RNA-containing antitumor agent and use thereof

Abstract

An anti-tumor agent which has a nucleotide sequence selected from the group consisting of the following (1) and (2), and which contains, as an active ingredient, an RNA having at least one nucleotide unit represented by formula (1) in a 3 '-terminal region within 20 bases from the 3'-terminal thereof and / or a 5 '-terminal region within 25 bases from the 5'-terminal thereof. (1) A nucleotide sequence represented by SEQ ID NO: 2 or a nucleotide sequence having 90% or more identity with the sequence (2) is a contiguous nucleotide sequence of 20 to 200 nucleotides in the nucleotide sequence (1): 20 # imgabs0 # (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 R3 represents a hydrogen atom or a halogen atom having a hydrogen atom substituted with an alkyl group or an alkenyl group; in the formula, the linking group is a divalent hydrocarbon group having one or more carbon atoms, 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, and the contents of the Japanese patent application are incorporated herein as part of this specification. The present specification relates to uses such as anti-tumor agents for inhibiting the metastasis of tumors containing RNA and methods for activating immune cells. Background Art

[0002] In recent years, potential anti-metastatic immune cells have been discovered. Specifically, B220 + CD11c + NK1.1 + Natural killer (NK) cells migrate from the liver to the lungs of tumor-bearing mice (Non-Patent Document 1). These NK cells accumulate in fibrinogen+ high permeability sites, remove concentrated fibrinogen, and have the ability to destroy tumors by producing interferon gamma (IFNγ) (Non-Patent Document 2).

[0003] B220 of the liver + CD11c + NK1.1 + NK cells and B220 in the lung + 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 to be a tumor suppressor gene in lymphoma and lung cancer patients (Non-patent Documents 3 and 4). Prior art literature Non-patent literature

[0004] Non-patent literature 1: EMBO Mol Med 10, doi: 10.15252 / emmm.201708643 (2018) Non-patent document 2: Nat Commun 4, 1853, doi: 10.1038 / ncomms2856 (2013) Non-patent document 3: Cancer Res 67, 93-99, doi: 10.1158 / 0008-5472.CAN-06-2723 (2007) Non-patent document 4: Br J Haematol 139, 161-163, doi: 10.1111 / j.1365-2141.2007.06752.x (2007) Summary of the invention

[0005] According to the present inventors, as of the current studies, it is presumed that the ZC3H12D protein interacts with extracellular RNA (exRNA) in the anti-metastatic function to activate NK cells.

[0006] The present specification provides an anti-tumor agent having RNA as an effective ingredient that can effectively interact with ZC3H12D protein and activate NK cells, and its use.

[0007] [1] An antitumor agent having a base sequence selected from the group consisting of the following (1) and (2), and containing, as an active ingredient, an 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 the 3' region within 20 bases from the 3' end and / or the 5' region within 25 bases from the 5' end. (1) The base sequence shown in SEQ ID NO: 2 or a base sequence having 90% or more identity with the sequence (2) A continuous base sequence of 20 or more but less than 200 bases in the base sequence of (1) Chemical formula 1 (In formula (1), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is replaced by an alkyl group or an alkenyl group, or a halogen atom, R 2 Denotes NHR with a linker 3 , the connecting group is a divalent hydrocarbon group having one or more carbon atoms, 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 Indicates OH (or O - ) or SH(or S - ), B represents a purine base or a pyrimidine base. ) Chemical formula 2 (In formula (2), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is replaced by an alkyl group or an alkenyl group, or a halogen atom, R 4 Denotes NHR with a linker 5 , the connecting group is a divalent hydrocarbon group having one or more carbon atoms, 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 Indicates OH (or O - ) or SH(or S - ), B represents a purine base or a pyrimidine base. ) [2] The antitumor agent according to [1], wherein the RNA has 1 to 20 nucleotide units in each of the 3' terminal region and / or the 5' terminal region. [3] The antitumor agent according to [1] or [2], wherein the first nucleotide unit is 1 is a methoxy group or a fluorine atom, R 2 It is an aminoethyl nucleotide unit. [4] The antitumor agent according to [1] or [2], wherein in the first nucleotide unit, n is 1, R 1 is a fluorine atom, R 2 is an aminoethyl nucleotide unit, There is at least one of the first nucleotide units within 2 bases from the 3' end of the 3' end region, and there is at least one of the first nucleotide units within 2 bases from the 5' end of the 5' end region. [5] The antitumor agent according to [1] or [2], wherein in the second nucleotide unit, n is 1, R 1 is methoxy, R 4 is an aminopropyl nucleotide unit, There is at least one second nucleotide unit within 2 bases from the 3' end of the 3' end region, and there is at least one second nucleotide unit within 2 bases from the 5' end of the 5' end region. [6] The antitumor agent according to [4] or [5], wherein the base sequence has 90% or more identity with a continuous base sequence of 30 to 70 bases in the base sequence shown in SEQ ID NO: 4. [7] The antitumor agent according to [4] or [5], wherein the base sequence has 90% or more identity with a continuous base sequence of 40 to 60 bases in the base sequence shown in SEQ ID NO: 4. [8] The antitumor agent according to [6] or [7], wherein the base sequence is a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 6 or 10. [9] The antitumor agent according to [8], wherein the base of the first or second nucleotide unit is cytosine or uracil.

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

[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-thiol-1-methyl-1-deazapseudouridine, 2-thiol-1-methylpseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxypseudouridine, 4-thiol-1-methylpseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine.

[12] An antitumor agent according to any one of [1] to

[11] , wherein the RNA is a variant of the RNA having the base sequence shown in SEQ ID NO: 1, and compared with the natural coding sequence, the coding sequence from positions 88 to 897 of the base sequence shown in SEQ ID NO: 1 has bases substituted by 1 to 5 additional stop codons.

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

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

[14] An immunomodulator having any base sequence selected from the group consisting of the following (1) and (2), and containing 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) as an active ingredient in the 3' end region within 20 bases from the 3' end and / or the 5' end region within 25 bases from the 5' end. (1) The base sequence shown in SEQ ID NO: 2 or a base sequence having 90% or more identity with the sequence (2) A continuous base sequence of 20 or more and 200 bases in the base sequence of (1) Chemical formula 3 (In formula (1), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is replaced by an alkyl group or an alkenyl group, or a halogen atom, R 2 Represents NHR with a linker 3 , the connecting group is a divalent hydrocarbon group having one or more carbon atoms, 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 Indicates OH (or O - ) or SH(or S -), B represents a purine base or a pyrimidine base. ) Chemical formula 4 (In formula (2), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is replaced by an alkyl group or an alkenyl group, or a halogen atom, R 4 Represents NHR with a linker 5 , the connecting group is a divalent hydrocarbon group having one or more carbon atoms, 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 Indicates OH (or O - ) or SH(or S - ), B represents a purine base or a pyrimidine base. )

[15] A tumor metastasis inhibitor having any base sequence selected from the group consisting of the following (1) and (2), and containing an 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) as an active ingredient in the 3' end region within 20 bases from the 3' end and / or the 5' end region within 25 bases from the 5' end. (1) The base sequence shown in SEQ ID NO: 2 or a base sequence having 90% or more identity with the sequence (2) A continuous base sequence of 20 or more and 200 bases in the base sequence of (1) Chemical formula 5 (In formula (1), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is replaced by an alkyl group or an alkenyl group, or a halogen atom, R 2 Represents NHR with a linker 3 , the connecting group is a divalent hydrocarbon group having one or more carbon atoms, 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 Indicates OH (or O - ) or SH(or S - ), B represents a purine base or a pyrimidine base. ) Chemical formula 6 (In formula (2), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is replaced by an alkyl group or an alkenyl group, or a halogen atom, R 4 Represents NHR with a linker 5, the connecting group is a divalent hydrocarbon group having one or more carbon atoms, 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 Indicates OH (or O - ) or SH(or S - ), B represents a purine base or a pyrimidine base. )

