can be removed after surgical treatment of the primary cancer, it is used alone or in combination with one or more of anticancer drug therapy, radiation therapy, electromagnetic therapy, etc. in order to suppress metastasis. (c) After surgical treatment of a primary cancer, when a new primary cancer or metastasis is found thereafter, the therapy may be used alone or in combination with one or more of surgical treatments, anticancer drug treatments, radiation therapy, electromagnetic therapy, etc., in order to suppress metastasis.

[0089] (Enhancer for enhancing the immunostimulating effect of immunostimulants and method for enhancing said effect) The enhancing agent of the present disclosure contains the RNA of the present disclosure as an active ingredient. The enhancing method disclosed herein can include administering the RNA of the present disclosure to a human or non-human animal in vitro. The RNA of the present disclosure is an RNA that can activate immune cells, increase the antitumor activity of cells, or suppress metastasis of tumor cells. Therefore, the above-mentioned embodiments of the antitumor agent and metastasis inhibitor are applicable to the activation of cells, immune cells, activation of immune cells, antitumor activity, and suppression of metastasis, which can be enhanced by using the RNA in combination with an immune cell activator, an antitumor agent, and a metastasis inhibitor to enhance the immune cell activation effect, antitumor activity effect, and metastasis suppression effect of these drugs.

[0090] The immunostimulant used in combination may be a known immunostimulant, and the metastasis inhibitor used in combination may be a known metastasis inhibitor.

[0091] The antitumor agent used in combination includes one or more antitumor agents selected from kinase inhibitors, apoptosis inducers, nuclear receptor regulators, immunomodulators, nuclear export signal inhibitors, proteasome regulators, DNA damaging agents, metabolic antagonists, platinum antitumor agents (platinum complexes), microtubule inhibitors, alkylating agents, and anthracycline antitumor agents, including salts of the compounds. These antitumor agents are well known, and commercially available ones can be used.

[0092] The administration method of the above-mentioned combined drug and the enhancer of the present disclosure is not particularly limited, and they can be administered simultaneously, sequentially, or at intervals, so long as the enhancer of the present disclosure exerts a predetermined enhancing effect. The administration order of these compositions is also not particularly limited, and the antitumor agent may be administered before, simultaneously, or after the RNA.

[0093] The concomitant drug and the enhancer of the present disclosure may be used concomitantly in the manner of use of individual drugs, or may be provided as a combination drug or drug kit with a specified usage method.

[0094] (Pharmaceutical composition, method for improving immunity, method for preventing or treating tumors) The pharmaceutical composition disclosed herein contains the present RNA as an active ingredient. The method for improving immunity and the method for preventing or treating tumors disclosed herein can include administering the RNA of the present disclosure to humans or non-human animals in vitro. The RNA of the present disclosure is capable of activating immune cells, increasing the antitumor activity of cells, or suppressing metastasis of tumor cells. Therefore, pharmaceutical compositions containing these are useful for preventing or improving various conditions and diseases by activating immune cells, and also for preventing or treating diseases such as tumors by their antitumor activity and tumor metastasis suppression activity. Examples of tumors include the various tumors already described.

[0095] The above-mentioned embodiments are applicable to the cells, immune cells, activation of immune cells, antitumor activity, and inhibition of metastasis of tumor cells.

[0096] When using the RNA of the present disclosure as an active ingredient of a medicine, a pharmaceutical carrier may be added as necessary, and various administration forms may be adopted according to the purpose of prevention or treatment. The form of the antitumor agent containing the RNA may be, for example, any of injections, suppositories, oral preparations, ointments, eye drops, etc., and preferably, injections (intravenous injections, etc.) are adopted. Each of these administration forms can be prepared by a formulation method known and commonly used by those skilled in the art.

[0097] As pharmaceutical carriers, various organic or inorganic carrier substances commonly used as formulation materials are used, and are incorporated as excipients, binders, disintegrants, lubricants, coating agents, etc. in solid preparations, and as solvents, solubilizing agents, suspending agents, isotonicity agents, pH regulators, pH buffers, soothing agents, etc. in liquid preparations. Furthermore, formulation additives such as preservatives, antioxidants, colorants, flavorings, odorants, stabilizers, etc. can also be used as necessary.

[0098] The subjects to which the various agents and pharmaceutical compositions according to the embodiments of the present invention are administered are mammals, preferably humans.

