WO2025105500A1 - Cell activating agent containing rna and uses thereof - Google Patents

Abstract

Provided are an agent containing RNA as an active ingredient and capable of strongly activating cells, and uses thereof. An RNA containing a base sequence represented by SEQ ID NO: 1 or a base sequence having 90% or more identity with the sequence has a cell activating ability.

Description

RNA-containing cell activator and its use

This application claims priority based on Japanese patent application No. 2023-196081, filed on November 17, 2023, the entire contents of which are incorporated herein by reference.
The present specification relates to an agent containing RNA for cell activation and the like, and uses thereof.

Recently, we have discovered potential anti-metastatic immune cells. Specifically, we found that B220 ⁺ CD11c ⁺ NK1.1 ⁺ natural killer (NK) cells migrated from the liver to the lungs of cancer-bearing mice (Non-Patent Document 1). These NK cells accumulate at fibrinogen ^- hyperpermeable sites, remove concentrated fibrinogen, and have the ability to destroy tumors by producing interferon gamma (IFNγ) (Non-Patent Document 2).

Comparison of gene expression analysis between B220 ⁺ CD11c ⁺ NK1.1 ⁺ NK cells in the liver and B220 ⁺ CD11c ⁺ NK1.1 ⁺ cells in the lung 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).

EMBOMolMed10,doi:10.15252/emmm.201708643(2018) NatCommun4,1853,doi:10.1038/ncomms2856(2013) CancerRes67,93-99,doi:10.1158/0008-5472.CAN-06-2723(2007) BrJHaematol139,161-163,doi:10.1111/j.1365-2141.2007.06752.x(2007)

According to the inventors, previous research has suggested that ZC3H12D protein interacts with extracellular RNA (exRNA) in tumor anti-metastatic function, activating NK cells. The inventors searched for RNA that can more potently activate such cells.

The present specification provides an agent containing RNA as an active ingredient that can effectively interact with ZC3H12D protein and strongly activate cells, and uses thereof.