[16] An enhancer of the immunomodulatory effect of an immunomodulator or the antitumor effect of an antitumor agent, comprising a base sequence selected from the group consisting of the following (1) and (2), and containing an 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) as an active ingredient in the 3' end region within 20 bases from the 3' end and / or the 5' end region within 25 bases from the 5' end. (1) The base sequence shown in SEQ ID NO: 2 or a base sequence having 90% or more identity with the sequence (2) A continuous base sequence of 20 or more and 200 bases in the base sequence of (1) Chemical formula 7 (In formula (1), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is replaced by an alkyl group or an alkenyl group, or a halogen atom, R 2 Represents NHR with a linker 3 , the connecting group is a divalent hydrocarbon group having one or more carbon atoms, 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 Indicates OH (or O - ) or SH(or S - ), B represents a purine base or a pyrimidine base. ) Chemical formula 8 (In formula (2), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is replaced by an alkyl group or an alkenyl group, or a halogen atom, R 4 Represents NHR with a linker 5 , the connecting group is a divalent hydrocarbon group having one or more carbon atoms, 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 Indicates OH (or O - ) or SH(or S - ), B represents a purine base or a pyrimidine base. ) Effects of the Invention

[0008] According to the anti-tumor agent disclosed in this specification, since the RNA has an RNA containing a nucleotide unit with a specific chemical modification as an effective ingredient in the 3' end region and / or the 5' end region of the RNA, it has excellent stability in vivo. In addition, the RNA disclosed in this specification can effectively interact with immune cells and activate immune cells. Thus, tumors can be effectively inhibited. According to this RNA, through the same action, immune cells can be effectively activated, the metastasis of tumor cells can be effectively inhibited, and immune cells can be effectively activated, for example, the anti-tumor effect of the anti-tumor agent used in combination can be enhanced. The RNA disclosed in this specification can be used to prevent and / or treat tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a diagram showing the outline of the synthetic RNA used in Examples. Figure 2 Graph showing the number of migrated cells when synthetic RNA was administered to 786-O cells overexpressing ZC3H12D. Figure 3 To express chemically modified RNA to human CD56 + CD3 - A diagram showing the results of evaluating the effect of IFNγ production in NK cells. Figure 4 To express chemically modified RNA to human CD56 + CD3 - A diagram showing the results of evaluating the effect of IFNγ production in NK cells. Figure 5 This is a diagram showing the outline of the synthetic RNA used in Examples. Figure 6 The graph shows the evaluation results of RNase resistance of chemically modified RNA in the serum of tumor-bearing mice. Figure 7 This is a graph showing the evaluation results of RNase resistance of chemically modified RNA in bovine serum. DETAILED DESCRIPTION

[0010] The present disclosure relates to synthetic RNA related to exILβ-mRNA and its use, wherein the exILβ-mRNA is a sequence near the untranslated region on the side of the 3' terminal region of IL-1β RNA. The inventors have found that even if a specific nucleotide unit is introduced as a chemical modification at the 3' end and 5' end of the synthetic RNA corresponding to exILβ-mRNA, the NK cell activation effect to the same extent as exIL1β-mRNA can be exerted. This synthetic RNA is believed to act as an intrinsic intercellular neurotransmitter substance with anti-tumor function or anti-metastasis function in vivo.

[0011] In the present specification, "chemical modification" of RNA may be any chemical modification performed on RNA, for example, it may include modifications in the ribose of nucleotides, phosphodiester binding parts and bases. With respect to bases, it includes not only substitution with a base sequence different from the natural base sequence, but also chemical modification of such bases even if the bases in the natural base sequence are of the same type as the bases.

[0012] Hereinafter, representative and non-limiting specific examples of the present invention will be described in detail with appropriate reference to the accompanying drawings. These detailed descriptions are only intended to provide those skilled in the art with details of preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Furthermore, the additional features and inventions disclosed below can be used alone or in combination with other features and inventions to provide further improved anti-tumor agents and uses thereof.

[0013] Furthermore, the combination of features and steps disclosed in the following detailed description is not necessary for implementing the invention in the broadest sense, and in particular, is only described to describe representative embodiments of the invention. In addition, the various features of the representative embodiments described above and below, as well as the various features described in the independent claims and dependent claims, do not have to be combined in the exact embodiments shown herein or in the order listed to provide additional and useful embodiments of the invention.

[0014] Independent of the composition of the features described in the embodiments and / or claims, all features described in this specification and / or the scope of protection of the invention are intended to be disclosed individually and independently of each other as limitations to the original disclosure and the specific subject matter claimed. In addition, all numerical ranges and descriptions of groups or groups are intended to disclose the intermediate compositions thereof as limitations to the original disclosure and the specific subject matter claimed.

[0015] In addition, the term "lower" in the substituent in the compound described in the present specification means that the number of carbon atoms constituting the substituent is at most 10. For example, it usually has 1 to 6 carbon atoms, or 1 to 5 carbon atoms, and, for example, 1 to 4 carbon atoms, and, for example, 1 to 3 carbon atoms.

[0016] Various embodiments disclosed in this specification are described in detail below. First, RNA used in this specification is described, and then embodiments using RNA are described.

[0017] (RNA) The RNA disclosed herein has any of the base sequences (1) and (2) below, and may have at least one first nucleotide unit represented by formula (1) or at least one second nucleotide unit represented by formula (2) below in the 3' region within 20 bases from the 3' end and / or in the 5' region within 25 bases from the 5' end.

[0018] The RNA of the present disclosure has the following sequence. (1) The base sequence shown in SEQ ID NO: 2 or a base sequence having 90% or more identity with the sequence (2) A continuous base sequence of 20 or more but less than 200 bases in the base sequence of (1)

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

[0020] The RNA disclosed herein may have the full length of the base sequence shown in SEQ ID NO: 2, and may also have a base sequence having an identity of 90% or more, such as 95% or more, such as 96% or more, such as 97% or more, such as 98% or more, such as 99% or more to the base sequence. In addition, for example, the identity of the base sequence can be determined by aligning two or more base sequences on the BLAST (BLAST: Basic Local Alignment Search Tool (nih.gov)) page of NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cg) using blastn under highly similar sequence (megablast) optimization conditions.

[0021] From the perspective of the present disclosure, a base sequence having 90% or more identity with the base sequence represented by 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 disclosed herein may be a portion of the base sequence shown in SEQ ID NO: 2 or a base sequence having 90% or more identity with the 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 portion of the base sequence shown in SEQ ID NO: 2, etc. may be, for example, a continuous sequence having a length of 20 bases or more and less and 200 bases or less in the base sequence shown in SEQ ID NO: 2, etc. The base length is, for example, more than 25 bases, for example, more than 30 bases, for example, more than 40 bases, for example, more than 45 bases, and the base length is less than 180 bases, for example, less than 160 bases, for example, less than 150 bases, for example, less than 140 bases, for example, less than 130 bases, for example, less than 120 bases, for example, less than 110 bases, for example, less than 100 bases, for example, less than 90 bases, for example, less than 80 bases, for example, less than 75 bases, for example, less than 70 bases, for example, less than 60 bases. The RNA disclosed in the present invention can be set in range by appropriately combining such lower limits and upper limits. For example, the length can be more than 20 bases and less than 100 bases, more than 30 bases and less than 100 bases, more than 30 bases and less than 80 bases, more than 40 bases and less than 60 bases, etc.

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

[0024] Such base sequences include, for example, base sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and 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, and for example, 99% or more identity with these sequences. In addition, from the perspective of the present invention, a part of the base sequence shown in SEQ ID NO: 2 has the ability to activate NK cells. The mRNA composed of the base sequence shown in SEQ ID NO: 5 is one of the fragments when human IL1β-mRNA is divided into 4 fragments containing 150 nucleotides. In addition, the base sequence shown in 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, and for example, 99% or more identity with the base sequence thereof is also included.