[0099] The daily dose of the RNA varies depending on the patient's symptoms, body weight, age, sex, etc. and cannot be determined in general, but the dose should be preferably 10 ng to 1 mg, more preferably 100 ng to 100 μg, and even more preferably 1 μg to 10 μg of the compound per day for a normal adult (body weight 50 kg).

[0100] The amount of RNA to be incorporated in each of the above-mentioned dosage unit forms is appropriately determined depending on the properties of the RNA used, the symptoms of the patient, the dosage form, etc.

[0101] The disclosures of all patent applications and publications cited herein are hereby incorporated by reference in their entireties.

[0102] The present disclosure will be described more specifically below with reference to examples, but the present disclosure is not limited thereto. EXAMPLES

[0103] Unless otherwise stated, the following methods were followed.

[0104] (1) Reagents The following primary antibodies and factors were used in this study: human anti-ZC3H12D antibody (ab1000862; Abcam), cell sorting against human CD56: PE anti-human CD56 (NCAM) antibody (BioLegend) and isotype control, mouse IgG1, κ (BioLegend). Cell sorting against human CD3: AlexaFluor488 anti-human CD3 antibody (BioLegend) and isotype control, mouse IgG1, κ (BioLegend). Mouse (D-17, sc-9344; SantaCruzBiotechnology) and human (B27; BioLegend) anti-IFNγ antibodies were used for cell staining. Human IL2 (BioLegend) and human IL12 (PeproTech) were used for cell culture.

[0105] (2) Tumor cell lines and tumor cell culture supernatants Human renal cancer cells 786-O were purchased from ATCC. The human cells were cultured in DMEM / Han's F-12 medium.

[0106] (3) Primary culture of human cells Primary human BMCs (HPBMCs; Lonza) were cultured in LGM-3 medium (Lonza) supplemented with 200 IU / mL IL2.

[0107] (4) Migration assay Cell migration was assessed using a chemotaxis Boyden chamber (NeuroProbe). The upper and lower wells were separated by a 5-μm pore-size polyvinylpyrrolidone-free polycarbonate filter (Nucleopore, Costar). Various RNAs were applied to the lower wells. Cell suspensions (2 × 10 5 From 2 × 10 6 An aliquot (50 μL) of 100 μl ...

[0108] (5) IFNγ induction assay After stimulation with 50ng / mL RNA for 48 hours in LGM-3 medium (Lonza) supplemented with 200IU / mL IL2, human CD56 + CD3 - NK cells were stained with anti-human IFNγ antibody. For a positive control of IFNγ induction, cells were incubated with 40 ng / mL hIL-12 for 48 hours.

[0109] (6) Vector construction and establishment of cells stably expressing ZC3H12D Human ZC3H12D expression vectors (hZC58 and hZC36) were purchased from OriGene Technologies Inc. (Rockville, MD). Mouse ZC3H12D was PCR amplified using the primer set 5′-GGTACCATGGAGCATCGGAGCAAGATGG-3′ (SEQ ID NO: 7) and 5′-CTCGAGTTAAGGATCCCCCAACGGAGCACC-3′ (SEQ ID NO: 8) and then cloned into pCRBluntII vector (Thermo). The cloned fragment was double-stranded digested with KpnI-XhoI and subcloned into pcDNA3 with a C-terminal FLAG-tag. To establish a cell line stably expressing human ZC3H12D, one of the constructs mentioned above was transfected into the cell line, and the cells were cultured in the presence of 400 μg / mL G418 for more than 2 weeks. After G418 selection, cell lysates were examined by Western blotting using a probe of DDDDK antibody (MBLCo., Ltd, Japan) to confirm the expression of ZC3H12D-FLAG protein.