（式（１）中、Ｒ^１は、水酸基、水素原子がアルキル基又はアルケニル基で置換された水酸基又はハロゲン原子を表し、Ｒ^２は、連結基を有するＮＨＲ^３を表し、連結基は炭素数１個以上の２価炭化水素基であり、Ｒ^３は、水素原子、アルキル基又はアルケニル基を表し、ｎは０又は１を表し、Ｘ^１は、酸素原子又は硫黄原子を表し、Ｘ^２は、ＯＨ（又はＯ^－）又はＳＨ（又はＳ^－）を表し、Ｂは、プリン塩基又はピリミジン塩基を表す。）
［４］前記ＲＮＡは、前記３’末端領域及び／又は前記５’末端領域に、これらの領域につき前記ヌクレオチド単位を１個以上５個以下有する、である、［３］に記載の抗腫瘍剤。
［５］前記ヌクレオチド単位は、前記式（１）中、Ｒ^１は、メトキシ基又はフッ素原子であり、Ｒ^２は、アミノエチル基であるヌクレオチド単位である、［３］又は［４］に記載の抗腫瘍剤。
［６］前記ヌクレオチド単位において、ｎは、１であり、前記ＲＮＡは、前記３’末端領域及び／又は前記５’末端領域の各領域に、前記式（１）中、Ｘ^１又はＸ^２のいずれかが、Ｓ又はＳＨ（Ｓ^－）であるヌクレオチド単位を有する、［３］～［５］のいずれかに記載の抗腫瘍剤。
［７］前記ＲＮＡにおけるウリジンヌクレオシドの総数のうちの８０％以上が、シュードウリジン、１－メチルシュードウリジン、１－エチルシュードウリジン、２－チオウリジン、４'－チオウリジン、５－メチルシトシン、２－チオ－１－メチル－１－デアザ－シュードウリジン、２－チオ－１－メチル－シュードウリジン、２－チオ－５－アザ－ウリジン、２－チオ－ジヒドロシュードウリジン、２－チオ－ジヒドロウリジン、２－チオ－シュードウリジン、４－メトキシ－２－チオ－シュードウリジン、４－メトキシ－シュードウリジン、４－チオ－１－メチル－シュードウリジン、４－チオ－シュードウリジン、５－アザ－ウリジン、ジヒドロシュードウリジン、５－メトキシウリジン、および２'－Ｏ－メチルウリジンからなる群より選択されるウリジン類似体を含む、［３］～［６］のいずれかに記載の抗腫瘍剤。
［８］以下の（１）、（２）、（３）及び（４）からなる群から選択されるいずれかの塩基配列を有効成分として含む、免疫賦活剤。
（１）配列番号１で表される塩基配列又は当該配列と９０％以上の同一性を有する塩基配列
（２）前記（１）の塩基配列のうちの連続する１０塩基以上の塩基配列
（３）配列番号２で表される塩基配列又は当該配列と９０％以上の同一性を有する塩基配列
（４）前記（３）の塩基配列のうちの連続する１０塩基以上の塩基配列
［９］以下の（１）、（２）、（３）及び（４）からなる群から選択されるいずれかの塩基配列を含むＲＮＡを有効成分として含む、腫瘍の転移抑制剤。
（１）配列番号１で表される塩基配列又は当該配列と９０％以上の同一性を有する塩基配列
（２）前記（１）の塩基配列のうちの連続する１０塩基以上の塩基配列
（３）配列番号１で表される塩基配列又は当該配列と９０％以上の同一性を有する塩基配列
（４）前記（１）の塩基配列のうちの連続する１０塩基以上の塩基配列
［１０］以下の（１）、（２）、（３）及び（４）からなる群から選択されるいずれかの塩基配列を含むＲＮＡを有効成分として含む、免疫賦活剤の免疫賦活効果又は抗腫瘍剤の抗腫瘍効果の増強剤。
（１）配列番号１で表される塩基配列又は当該配列と９０％以上の同一性を有する塩基配列
（２）前記（１）の塩基配列のうちの連続する１０塩基以上の塩基配列
（３）配列番号２で表される塩基配列又は当該配列と９０％以上の同一性を有する塩基配列
（４）前記（３）の塩基配列のうちの連続する１０塩基以上の塩基配列 [1] A cell activator comprising, as an active ingredient, RNA having any one of the base sequences selected from the group consisting of (1), (2), (3) and (4) below:
(1) A base sequence represented by SEQ ID NO: 1 or a base sequence having 90% or more identity to said sequence; (2) A base sequence of 10 or more consecutive bases among the base sequence of (1); (3) A base sequence of 2 or a base sequence having 90% or more identity to said sequence; (4) A base sequence of 10 or more consecutive bases among the base sequence of (3); [2] An antitumor agent comprising RNA as an active ingredient containing any of the base sequences selected from the group consisting of (1), (2), (3) and (4) below.
(1) A base sequence represented by SEQ ID NO: 1 or a base sequence having 90% or more identity thereto; (2) A base sequence of 10 or more consecutive bases among the base sequence of (1); (3) A base sequence represented by SEQ ID NO: 2 or a base sequence having 90% or more identity thereto; (4) A base sequence of 10 or more consecutive bases among the base sequence of (3); [3] An antitumor agent according to [2], which contains the base sequence and, as an active ingredient, RNA having at least one nucleotide unit represented by the following formula (1) in its 3'-terminal region within 10 bases from the 3'-terminus and/or in its 5'-terminal region within 10 bases from the 5'-terminus.

(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 represents NHR ³ having a linking group, the linking group being 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 represents OH (or O ⁻ ) or SH (or S ⁻ ); and B represents a purine base or a pyrimidine base.)
[4] The antitumor agent according to [3], wherein the RNA has 1 to 5 of the nucleotide units in the 3'-terminal region and/or the 5'-terminal region.
[5] The antitumor agent according to [3] or [4], wherein the nucleotide unit is a nucleotide unit represented by formula (1), in which R ¹ is a methoxy group or a fluorine atom, and R ² is an aminoethyl group.
[6] The antitumor agent according to any one of [3] to [5], wherein, in the nucleotide unit, n is 1, and the RNA has a nucleotide unit in which either ^X1 or ^X2 in the formula (1) is S or SH (S ^- ) in each of the 3'-terminal region and/or the 5'-terminal region.
[7] The antitumor agent according to any of [3] to [6], 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.
[8] An immunostimulant comprising, as an active ingredient, any one of base sequences selected from the group consisting of (1), (2), (3) and (4) below.
(1) A base sequence represented by SEQ ID NO: 1 or a base sequence having 90% or more identity to said sequence; (2) A base sequence of 10 or more consecutive bases among the base sequence of (1); (3) A base sequence represented by SEQ ID NO: 2 or a base sequence having 90% or more identity to said sequence; (4) A base sequence of 10 or more consecutive bases among the base sequence of (3) [9] A tumor metastasis inhibitor comprising RNA as an active ingredient containing any of the base sequences selected from the group consisting of (1), (2), (3) and (4) below.
(1) A base sequence represented by SEQ ID NO: 1 or a base sequence having 90% or more identity to said sequence; (2) A base sequence of 10 or more consecutive bases among the base sequence of (1); (3) A base sequence represented by SEQ ID NO: 1 or a base sequence having 90% or more identity to said sequence; (4) A base sequence of 10 or more consecutive bases among the base sequence of (1) [10]. An agent for enhancing the immunostimulating effect of an immunostimulant or the antitumor effect of an antitumor agent, comprising as an active ingredient RNA containing any base sequence selected from the group consisting of (1), (2), (3) and (4) below.
(1) A base sequence represented by SEQ ID NO: 1 or a base sequence having 90% or more identity thereto. (2) A base sequence of 10 or more consecutive bases from the base sequence of (1). (3) A base sequence represented by SEQ ID NO: 2 or a base sequence having 90% or more identity thereto. (4) A base sequence of 10 or more consecutive bases from the base sequence of (3).