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

[0026] Chemical formula 9

[0027] Alkyl groups include saturated hydrocarbon groups that are linear, branched, cyclic, or a combination thereof. Generally, lower alkyl groups are preferred, for example, more preferred examples include lower alkyl groups with 1 to 6 carbon atoms, or lower alkyl groups with 1 to 5 carbon atoms, and further, particularly preferred examples include lower alkyl groups with 1 to 4 carbon atoms or 1 to 3 carbon atoms. Suitable examples of linear alkyl groups with 1 to 4 carbon atoms include methyl, ethyl, n-propyl, or n-butyl, among which methyl, ethyl, n-propyl are preferred, and, for example, methyl and ethyl are preferred, and, for example, methyl is preferred. Furthermore, branched alkyl groups with 1 to 4 carbon atoms include isopropyl, isobutyl, sec-butyl, tert-butyl, etc., among which isopropyl is particularly preferred. Furthermore, cyclic alkyl groups with 1 to 4 carbon atoms include cyclopropyl, cyclobutyl, or cyclopropylmethyl, etc.

[0028] Alkenyl includes straight chain, branched chain, cyclic, or a combination thereof. Usually, lower alkenyl is preferred, and lower alkenyl includes, for example, vinyl, 1-propenyl, 2-propenyl, 1-methyl-2-propenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, etc.

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

[0030] R 2 Can represent NHR with a linker 3 That is, NHR 3 It is bonded to the 4'-carbon atom via a linking group bonded to the nitrogen atom.

[0031] The linking group may be, for example, a divalent hydrocarbon group having 1 or more carbon atoms, that is, an alkylene group having 1 to 8 carbon atoms, an alkenylene group having 2 to 8 carbon atoms, and the like.

[0032] The alkylene group as a connecting group may be straight chain or branched chain, but is preferably straight chain. For example, a lower alkylene group is preferred, such as a lower alkylene group with 1 to 6 carbon atoms, and a lower alkylene group with 2 to 6 carbon atoms. For example, a lower alkylene group with 2 to 4 carbon atoms or 2 to 3 carbon atoms is preferred. Alkylene groups with 1 to 4 carbon atoms include methylene, ethylene, propane-1, 3-diyl, n-butane-1, 1-diyl, n-pentyl-1-5, -diyl, n-hexyl-1, 6-diyl, etc. And, for example, butane-1, 2-diyl, etc. are included. For example, particularly preferred examples include ethylene, propane-1, 3-diyl, and n-butane-1, 1-diyl.

[0033] The alkenylene group as the linking group is straight chain or branched chain, preferably straight chain, for example, preferably lower alkenylene, such as ethylene-1,2-diyl, propylene-1,3-diyl, butene-1,4-diyl, etc.

[0034] From the viewpoint of the NK cell activating ability of the RNA disclosed herein, the nucleotide unit represented by formula (1) preferably includes a divalent hydrocarbon group such as an alkylene group having 1 to 3 carbon atoms, such as a methylene group, an ethylene group, or a propylene group.

[0035] R 3 Including hydrogen atoms, alkyl groups or alkenyl groups. In addition to the alkyl groups already described, the alkyl groups preferably include lower alkyl groups, for example, alkyl groups having 1 to 3 carbon atoms, alkyl groups having 1 or 2 carbon atoms, and methyl groups. In addition to the alkenyl groups already described, the alkenyl groups preferably include lower alkenyl groups. 3 When it is a hydrogen atom or the like, the number of carbon atoms of the linking group is preferably 2 or more, for example 3 or more, for example 4 or more, for example 6 or less, further for example 5 or less, further for example 4 or less alkylene.

[0036] And, R 3 When it is a hydrogen atom, R 2 is NH2 (amino) with a connecting group, that is, when the connecting group is an alkylene group, it becomes an aminoalkyl group. 2Aminoalkyl groups such as aminoethyl and aminopropyl are considered 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 becomes O-, for example, it can be OH. When n is 1, X 1 represents an oxygen atom or a sulfur atom, X 2 Indicates OH (or O - ) or SH(or S - ). 1 or X 2 When n is a sulfur atom or SH (S-), the nucleotide unit will have the next nucleotide unit at the 3' side through a phosphorothioate bond. When n is 1, the nucleotide unit may have a phosphate group or a phosphorothioate group at the 3' position. In addition, when it is a phosphate group, etc., it can also form a salt with an appropriate base.

[0038] B represents a purine base or a pyrimidine base. The purine base is not particularly limited, for example, in addition to purine-9-base, various substituted purine-9-bases such as 2,6-dimethoxypurine-9-base and 2,6-dichloropurine-9-base are included. In addition, pyrimidine bases include 2-oxo-pyrimidine-1-base and 2-oxo-pyrimidine-1-base. In addition, 2-oxo-4-methoxy-pyrimidine-1-base, 4-(1H-1,2,4-triazol-1-base)-pyrimidine-1-base and other substituted 2-oxo-pyrimidine-1-base are included.

[0039] In addition, B may be a modified base described below.

[0040] (Second nucleotide unit: nucleotide unit represented by formula (2)) More specifically, the second nucleotide unit shown in the following formula (2) is a nucleotide unit represented by any one or both of the following formulas (2a) and 2 (b). The nucleotide unit represented by formula (2a) involves an asymmetric carbon atom at the 5' position and is an R body, and the nucleotide unit represented by formula (2b) involves an asymmetric carbon atom at the 5' position and is an S body. In addition, in the RNA disclosed in this specification, these nucleotide units can be used in combination with R bodies and S bodies, or only any one of them can be used. For example, from the viewpoint of nuclease resistance, stability in serum, and thermal stability of RNA / RNA duplexes, the RNA disclosed in this specification preferably uses only S bodies as the nucleotide unit shown in formula (2). In addition, in the present specification, in particular, as long as the S body and R body are not clearly stated, the nucleotide unit shown in formula (2) and the second nucleotide unit represent S bodies and / or R bodies.

[0041] Chemical formula 10

[0042] Chemical formula 11

[0043] In the second nucleotide unit, R 1 has the same meaning as in formula (1). R 1 Preferably, it represents a hydroxyl group or a halogen atom in which a hydrogen atom is replaced by an alkyl group or an alkenyl group. 1 The hydroxyl group in which the hydrogen atom is replaced by an alkyl group having 1 to 3 carbon atoms is preferred. For example, a methyl group, an ethyl group, and an n-propyl group are preferred. For example, a methyl group and an ethyl group are preferred, and a methyl group is preferred. The halogen atom is not particularly limited, and for example, a fluorine atom is preferred.

[0044] In the second nucleotide unit, R 4 Can represent NHR with a linker 5 That is, NHR 5 It is bonded to the carbon atom at the 5' position via a linking group bonded to the nitrogen atom.

[0045] In the second nucleotide unit, the linker and R 5 With R in formula (1) 2 The connecting group and R 3 have the same meaning. The linking group may preferably be, for example, a lower alkylene group, such as a lower alkylene group having 1 to 6 carbon atoms, such as a lower alkylene group having 2 to 6 carbon atoms, such as a lower alkylene group having 2 to 4 carbon atoms or 2 to 3 carbon atoms. For example, methylene, ethylene, propane-1,3-diyl, n-butane-1,1-diyl, n-pentyl-1-5,-diyl, n-hexyl-1,6-diyl, etc. are included. And, for example, butane-1,2-diyl, etc. are included. Particularly preferred are, for example, ethylene, propane-1,3-diyl, n-butane-1,1-diyl, and propane-1-,3-diyl may be preferred.

[0046] The alkenylene group as the linking group is straight-chain or branched, preferably straight-chain. For example, it may be preferably a lower alkenylene group, such as ethylene-1,2-diyl, propylene-1,3-diyl, butene-1,4-diyl, etc.

[0047] In the second nucleotide unit, from the viewpoint of the NK cell activating ability of the RNA disclosed herein, a divalent hydrocarbon group such as an alkylene group having 1 to 3 carbon atoms, such as methylene, ethylene, and propane-1,3-diyl, is preferred.

[0048] In the second nucleotide unit, R 5 Preferably, it is 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. 5When it is a hydrogen atom or the like, the connecting group may preferably be an alkylene group having 2 or more carbon atoms, further such as 3 or more, further such as 4 or more, further such as 6 or less, further such as 5 or less, further such as 4 or less, and may preferably be ethylene, propane-1-, or 3-diyl.