[0110] (7) Immunohistochemistry and cell counting Anti-ZC3H12D (ab1000862, Abcam) antibody was used to stain CD56+CD3- NK cells. The values ​​of immunostained cell area are shown as pixel counts normalized to DAPI signal. Labeled cells were detected by confocal or fluorescence microscopy and normalized to total surface area. EXAMPLES

[0111] (Synthesis of chemically modified RNA) Two types of RNA (HumanIL1β_AE, HumanIL1β_Me) having the base sequence (partial sequence of human IL1β (SEQ ID NO: 6)) and chemical modification shown in FIG. 1 were synthesized according to a conventional method. The nucleotide unit having a fluorine atom at the 2' position and an aminoethyl group at the 4' position was synthesized in full length according to the phosphoramidite method in accordance with JP 2019-10035 A and the like. As a control, a 50mer PolyA (PolyA_50mer_Me_AE) (SEQ ID NO: 9) that was chemically modified as shown in FIG. 1 was also synthesized. EXAMPLES

[0112] (Confirmation of cell migration activation effect of chemically modified RNA in ZC3H12D overexpressing cells) The two types of chemically modified RNAs synthesized in Example 1 and the 50mer polA_50mer_Me_AE as a control were administered to a medium containing 786-O cells overexpressing ZC3H12D, and the cell migration ability was measured by the average number of migrated cells. The concentration of RNA was 100 ng / mL. The results are shown in Figure 2.

[0113] As shown in FIG. 2, the two types of synthetic RNA showed a significant improvement in migration activity compared to the control and no addition. EXAMPLES

[0114] (Confirmation of IFN-γ induction by chemically modified RNA in human NK cells) The two types of chemically modified RNA synthesized in Example 1 and 50mer polyA_50mer_Me_AE as a control were used to detect human CD56. + CD3 - The effect of chemically modified RNA on IFNγ production in NK cells was evaluated, and the results are shown in Figures 3 and 4.

[0115] Figure 3 shows the signal intensity obtained from INFγ by DAPI staining. As shown in Figure 3, it was found that both types of chemically modified RNA significantly produced INFγ compared to the case without addition.

[0116] The number of INFγ-positive NK cells per total NK cells is shown in Figure 4. As shown in Figure 4, the two types of chemically modified RNAs showed a high number of INFγ-producing cells.

[0117] From the above, it was found that the RNA of the present disclosure, although partially chemically modified, has migration activation ability and INFγ induction activation ability. EXAMPLES

[0118] (Synthesis of chemically modified RNA) Seven types of RNA (HumanIL1β_OME_AE, MouseIL1β_OMe_AE, MouseIL1β_OMe_AE_F, MouseIL1β_F, HumanIL1β_OME_AP, HumanIL1β_OME_AP_F, HumanIL1β_F) having the base sequence shown in FIG. 5 (partial sequence of human IL1β (SEQ ID NO: 10) and partial sequence of mouse IL1β (SEQ ID NO: 11)) and chemical modification were synthesized according to a conventional method. The nucleotide unit having a fluorine atom at the 2' position and an aminoethyl group at the 4' position was synthesized in full length according to the phosphoramidite method in accordance with JP 2019-10035 A and the like. The nucleotide unit having a methoxy group at the 2' position and an aminopropyl group at the 5' position was synthesized in full length according to the phosphoramidite method in accordance with the pamphlet of International Publication No. WO 2019 / 088179 and the like. As a control, we also synthesized a 50-mer PolyA (PolyA_50_OMe_AE) (SEQ ID NO: 9) that was chemically modified as shown in Figure 5. MouseIL1β_OMe_AE_F, MouseIL1β_F, HumanIL1β_OME_AP_F and HumanIL1β_F are labeled with fluorescein at their 5' ends. EXAMPLES

[0119] (RNase resistance evaluation of chemically modified RNA) Using fluorescein-labeled MouseIL1β_OME_AE_F and MouseIL1ββ_F synthesized in Example 4, the nuclease (RNase) resistance in the serum of tumor-bearing mice was evaluated for synthetic RNA having one nucleotide unit (2'-F-4'-AE) with a fluorine atom at the 2' position and an aminoethyl group at the 4' position at each end, and all other nucleotide units having a methoxy group at the 2' position (2'-OMe). The tumor-bearing mice were 8-12 week-old C57BL / 6 mice, which were intramammary glands were injected with 1×10 6 Tumor-bearing mouse serum was prepared by injecting E0771 cells at a dose of 100 μg / mouse. Tumor-bearing mouse serum was obtained by intracardiac blood collection from mice that had developed mammary tumors measuring approximately 1 cm in size one week after the injection of E0771 cells.