[0049] And, when R 5 When it is a hydrogen atom, R 4 becomes NH2 (amino) with a connecting group, that is, when the connecting group is an alkylene group, it becomes an aminoalkyl group. 4 Since it 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 2 has the same meaning as in the first nucleotide unit and can take the same form. In the second nucleotide unit, n represents 0 or 1. The form of the second nucleotide unit when N is 0 or 1 is the same as that in the first nucleotide unit. Furthermore, B has the same meaning as B in the first nucleotide unit and can take the same form.

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

[0052] Furthermore, the RNA of the present disclosure may have a combination of the first nucleotide unit and the second nucleotide unit in the 5' terminal region. Furthermore, the RNA of the present disclosure may have a combination of the first nucleotide unit and the second nucleotide unit in the 3' terminal region. In addition, the RNA of the present disclosure may 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 first nucleotide unit and the second nucleotide unit may each be present in the 5' terminal region and the 3' terminal region, or vice versa.

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

[0054] The RNA disclosed in the present invention has at least one nucleotide unit shown in formula (1) and / or a nucleotide unit shown in formula (2) in the 5' end region. The number of nucleotide units represented by the nucleotide units shown in formula (1) and formula (2) in the region is not particularly limited, for example, more than 1 and less than 6, more than 1 and less than 5, more than 1 and less than 4, more than 1 and less than 3, 1 or 2. For example, the nucleotide unit shown in formula (1) and the nucleotide unit shown in formula (2) are arranged in a region within 6 bases from the 5' end. It is believed that by doing so, the 5' end side can be effectively protected. The nucleotide unit shown in formula (1) and the nucleotide unit shown in formula (2) can be effectively arranged, for example, at least at the position of the base at the 5' end or 2 bases from the 5' end.

[0055] The RNA disclosed in the present invention has at least one nucleotide unit represented by formula (1) and / or a nucleotide unit represented by formula (2) in the 3' end region. The number of nucleotide units represented by formula (1) and nucleotide units represented by formula (2) in the region is not particularly limited, for example, more than 1 and less than 6, more than 1 and less than 5, more than 1 and less than 4, more than 1 and less than 3, 1 or 2. The nucleotide unit represented by formula (1) and the nucleotide unit represented by formula (2) are arranged in a region within 6 bases from the 3' end, for example. It is believed that by doing so, the 3' end side can be effectively protected. The nucleotide unit represented by formula (1) and the nucleotide unit represented by formula (2) can be effectively arranged, for example, at least at the position of the base at the 3' end or 2 bases away from the 5' end.

[0056] The RNA disclosed herein has at least one nucleotide unit shown in formula (1) and / or a nucleotide unit shown in formula (2) in both the 3' end region and the 5' end region. Since such RNA has these nucleotide units in these terminal regions, it has excellent nuclease resistance, and as a result, it exerts excellent anti-tumor and metastasis inhibition effects. The nucleotide units shown in formula (1) and the nucleotide units shown in formula (2) arranged in each of these terminal regions are, for example, more than 1 and less than 5, for example, more than 1 and less than 4, for example, more than 1 and less than 3, for example, more than 1 and less than 2. In each of these terminal regions, it is preferred to have 1 or 2 nucleotide units shown in formula (1) and / or nucleotide units shown in formula (2) within 3 bases or within 2 bases from each end. In addition, at least one or all of the nucleotide units represented by formula (1) provided 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) provided 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, the nucleotide unit represented by formula (2) having an aminopropyl group at the 5' position is present in one or two regions from the 5' end of the 5' end region of the RNA, and the nucleotide unit represented by formula (1) having an aminopropyl group at the 4' position is present in one or two regions from the 3' end of the 3' end region of the RNA. This is because, when it is in this form, the aminoalkyl group is closer to the 5' end and the 3' end exposed to the nuclease, respectively. In addition, the types of bases in these nucleotide units are not particularly limited, and for example, bases having a pyrimidine ring may be preferred, for example, cytosine or uracil may be preferred.

[0057] According to the above, for example, the following approaches may be suitable for the RNA of the present disclosure. [1] The nucleotide unit represented by formula (1) is that n is 1, R 1 is a fluorine atom, R 2 is an aminoethyl nucleotide unit, The nucleotide unit represented by formula (2) is R 1 is methoxy, R 4 is an aminopropyl nucleotide unit, There is at least one nucleotide unit represented by formula (1) and / or a nucleotide unit represented by formula (2) within 2 bases from the 3' end of the 3' end region, and there is at least one nucleotide unit represented by formula (1) and / or a nucleotide unit represented by formula (2) within 2 bases from the 5' end of the 5' end region. [2] In [1], the base sequence has 90% or more identity with a contiguous base sequence of 30 to 70 bases in the base sequence shown in SEQ ID NO: 4. [3] In [1], the base sequence has 90% or more identity with a contiguous base sequence of not less than 40 bases and not more than 60 bases in the base sequence shown in SEQ ID NO: 4. [4] In [1] to [3], the base sequence is a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 6 or 10. [5] In [1] to [4], the base of the nucleotide unit is cytosine or uracil.

[0058] In the 5' end region and the 3' end region of the RNA disclosed herein, in addition to the nucleotide unit represented by formula (1) and / or the nucleotide unit represented by formula (2), there may also be a structure in which ribose is connected by a phosphorothioate bond. The nucleotide unit represented by formula (1) and the nucleotide unit represented by formula (2) may also have a connection by a phosphorothioate bond, but it may be advantageous if the nucleotides corresponding to other bases not replaced by the nucleotide unit are also connected by a phosphorothioate bond.

[0059] The position or number of the phosphorothioate bonds introduced into the 5'-end region and the 3'-end region is not particularly limited. For example, each of the first connecting parts starting from the 5'-end and the 3'-end may have 1 or more and 8 or less, 1 or more and 6 or less, 1 or more and 5 or less, 1 or more and 4 or less, 1 or more and 3 or less, etc., in succession, and in addition to the first connecting part starting from the end, for example, 1 or more and 4 or less may be included in 1 or more and 2 or less bases.

[0060] In addition to having at least one nucleotide unit represented by formula (1) and / or a nucleotide unit represented by formula (2) in the 5' terminal region and / or the 3' terminal region, the other nucleotide units of the RNA disclosed herein may be natural nucleotides, i.e., nucleotide units having natural ribose, or nucleotide units in which the OH group of the ribose is modified, for example, by introducing a methoxy group at the 2' position of the ribose, or may be nucleotide units in which other riboses are subjected to known chemical modifications. For example, the RNA disclosed herein may be a chemically modified ribose RNA in which all nucleotide units except the nucleotide unit represented by formula (1) disposed in the 5' terminal region and / or the 3' terminal region thereof have a methoxy group at the 2' position of the ribose.

[0061] According to the above, for example, as the RNA of the present disclosure, in any one of the above forms (1) to (5), preferably, all nucleotide units other than the above nucleotide units have a chemically modified ribose having a methoxy group at the 2' position of the ribose.

[0062] The RNA disclosed herein may also be chemically modified to convert uridine in the RNA into a uridine analog. The chemical modification of replacing uridine with a uridine analog, for example, the proportion of uridine to the total number of uridines in the RNA disclosed herein is not particularly limited. For example, more than 80% of the total number of uridines, and more than 90% of the total number of uridines. For example, more than 100% of the total number of uridines can be uridine analogs.

[0063] Nucleobases and nucleosides comprising uridine analogs include, but are not limited to, uridine pseudouridine, 1-methyl pseudouridine, 1-ethyl pseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine. For example, the uridine analog can be 1-methyl-pseudouridine.

[0064] The RNA disclosed herein may also be chemically modified to convert cytidine in the RNA into a cytidine analog. The chemical modification of replacing cytidine with a cytidine analog, for example, the proportion of cytidine in the RNA disclosed herein to the total number of cytidines is not particularly limited. For example, more than 80% of the total number of cytidines, more than 90% of the total number of cytidines, and more than 100% of the total number of cytidines may be cytidine analogs.

[0065] Nucleobases and nucleosides comprising cytidine analogs include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, 4-acetyl-cytidine, 5-formyl-cytidine, 4-methyl-cytidine, 5-methyl-cytidine, 5-halo-cytidine, 5-hydroxymethyl-cytidine, 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, zebulin, 5-aza-zebulin, 5-methyl-zebulin lin, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysine, 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 may also have a chemical modification that converts adenine in the RNA into an adenine analog. The chemical modification of replacing adenine with an adenine analog, for example, the proportion of adenine in the RNA of the present disclosure to the total number of adenines is not particularly limited. For example, more than 80% of the total number of adenines, more than 90% of the total number of adenines, and more than 100% of the total number of adenines can be adenine analogs.

[0067] Nucleobases and nucleosides comprising 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-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, 2-methyladenine, 6-methyladenosine, 2-methylthio-6-methyladenosine, 6-isopentenyladenosine, 2-methylthio-6-isopentenyladenosine, 6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-6-(cis-hydroxyisopentenyl)adenosine, 6-glycylaminoformyladenosine glycosides, N6-threonylaminoformyl adenosine, 6-methyl-N6-threonylaminoformyl-adenosine, 2-methylthio-6-threonylaminoformyl-adenosine, 6,6-dimethyl-adenosine, 6-hydroxy-n-valerylaminoformyl-adenosine, 2-methylthio-6-hydroxy-n-valerylaminoformyl-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-ribosyl-adenosine (phosphate), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-arabino-adenosine, 2'-F-adenosine, 2'-OH-arabino-adenosine, and 6-(19-amino-pentaoxydodecyl)-adenosine.

[0068] The RNA of the present disclosure may also have a chemical modification that converts guanine in the RNA into a guanine analog. The chemical modification of replacing guanine with a guanine analog, for example, the proportion of guanine in the RNA of the present disclosure to the total number of guanines is not particularly limited. For example, more than 80% of the total number of guanines, more than 90% of the total number of guanines, and more than 100% of the total number of guanines can be guanine analogs.

[0069] Nucleobases and nucleosides comprising guanine analogs include inosine, 1-methyl-inosine, inosine, methylinosine, 4-demethyl-inosine, isoinosine, whitinosine, peroxywhitinosine, hydroxywhitinosine, unmodified hydroxywhitinosine, 7-deaza-guanosine, lignin, epoxylignin, galactosyl-lignin, mannosyl-lignin, 7-cyano-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, 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-arabino-guanosine, and 2'-F-guanosine.

[0070] When the base sequence defined in (1) and (2) is used as the first region, the RNA of the present disclosure may have a 5' untranslated region (UTR) at the 5' end of the first region. The 5'UTR may be the natural 5'UTR of the polypeptide of interest encoded by the first region, or may be a 5'UTR different from the encoded polypeptide of interest. For example, the 5'UTR may include at least one translation initiation sequence, such as a Kozak sequence, an intra-sequence 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 also include a 5' cap and a poly A tail. The 5' cap is generally a modified nucleotide component, generally referring to a structure added to the 5' end of a mature mRNA.

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

[0073] The poly A tail is also called a poly A tail, and refers to a continuous adenine polynucleotide. The base length of the poly A is not particularly limited, but is preferably 20 or more, preferably 50 or more. The upper limit of the base length is not particularly limited, but is, for example, 200 or less.

[0074] The RNA disclosed herein may be an mRNA or a portion thereof. In the case of an mRNA or a portion thereof, a 5'UTR and / or a poly A tail may be included. When the RNA of the present specification is an mRNA, the coding region, for example, an RNA variant having a base sequence shown in SEQ ID NO: 1, when including a coding sequence from positions 88 to 897 of the base sequence shown in SEQ ID NO: 1, may include a variation containing about 1 to 5 stop codons. For example, the base sequence represented by sequence number 3 (including 3 stop codons) may be included. Furthermore, if necessary, the RNA disclosed herein may include a G→A variation to avoid consecutive bases such as G.

[0075] The RNA disclosed herein may be an RNAi inducer, an RNAi agent, siRNA, shRNA, miRNA, antisense RNA, 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, a colored substance, etc. attached to a portion thereof.

[0077] The above-mentioned various chemical modifications are known or well-known to those skilled in the art, and those skilled in the art can obtain RNA using the desired chemical modification based on the disclosure of this specification and the common knowledge at the time of this application. RNA having a nucleotide unit represented by formula (1) and / or a nucleotide unit represented by formula (2) can be manufactured by the method described in the pamphlet of International Publication No. 2018 / 110678.

[0078] When the RNA of the present disclosure is administered to cells overexpressing the ZC3H12D protein, when the cells are natural killer (NK) cells, it can activate the migration ability of the cells, induce the anti-metastatic activity of the cells, and increase the tumor destruction activity, etc.

[0079] The various embodiments of the RNA of the present disclosure can be applied alone or in combination to the following various forms of the RNA of the present disclosure.

[0080] (Antitumor agent and method for inhibiting tumor growth) The anti-tumor agent of the present invention contains the RNA of the present invention as an active ingredient. The method of inhibiting tumor cell proliferation of the present invention administers the RNA of the present invention to humans or non-human animals in vitro. By administering the RNA of the present invention to humans or non-human animals in vitro, the RNA of the present invention has the ability to activate immune cells, etc., and thus can exert an anti-tumor effect.

[0081] The RNA disclosed herein as an active ingredient of an anti-tumor agent is an RNA that can activate immune cells or improve the anti-tumor activity of cells. In addition, the cell is a cancer cell, a tumor cell or an immune cell, and the immune cell includes a natural killer cell (NK cell), a macrophage, an eosinophil, a neutrophil, a basophil, a dendritic cell, a lymphocyte, etc. Preferably, the immune cell is a natural killer cell (NK cell), a macrophage, 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, endometrial cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, osteosarcoma, multiple myeloma, skin cancer, brain tumors, mesothelioma, etc.

[0083] The activation of immune cells can be evaluated by the production of IFNγ in immune cells, the 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] (Immune cell activator (activator) and immune cell activation (activation) method) The immune cell activator disclosed herein comprises the RNA disclosed herein as an effective ingredient. The immune cell activation method disclosed herein may include administering the RNA disclosed herein to a human or non-human animal in vitro. With respect to cells, immune cells, and activation of immune cells, the embodiments described above for antitumor agents are administered.

[0085] (Tumor cell metastasis inhibitor and method for inhibiting tumor cell metastasis) The tumor cell metastasis inhibitor disclosed herein comprises the RNA disclosed herein as an active ingredient. The method for inhibiting tumor cell metastasis disclosed in this specification may include administering the RNA disclosed herein to a human or non-human animal in vitro. The RNA disclosed herein may increase the anti-tumor activity of cells and inhibit the metastasis of tumor cells by activating immune cells. With respect to cells, immune cells, activation of immune cells and anti-tumor activity, the embodiments described for anti-tumor agents are applicable.

[0086] The inhibition of tumor cell metastasis can be evaluated by counting the number of metastatic tumor cells, which is known.

[0087] The treatment for inhibiting the metastasis of primary cancer using a metastasis inhibitor is performed in combination with the treatment of the primary cancer or independently. For example, when a metastasis inhibitor is used in combination, for example, one or more of them can be used before or after or simultaneously with surgical treatment, anticancer agent treatment, radiotherapy (X-ray, proton beam, heavy ion beam) and electromagnetic wave therapy (radio wave) for the primary cancer. Combination with surgical treatment is one of the preferred forms.

[0088] Typically, for example, the following usage forms may be included. (a) When the primary cancer still exists after surgical treatment of the primary cancer, it is used in combination with one or more of anticancer drug therapy, radiotherapy and electromagnetic wave therapy. (b) When the primary cancer can be removed after surgical treatment, it can be used alone or in combination with one or more of anticancer drug therapy, radiotherapy, and electromagnetic wave therapy to inhibit metastasis. (c) After surgical treatment of primary cancer, when new primary cancer or metastasis is discovered later, it can be used alone or in combination with one or more of surgical treatment, anticancer drug treatment, radiotherapy and electromagnetic wave treatment to inhibit metastasis.