[0120] Using fluorescein-labeled HumanIL1β_OMe_AP_F and HumanIL1β_F synthesized in Example 4, the nuclease (RNase) resistance in bovine serum of synthetic RNA was evaluated, which has one nucleotide unit (2'-OMe-5'-AP) with a methoxy group at the 2' position and an aminopropyl group at the 5' position at each end, and all other nucleotide units are nucleotide units (2'-OMe) with a methoxy group at the 2' position.

[0121] Nuclease resistance was measured by dissolving 300 pmol of each RNA in 37.5 μL of OPTI-MEM, dispensing 1.1 μL of the RNA, and adding 15 μL of loading buffer (10 mM EDTA, 90% formamide) to prepare a 0-minute sample. In addition, 4 μL (10%) of tumor-bearing mouse serum or 4 μL (10% and 20%) or 9.1 μL (20%) of bovine serum was added thereto and incubated at 37°C. After 5 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, and 48 hours, 2 μL of the reaction solution was added to 10 μL of loading solution that had been dispensed in advance into a separate sampling tube to stop the reaction. This reaction solution was electrophoresed on 20% denaturing PAGE (500 V, 20 mA) and then analyzed using an Image analyzer LAS-4000 (Fuji Film Corporation). The results are shown in Figures 6 and 7. FIG. 6 shows the amount of RNA (%) after 5 minutes to 48 hours relative to the amount of RNA at 0 minutes based on the electrophoresis results.

[0122] The denaturing PAGE was prepared as follows: 40% acrylamide (19:1) solution (20 ml), urea (16.8 g), and 10×TBE buffer (4 ml) were added and dissolved, and then denatured in H 2 O was added to make the volume 40 ml. Finally, APS (27.5 mg) was added and dissolved, TEMED (20 μl) was added and shaken, and the mixture was poured between two glass plates fixed with a spacer in between, and left to stand for more than 1 hour to solidify. 1×TBE buffer was used as the electrophoresis buffer.

[0123] As shown in Figure 6, the chemically modified RNA with one (2'-F-4'-AE) at each end and all other nucleotide units being (2'-OMe) maintained 80% of the RNA amount even after 48 hours in serum. In contrast, it was found that the RNA in which all nucleotide units were natural nucleotide units had almost completely disappeared after 5 minutes. From the above, it was found that the RNA with (2'-F-4'-AE) at the end has excellent resistance to RNase in serum.

[0124] Furthermore, as shown in Figure 7, chemically modified RNA with one (2'-OMe-5'-AP) at each end and all other nucleotide units being (2'-OMe) maintained nearly 100% RNA amount even after 48 hours in 20% serum. In contrast, it was found that in RNA in which all nucleotide units were natural nucleotide units, almost all RNA had disappeared after 5 minutes. From the above, it was found that RNA with (2'-OMe-5'-AP) at the end has excellent resistance to RNase in serum. EXAMPLES

[0125] (Confirmation of cell migration activation effect by chemically modified RNA) HumanIL1β_OME_AE synthesized in Example 4 and PolyA_50_OMe_AE as a control were administered through a filter to a cell suspension containing 786-O cells overexpressing ZC3H12D protein, and the number of migrated cells was counted. A Boyden chamber (Neuro Probe) was used as the apparatus. The upper and lower wells were separated by a polycarbonate filter (Nucleopore; Costar) with a pore size of 5 μm. The upper well contained the cell suspension (1 × 10 6 The medium containing the above RNA at concentrations of 1 ng / ml, 10 ng / ml, 100 ng / ml, and 1 μg / ml was placed in the upper well, and incubated for 3 hours, after which the number of cells that had migrated to the lower well was counted. The results are shown in Table 1.

[0126] [Table 1]

[0127] As shown in Table 1, when HumanIL1β_OME_AE was administered, the number of migratory cells was 150 on average at each RNA concentration, with the number without addition being set at 100. The number also increased significantly depending on the RNA concentration, reaching approximately 200 at 100 ng / ml. On the other hand, when PolyA_50_OMe_AE was administered, the number was 110 on average, the same as without addition, regardless of the concentration. From the above, it was found that HumanIL1β_OME_AE, which has one (2'-F-4'-AE) at each end, contributes to increasing the migration ability of NK cells expressing the ZC3H12D protein. EXAMPLES

[0128] (Confirmation of IFNγ induction) Human IL1β_OME_AE synthesized in Example 4 and PolyA_50_OMe_AE as a control were used to synthesize human CD56 + CD3 - The effect of chemically modified RNA on IFNγ production in NK cells was evaluated. + CD3 - NK cells were collected from cryopreserved human PBMCs by FACS. 24 hours after priming with 10 ng / mL of each RNA in HPLM medium (Gibco) in the presence of 200 U / mL IL-2 (BioLegend), the cells were stained with anti-human IFN-γ antibody (BioLegend). IL-12 (Proteintech) was used as a positive control for IFN-γ induction. The results are shown in Table 2.