[0089] (Enhancer of immunomodulatory effect, etc. of immunomodulator and method for enhancing the effect, etc.) The enhancer of the present invention contains the RNA of the present invention as an active ingredient. The enhancement method disclosed in this specification may include administering the RNA of the present invention to humans or non-human animals in vitro. The RNA of the present invention is an RNA that can activate immune cells, increase the anti-tumor activity of cells, or inhibit tumor cell metastasis. Therefore, when used in combination with immune cell activators, anti-tumor agents and metastasis inhibitors, the embodiments of anti-tumor agents and metastasis inhibitors can be applied to enhance the immune cell activation effect, anti-tumor activity effect, metastasis inhibition effect of these agents, cells, immune cells, immune cell activation, anti-tumor activity and inhibition of metastasis.

[0090] As the immunomodulator to be used in combination, a known immunomodulator can be used. Also, as the metastasis inhibitor to be used in combination, a known metastasis inhibitor can be used.

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

[0092] There is no particular limitation on the method of administration of the above-mentioned combined agent and the enhancer disclosed herein, as long as the predetermined enhancing effect of the enhancer disclosed herein is achieved, and they can be administered simultaneously, sequentially, or at intervals. There is no particular limitation on the order of administration of these compositions, and the antitumor agent can be administered before, simultaneously with, or after the RNA.

[0093] The combined agent and the enhancer of the present disclosure may be used in combination as separate agents, or may be provided as a combined agent or a pharmaceutical kit according to a prescribed usage.

[0094] (Pharmaceutical composition, immune improvement method, tumor prevention or treatment method) The pharmaceutical composition disclosed in this specification contains this RNA as an effective ingredient. The immune improvement method, tumor prevention or treatment method disclosed in this specification may include administering the RNA disclosed in vitro to humans or non-human animals. The RNA disclosed in this specification is an RNA that can activate immune cells, increase the anti-tumor activity of cells, or inhibit the metastasis of tumor cells. Therefore, according to the pharmaceutical composition containing these, in addition to preventing or improving various conditions or diseases by activating immune cells, etc., it can also be used to prevent or treat diseases such as tumors due to anti-tumor activity or tumor metastasis inhibition activity. Tumors include, for example, the various tumors described.

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

[0096] When the RNA disclosed herein is used as an active ingredient of a drug, a drug carrier may be added as needed, and various administration forms may be used according to the purpose of prevention or treatment. The anti-tumor agent containing the above RNA may be in the form of, for example, an injection, a suppository, an oral preparation, an ointment, an eye drop, etc., preferably an injection (intravenous injection, etc.). These administration forms may be manufactured by conventional formulation methods known to those skilled in the art.

[0097] As drug carriers, various organic or inorganic carrier substances commonly used as preparation materials are prepared into excipients, binders, disintegrants, lubricants, coating agents, etc. in solid preparations, solvents, solubilizers, suspending agents, tonicity agents, pH regulators, pH buffers, analgesics, etc. in liquid preparations. In addition, as needed, preparation additives such as preservatives, antioxidants, colorants, flavoring agents, deodorants, stabilizers, etc. can also be used.

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

[0099] Furthermore, the daily dose of the above RNA varies depending on the patient's symptoms, weight, age, gender, etc., but cannot be determined unconditionally. For an average adult (weight 50 kg), the compound may preferably be 10 ng to 1 mg per day, more preferably 100 ng to 100 μg, and further preferably 1 μg to 10 μg.

[0100] The amount of RNA formulated into each of the above-mentioned dosage units is appropriately determined depending on the properties of the RNA used, the patient's symptoms, the drug, and the like.

[0101] The disclosures of all patent applications and documents cited in this specification are incorporated by reference in their entirety.

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

[0103] Unless otherwise stated, follow the methods below.

[0104] (1) Reagents The following primary antibodies and factors were used in this study: human anti-ZC3H12D antibody (ab1000862; Abcam), cell sorting for human CD56: PE anti-human CD56 (NCAM) antibody (BioLegend) and isotype control, mouse IgG1, κ (BioLegend). Cell sorting for 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 carcinoma cell line 786-O was 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 of IL2.

[0107] (4) Migration assay Cell migration was evaluated 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. Aliquots (50 μl) of the cell suspension (2 × 10 5 Up to 2×10 6 cells / mL) were seeded into each upper well and incubated for 3.5 hours.

[0108] (5) IFNγ induction assay After stimulating with 50 ng / mL RNA for 48 h in LGM-3 medium (Lonza) supplemented with 200 IU / mL IL2, human CD56 was stained with anti-human IFNγ antibody. + CD3 - NK cells were stained. For positive control of IFNγ induction, cells were incubated with 40 ng / mL of 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 USA) and cloned into the human ZC3H12D coding region pCMV6-entry vector (C-terminalmyc-FLAGtag). Mouse ZC3H12D was PCR amplified using primer sets 5′-GGTACCATGGAGCATCGGAGCAAGATGG-3′ (SEQ ID NO: 7) and 5′-CTCGAGTTAAGGATCCCCCAACGGAGCACC-3′ (SEQ ID NO: 8), and then cloned using pCRBluntII vector (Thermo). The cloned fragment was digested into double strands by KpnI-XhoI and cloned using pcDNA3 with C-terminalFLAG-tag. To establish a cell line stably expressing human ZC3H12D, one of the above constructs 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 tested by Western blotting using a probe with DDDDK antibody (MBLCo., Ltd, Japan) to confirm the expression of ZC3H12D-FLAG protein.

[0110] (7) Immunohistochemistry and cell counting CD56 was stained with anti-ZC3H12D (ab1000862, Abcam) antibody+ CD3 - NK cells were stained. The values ​​of the immunostained cell area are shown as the number of pixels normalized to the DAPI signal. Labeled cells were detected by confocal microscopy or fluorescence microscopy and normalized to the total surface area. Example 1

[0111] (Synthesis of Chemically Modified RNA) According to the conventional method, Figure 1 The base sequence shown (partial sequence of human IL1β (SEQ ID NO: 6)) and two types of chemically modified RNA (human IL1β_AE, human IL1β_Me). In addition, a nucleotide unit having a fluorine atom at the 2' position and an aminoethyl group at the 4' position was synthesized by the phosphoramidite method according to Japanese Patent Publication No. 2019-10035. In addition, as a control, a nucleotide unit having been subjected to Figure 1 The chemically modified 50mer poly A (PolyA_50mer_Me_AE) (SEQ ID NO: 9) is shown. Example 2

[0112] (Confirmation of the cell migration activation effect of chemically modified RNA in ZC3H12D overexpressing cells) The two types of chemically modified RNA synthesized in Example 1 and the 50mer poly A_50mer_Me_AE as a control were applied to the culture medium containing 786-O cells overexpressing ZC3H12D, and the cell migration ability was determined by the average value of migrating cells. The concentration of RNA was 100 ng / mL. The results are shown in Figure 2 shown.

[0113] like Figure 2 As shown, both types of synthetic RNA showed significantly improved migration activity compared to no addition and control. Example 3

[0114] (Confirmation of IFNγ 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 evaluate the effects of chemically modified RNA on human CD56. + CD3 - The results, such as Figure 3 and Figure 4 shown.

[0115] Figure 3 The signal intensity obtained from INFγ by DAPI staining is shown. Figure 3As shown, it was found that both types of chemically modified RNAs significantly produced INFγ compared to the case where no RNA was added.

[0116] Figure 4 The number of INFγ-positive NK cells in each total NK cell population is shown. Figure 4 As shown, both types of chemically modified RNA showed a high number of INFγ-producing cells.

[0117] According to the above, although the RNA of the present disclosure was partially chemically modified, it was found to have migration activation ability and INFγ induction activation ability. Example 4

[0118] (Synthesis of Chemically Modified RNA) According to conventional methods, Figure 5 The base sequences shown (partial sequence of human IL1β (SEQ ID NO: 10) and partial sequence of mouse IL1β (SEQ ID NO: 11)) and seven types of chemically modified RNA (human IL1β_OME_AE, mouse IL1β_OMe_AE, mouse IL1β_OMe_AE_F, mouse IL1β_F, human IL1β_OME_AP, human IL1β_OME_AP_F, human IL1β_F). In addition, the nucleotide unit having a fluorine atom at the 2' position and an aminoethyl group at the 4' position was synthesized as a whole according to the phosphoramidite method based on Japanese Patent Publication No. 2019-10035. In addition, the nucleotide unit having a methoxy group at the 2' position and an aminopropyl group at the 5' position was synthesized as a whole according to the phosphoramidite method based on International Publication No. WO2019 / 088179 pamphlet. In addition, as a control, the nucleotide unit that has been synthesized was combined and Figure 5 The chemically modified 50mer poly A (PolyA_50_OMe_AE) (SEQ ID NO: 9) is shown. In addition, mouse IL1β_OMe_AE_F, mouse IL1β_F, human IL1β_OME_AP_F and human IL1β_F are labeled with fluorescein at their 5' end. Example 5

[0119] (Evaluation of RNase resistance of chemically modified RNA) The fluorescein-labeled mouse IL1β_OME_AE_F and mouse IL1ββ_F synthesized in Example 4 were used to evaluate the nuclease (RNase) resistance of synthetic RNA in the serum of tumor-bearing mice. The synthetic RNA had a nucleotide unit with a fluorine atom at the 2' position and an aminoethyl group at the 4' position (2'-F-4'-AE) at both ends, and the other nucleotide units were all nucleotide units with a methoxy group at the 2' position (2'-OMe). In addition, by 6E0771 cells / mouse were injected into the mammary glands of 8-12 week old C57BL / 6 mice to produce tumor-bearing mice. One week after the injection of E0771 cells, blood was collected from the mice with approximately 1 cm tumors formed in the mammary glands to obtain serum from tumor-bearing mice.

[0120] The nuclease (RNase) resistance of synthetic RNA in the serum of tumor-bearing mice was evaluated using fluorescein-labeled human IL1β_OMe_AP_F and human IL1β_F synthesized in Example 4. The synthetic RNA has a nucleotide unit with a methoxy group at the 2' position and an aminopropyl group at the 5' position (2'-OMe-5'-AP) at both ends, and the other nucleotide units are all nucleotide units with a methoxy group at the 2' position (2'-OMe).

[0121] For nuclease resistance, 300 pmol of each RNA was dissolved in 37.5 μl of OPTI-MEM, 1.1 μl was dispensed from it, and 15 μl of loading buffer (10 mM EDTA, 90% formamide) was added to prepare a 0-minute sample. In addition, 4 μl (10%) of tumor-bearing mouse serum or 4 μl (10% and 20%) and 9.1 μl (20%) of bovine serum were added thereto, and incubated at 37°C. After 5 minutes, 1 time, 3 times, 6 times, 12 times, 24 times, and 48 times, 2 μl of the reaction solution was added to 10 μl of the loading solution pre-packed in a separate sampling tube to terminate the reaction. The reaction solution was subjected to 20% denaturing PAGE (500 V, 20 mA) electrophoresis and analyzed using an image analyzer LAS-4000 (Fuji Film Co., Ltd.). The results are as shown below. Figure 6 and Figure 7 In addition, Figure 6 In the figure, the RNA amount (%) after 5 minutes to 48 hours relative to the RNA amount at 0 minutes is shown based on the electrophoresis results.

[0122] In addition, denaturing PAGE was prepared by the following method. 40% acrylamide (19:1) solution (20 ml), urea (16.8 g), 10×TBE buffer (4 ml) were added to dissolve, and H2O was added to make the volume 40 ml. Finally, APS (27.5 mg) was added to dissolve, TEMED (20 μl) was added and shaken, and poured between two glass plates sandwiching a fixed gasket, and allowed to stand for more than 1 hour to solidify. In addition, 1×TBE buffer was used as the electrophoresis buffer.

[0123] like Figure 6As shown, the chemically modified RNA with one (2'-F-4'-AE) at each end and all other nucleotide units (2'-OMe) still maintained 80% RNA even after 48 hours in serum. And, on the contrary, for RNA in which all nucleotide units are natural nucleotide units, it was found that about all RNA disappeared after 5 minutes. From the above, it can be seen that RNA with (2'-F-4'-AE2'-F-4'-AE) at the end has excellent resistance to RNase in serum.

[0124] And, if Figure 7 As shown, the chemically modified RNA with one (2'-OMe-5'-AP) at each end and all other nucleotide units (2'-OMe) maintained almost 100% RNA even after 48 hours in 20% serum. And, on the contrary, for RNA in which all nucleotide units are natural nucleotide units, it was found that almost all RNA disappeared after 5 minutes. From the above, it can be seen that RNA with (2'-OMe-5'-AP) at the end has excellent resistance to RNase in serum. Example 6

[0125] (Confirmation of the effect of chemically modified RNA on activating cell migration ability) The human IL1β_OME_AE synthesized in Example 4 and the poly A_50_OMe_AE as a control were applied through a filter to a cell suspension containing 786-O cells overexpressing the ZC3H12D protein, and the number of migrating cells was counted. A Boyden chamber (manufactured by Neuro Probe) was used as an apparatus. The upper and lower wells were separated using a polycarbonate filter with a pore size of 5 μm (Nucleopore; Costar). A cell suspension (1×10 6 / ml), the culture medium containing the above RNA was added to the lower wells at concentrations of 1ng / ml, 10ng / ml, 100ng / ml and 1μg / ml, respectively, and incubated for 3 hours, and the number of cells that migrated to the lower wells was counted. The results are shown in Table 1.

[0126] Table 1 none IL1β_OMe_AE Poly A_OMe_AE Number of migrated cells 100 150 100

[0127] As shown in Table 1, when human IL1β_OME_AE was administered, the average number of migrating cells at each RNA concentration was 150, when the number of migrating cells when not added was set to 100. In addition, it increased significantly with the increase in RNA concentration, and was about 200 at 100ng / ml. On the other hand, when poly A_50_OMe_AE was administered, regardless of the concentration, it was the same as when not added, with an average of 110. From the above, it can be seen that human IL1β_OME_AE with one (2'-F-4'-AE) at each end helps to improve the migration ability of NK cells expressing ZC3H12D protein. Example 7

[0128] (Confirmation of IFNγ induction) The human IL1β_OME_AE synthesized in Example 4 and poly A_50_OMe_AE as a control were used to evaluate the effects of chemically modified RNA on human CD56 + CD3 - Effect of IFNγ production in NK cells. Human CD56 cells were collected from cryopreserved human PBMCs by FACS + CD3 - NK cells. After priming with 10 ng / mL of each RNA in HPLM medium (Gibco) for 24 hours in the presence of 200 U / mL of IL-2 (BioLegend), they were stained with anti-human IFN-γ antibody (BioLegend). In addition, IL-12 (Proteintech) was used as a positive control for IFNγ induction. The results are shown in Table 2.

[0129] Table 2

[0130] As shown in Table 2, when human IL1β_OME_AE was used, the amount of INFγ was induced 2.3 times compared to the amount of IFNγ when it was not added. This is comparable to the positive control. On the other hand, when poly A_50_OMe_AE was used, the induction of IFNγ was only the same as when it was not added. From the above, it can be seen that human IL1β_OME_AE with one (2'-F-4'-AE) at each end helps to increase the amount of INFγ produced by human NK cells. Example 8

[0131] (Evaluation of the cytotoxicity of human NK cells) For the human IL1β_OME_AE synthesized in Example 4 and the poly A_50_OMe_AE as a control, the Zombie Green Fixable Viability Kit (BioLegend) was used to evaluate the activity of human CD56 + CD3 -The cell killing ability of NK cells. That is, human CD56 + CD3 - After NK cells and cancer cells (Caco-2: colon cancer cell line) were co-cultured at a ratio of 1:1 for 24 hours, the proportion 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 human IL1β_OME_AE was used, the amount of cell killing observed was 2.5 times that when it was not added. On the other hand, when poly A_50_OMe_AE was used, the same degree of cell killing was observed as when it was not added. From the above, it can be seen that human IL1β_OME_AE with one (2'-F-4'-AE) at each end helps to improve the cell killing ability of human NK cells. Example 9