[0129] [Table 2]

[0130] As shown in Table 2, when HumanIL1β_OME_AE was used, IFNγ was induced at a level 2.3 times higher than the amount of IFNγ when no addition was made. This was comparable to the positive control. On the other hand, when PolyA_50_OMe_AE was used, IFNγ was only induced at a level comparable to that when no addition was made. From the above, it was found that HumanIL1β_OME_AE, which has one (2'-F-4'-AE) at each end, contributes to increasing the amount of IFNγ produced by human NK cells. EXAMPLES

[0131] (Evaluation of the cytotoxicity of human NK cells) Human IL1β_OME_AE synthesized in Example 4 and PolyA_50_OMe_AE as a control were subjected to human CD56 immunofluorescence using Zombie Green Fixable Viability Kit (BioLegend). + CD3 - The cytotoxicity of NK cells was evaluated. + CD3 - NK cells and cancer cells (Caco-2: colon cancer cell line) were co-cultured at a ratio of 1:1, and 24 hours later, the percentage of tumor cells stained dark green was observed. The results are shown in Table 3.

[0132] [Table 3]

[0133] As shown in Table 3, when HumanIL1β_OME_AE was used, 2.5 times more cell killing was observed than when no polyA_50_OMe_AE was added. On the other hand, when PolyA_50_OMe_AE was used, the same level of cell killing was observed as when no polyA_50_OMe_AE was added. From the above, it was found that HumanIL1β_OME_AE, which has one (2'-F-4'-AE) at each end, contributes to increasing the cell killing ability of human NK cells. EXAMPLES

[0134] (Evaluation of anti-metastatic activity in a mouse model of simulated lung metastasis) The anti-metastatic activity of HumanIL1β_OME_AE synthesized in Example 4 and PolyA_50_OMe_AE as a control was evaluated. Each RNA (HumanIL1β_OME_AE and PolyA_50_OMe_AE, 1 μg each) and PBS as a negative control were injected into the tail vein of mice induced to bear E0771 breast cancer cells every few days. Tumor cells (E0771 breast cancer cells) stained with PKH-26 (Sigma-Aldrich) were injected into the tail vein 48 hours after which the mice were dissected and the number of PKH-26 positive cells in the lungs was observed. The results are shown in Table 4.

[0135] [Table 4]

[0136] As shown in Table 4, when HumanIL1β_OME_AE was used, 65% of the number of positive cells was observed compared to when PBS was added. On the other hand, when PolyA_50_OMe_AE was used, the number of positive cells observed was about the same as when PBS was added. From the above, it was found that HumanIL1β_OME_AE, which has one (2'-F-4'-AE) at each end, contributes to the inhibition of tumor cell metastasis even in vivo. [Sequence List Free Text]

[0137] SEQ ID NOs: 7 and 8: primers SEQ ID NO: 9: Control RNA

Claims

1. An antitumor agent comprising a base sequence including the base sequence represented by SEQ ID NO: 6 or 10 among the base sequence represented by SEQ ID NO: 2, a total length of 130 bases or less, and a base sequence other than SEQ ID NO: 6 or 10 that has 90% or more identity to the base sequence represented by SEQ ID NO: 2, and comprising RNA as an active ingredient having at least one first nucleotide unit represented by the following formula (1) in its 3'-terminal region within 20 bases from the 3'-terminus and / or in its 5'-terminal region within 25 bases from the 5'-terminus. 【Chemistry 12】 (In formula (1), R １ represents a hydroxyl group or a halogen atom in which a hydrogen atom has been substituted with a methyl group, R ２ represents an aminoethyl group, n represents 0 or 1, X １ represents an oxygen atom, X ２ represents OH or O − , and B represents a purine base or a pyrimidine base.

2. The antitumor agent of claim 1, wherein the RNA has a total length of 70 bases or less, the 3'-terminal region is within 15 bases from the 3'-terminal, and the 5'-terminal region is within 15 bases from the 5'-terminal and comprises the first nucleotide unit.

3. The antitumor agent of claim 1, wherein the RNA has a total length of 60 bases or less, the 3'-terminal region is within 10 bases from the 3'-terminal, and the 5'-terminal region is within 10 bases from the 5'-terminal and comprises the first nucleotide unit.

4. The antitumor agent according to claim 2 or 3, wherein the RNA has at least one first nucleotide unit within 2 bases from the 3' end of the 3' terminal region and at least one first nucleotide unit within 2 bases from the 5' end of the 5' terminal region.

5. The first nucleotide unit is represented by the formula (1), １ The antitumor agent according to claim 2 or 3, wherein is a nucleotide unit which is a fluorine atom.

6. The antitumor agent according to claim 1, wherein the RNA comprises a base sequence represented by SEQ ID NO: 6 or 10 among the base sequences represented by SEQ ID NO: 4, and is composed of a base sequence other than SEQ ID NO: 6 or 10 that has 90% or more identity with the base sequence represented by SEQ ID NO:

4.

7. The antitumor agent according to claim 1 , wherein the base of the first nucleotide unit is cytosine or uracil.

8. The antitumor agent of claim 1, wherein 80% or more of the total number of uridine nucleosides in the RNA comprises a uridine analog selected from the group consisting of pseudouridine, 1-methylpseudouridine, 1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine.

9. An immunostimulant comprising a base sequence including the base sequence represented by SEQ ID NO: 6 or 10 among the base sequence represented by SEQ ID NO: 2, a total length of 130 bases or less, and a base sequence other than SEQ ID NO: 6 or 10 that has 90% or more identity to the base sequence represented by SEQ ID NO: 2, and comprising RNA as an active ingredient having at least one first nucleotide unit represented by the following formula (1) in its 3'-terminal region within 20 bases from the 3'-terminus and / or in its 5'-terminal region within 25 bases from the 5'-terminus. 【Chemistry 13】 (In formula (1), R １ represents a hydroxyl group or a halogen atom in which a hydrogen atom has been substituted with a methyl group, R ２ represents an aminoethyl group, n represents 0 or 1, X １ represents an oxygen atom, X ２ represents OH or O − , and B represents a purine base or a pyrimidine base.

10. An agent for suppressing tumor metastasis, comprising a base sequence including the base sequence represented by SEQ ID NO: 6 or 10, out of the base sequence represented by SEQ ID NO: 2, and having a total length of 130 bases or less, and a base sequence other than SEQ ID NO: 6 or 10 that has 90% or more identity to the base sequence represented by SEQ ID NO: 2, and comprising as an active ingredient RNA having at least one first nucleotide unit represented by the following formula (1) in its 3'-terminal region within 20 bases from the 3'-terminus and / or in its 5'-terminal region within 25 bases from the 5'-terminus. 【Chemistry 14】 (In formula (1), R １ represents a hydroxyl group or a halogen atom in which a hydrogen atom has been substituted with a methyl group, R ２ represents an aminoethyl group, n represents 0 or 1, X １ represents an oxygen atom, X ２ represents OH or O − , and B represents a purine base or a pyrimidine base.

11. An agent for enhancing the immunostimulating effect of an immunostimulant or the antitumor effect of an antitumor agent, comprising a base sequence that includes the base sequence represented by SEQ ID NO: 6 or 10 out of the base sequence represented by SEQ ID NO: 2, and has a total length of 130 bases or less, and in base sequences other than SEQ ID NO: 6 or 10, has 90% or more identity to the base sequence represented by SEQ ID NO: 2, and contains as an active ingredient RNA having at least one first nucleotide unit represented by the following formula (1) in its 3'-terminal region within 20 bases from the 3'-terminus and / or its 5'-terminal region within 25 bases from the 5'-terminus. 【Chemistry 15】 (In formula (1), R １ represents a hydroxyl group or a halogen atom in which a hydrogen atom has been substituted with a methyl group, R ２ represents an aminoethyl group, n represents 0 or 1, X １ represents an oxygen atom, X ２ represents OH or O − , and B represents a purine base or a pyrimidine base.

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