[0134] (Evaluation of anti-metastatic activity in mice simulating lung metastasis model) The anti-metastatic activity was evaluated for the human IL1β_OME_AE synthesized in Example 4 and the poly A_50_OMe_AE as a control. Each RNA (human IL1β_OME_AE and poly A_50_OMe_AE, 1 μg each) and PBS as a negative control were injected into the tail vein of mice bearing tumors made with E0771 breast cancer cells every few days. The mice were dissected 48 hours after the tail vein injection of tumor cells (E0771 breast cancer cells) stained with PKH-26 (Sigma-Aldrich), 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 human IL1β_OME_AE was used, 65% of the positive cells were observed compared to when PBS was added. On the other hand, when poly A_50_OMe_AE was used, the same number of positive cells as when PBS was added were observed. As shown above, human IL1β_OME_AE with one (2'-F-4'-AE) at each end helps inhibit the metastasis of tumor cells in vivo. Sequence Listing Free Text

[0137] SEQ ID NO: 7, 8: Primer SEQ ID NO: 9: Control RNA

Claims

1. An antitumor agent, characterized in that: A method of producing a RNA having any base sequence selected from the group consisting of the following (1) and (2), wherein the RNA comprises as an active ingredient 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 a 3' region within 20 bases from the 3' end and / or a 5' region within 25 bases from the 5' end: (1) the base sequence shown in SEQ ID NO: 2 or a base sequence having 90% or more identity thereto, (2) a continuous base sequence of 20 to 200 bases in the base sequence of (1), Chemical formula 12: In formula (1), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is replaced by an alkyl group or an alkenyl group, or a halogen atom, R 2 Denotes NHR with a linker 3 , the connecting group is a divalent hydrocarbon group having one or more carbon atoms, 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 Indicates OH (or O - ) or SH(or S - ), B represents a purine base or a pyrimidine base, Chemical formula 13: In formula (2), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is replaced by an alkyl group or an alkenyl group, or a halogen atom, R 4 Denotes NHR with a linker 5 , the connecting group is a divalent hydrocarbon group having one or more carbon atoms, 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 Indicates OH (or O - ) or SH(or S - ), B represents a purine base or a pyrimidine base.

2. The antitumor agent according to claim 1, characterized in that In the 3' terminal region and / or the 5' terminal region, the RNA has 1 to 20 of the first or second nucleotide units in each of these regions.

3. The antitumor agent according to claim 1, characterized in that The first or second nucleotide unit is in the formula (1), R 1 is a methoxy group or a fluorine atom, R 2 It is an aminoethyl nucleotide unit.

4. The antitumor agent according to claim 1, characterized in that In the first nucleotide unit, n is 1, R 1 is a fluorine atom, R 2 is an aminoethyl nucleotide unit, There is at least one of the first nucleotide units within 2 bases from the 3' end of the 3' end region, and there is at least one of the first nucleotide units within 2 bases from the 5' end of the 5' end region.

5. The antitumor agent according to claim 1, characterized in that In the second nucleotide unit, n is 1, R 1 is methoxy, R 4 is an aminopropyl nucleotide unit, There is at least one second nucleotide unit within 2 bases from the 3' end of the 3' end region, and there is at least one second nucleotide unit within 2 bases from the 5' end of the 5' end region.

6. The antitumor agent according to claim 4 or 5, characterized in that The base sequence has 90% or more identity with a continuous base sequence of 30 to 70 bases in the base sequence shown in SEQ ID NO:

4.

7. The antitumor agent according to claim 4 or 5, characterized in that The base sequence has 90% or more identity with a continuous base sequence of 40 to 60 bases in the base sequence shown in SEQ ID NO:

4.

8. The antitumor agent according to claim 4 or 5, characterized in that The base sequence is a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 6 or 10.

9. The antitumor agent according to claim 8, characterized in that The base of the nucleotide unit is cytosine or uracil.

10. The antitumor agent according to claim 1, characterized in that In the first or second nucleotide unit, n is 1, and the RNA has X in the formula (1) in each region of the 3' terminal region and / or the 5' terminal region: 1 or X 2 Any one of S or SH (S - )'s first or second nucleotide unit.

11. The antitumor agent according to claim 1, characterized in that More than 80% of the total number of uridine nucleosides in the RNA comprise a uridine analog selected from the group consisting of pseudouridine, 1-methylpseudouridine, 1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine.

12. The antitumor agent according to claim 1, characterized in that The RNA is a variant of the RNA having the base sequence shown in SEQ ID NO:

1. Compared with the natural coding sequence, the coding sequence from positions 88 to 897 of the base sequence shown in SEQ ID NO: 1 has bases substituted by 1 to 5 additional stop codons.

13. The antitumor agent according to claim 1, characterized in that The length of the RNA is 20 to 100 bases.

14. An immunomodulator, characterized in that A method of producing a RNA having any base sequence selected from the group consisting of the following (1) and (2), wherein the RNA comprises as an active ingredient 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 a 3' region within 20 bases from the 3' end and / or a 5' region within 25 bases from the 5' end: (1) the base sequence shown in SEQ ID NO: 2 or a base sequence having 90% or more identity thereto, (2) a continuous base sequence of 20 to 200 bases in the base sequence of (1), Chemical formula 14: In formula (1), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is replaced by an alkyl group or an alkenyl group, or a halogen atom, R 2 Denotes NHR with a linker 3 , the connecting group is a divalent hydrocarbon group having one or more carbon atoms, 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 Indicates OH (or O - ) or SH(or S - ), B represents a purine base or a pyrimidine base, Chemical formula 15: In formula (2), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is replaced by an alkyl group or an alkenyl group, or a halogen atom, R 4 Denotes NHR with a linker 5 , the connecting group is a divalent hydrocarbon group having one or more carbon atoms, 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 Indicates OH (or O - ) or SH(or S - ), B represents a purine base or a pyrimidine base.

15. A tumor metastasis inhibitor, characterized in that: A method of producing a RNA having any base sequence selected from the group consisting of the following (1) and (2), wherein the RNA comprises as an active ingredient 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 a 3' region within 20 bases from the 3' end and / or a 5' region within 25 bases from the 5' end: (1) the base sequence shown in SEQ ID NO: 2 or a base sequence having 90% or more identity thereto, (2) a continuous base sequence of 20 to 200 bases in the base sequence of (1), Chemical formula 16: In formula (1), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is replaced by an alkyl group or an alkenyl group, or a halogen atom, R 2 Denotes NHR with a linker 3 , the connecting group is a divalent hydrocarbon group having one or more carbon atoms, 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 Indicates OH (or O - ) or SH(or S - ), B represents a purine base or a pyrimidine base, Chemical formula 17: (In formula (2), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is replaced by an alkyl group or an alkenyl group, or a halogen atom, R 4 Denotes NHR with a linker 5 , the connecting group is a divalent hydrocarbon group having one or more carbon atoms, 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 Indicates OH (or O - ) or SH(or S - ), B represents a purine base or a pyrimidine base.

16. An enhancer for the immunomodulatory effect of an immunomodulator or the antitumor effect of an antitumor agent, characterized in that: A method of producing a RNA having any base sequence selected from the group consisting of the following (1) and (2), wherein the RNA comprises as an active ingredient 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 a 3' region within 20 bases from the 3' end and / or a 5' region within 25 bases from the 5' end: (1) the base sequence shown in SEQ ID NO: 2 or a base sequence having 90% or more identity thereto, (2) a continuous base sequence of 20 to 200 bases in the base sequence of (1), Chemical formula 18: In formula (1), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is replaced by an alkyl group or an alkenyl group, or a halogen atom, R 2 Denotes NHR with a linker 3 , the connecting group is a divalent hydrocarbon group having one or more carbon atoms, 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 Indicates OH (or O - ) or SH(or S - ), B represents a purine base or a pyrimidine base, Chemical formula 19: In formula (2), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is replaced by an alkyl group or an alkenyl group, or a halogen atom, R 4 Denotes NHR with a linker 5 , the connecting group is a divalent hydrocarbon group having one or more carbon atoms, 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 Indicates OH (or O - ) or SH(or S - ), B represents a purine base or a pyrimidine base.

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