FIG. 1 shows an overview of RNA of the present disclosure. FIG. 1 shows an overview of RNA of the present disclosure. FIG. 1 shows an outline of synthetic RNA used in the examples. FIG. 1 shows the number of migrating cells when various synthetic RNAs were administered to 786-O cells in which the ZC3H12D protein was overexpressed. FIG. 13 shows the number of migrating cells when various synthetic RNAs were administered to 786-O cells in which the ZC3H12D protein was overexpressed. FIG. 1 shows the results of a killing assay of human colon cancer cell line DLD-1 by CD56 ⁺ CD3 ^- NK cells prepared from human peripheral blood using a cell sorter.

The present disclosure relates to the use of sequences near the untranslated regions on the 3'-terminal side of the RNA of IL-2 and IL-10. The inventors have discovered that synthetic RNAs made by selecting portions of these 3'-UTRs have stronger cell migration activity, in other words, NK cell activation activity, than conventional synthetic RNAs of the same type, and have found that such synthetic RNAs act in vivo as unique intercellular neurotransmitters with anti-tumor or anti-metastasis functions.

According to the research of the present inventors, the synthetic RNA disclosed herein can efficiently interact with and activate immune cells, thereby effectively suppressing tumors. By the same action, such synthetic RNA can 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 synthetic RNA disclosed herein is useful for preventing and/or treating tumors.

IL-2 is generally considered to be a multifaceted cytokine that exerts both immunostimulatory and immunosuppressive activities on target cells. IL-10 is known as an inhibitory cytokine, and is thought to suppress the production of IFN-γ from Th1 cells as well as the production of IL-1, IL-6, IL-12, and TNF-α from macrophages. It is also thought to act on macrophages to suppress the expression of the auxiliary signal molecules CD80/CD86, thereby suppressing the T cell activation auxiliary function.

In this specification, when RNA or synthetic RNA is mentioned without any particular limitation, it means RNA that is a part of natural RNA but is artificially obtained by genetic engineering or chemical synthesis. Furthermore, in this specification, "chemical modification" of RNA may be any chemical modification applied to RNA, and may include, for example, known modifications of ribose, phosphodiester bond moieties, and bases of nucleotides. With regard 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.

In addition, in this specification, the base sequences represented by SEQ ID NOs: 1 to 7 are essentially RNA, but are described in the sequence table using DNA bases.

Below, representative but non-limiting 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 implementing 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 uses thereof.

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 described only to illustrate representative examples of the invention. Furthermore, the various features of the representative examples described above and below, as well as the various features of those described in the independent and dependent claims, do not have to be combined in the exact specific examples described herein, or in the exact order listed, in order to provide additional and useful embodiments of the invention.

All features described in this 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 intermediate configurations thereof as limitations to the original disclosure and claimed particulars.

The meaning of "lower" in the substituents of the compounds described in this specification hereinafter means that the number of carbon atoms constituting the substituent is up to 10. For example, typically, the number of carbon atoms is 1 to 6, or 1 to 5, and also, for example, the number of carbon atoms is 1 to 4, or 1 to 3.

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

(RNA)
The RNA of the present disclosure has any one of the following base sequences (1), (2), (3), and (4).
The RNA of the present disclosure has the following sequence:
(1) A base sequence represented by SEQ ID NO: 1 or a base sequence having 90% or more identity thereto. (2) A base sequence of 10 or more consecutive bases from the base sequence of (1). (3) A base sequence represented by SEQ ID NO: 2 or a base sequence having 90% or more identity thereto. (4) A base sequence of 10 or more consecutive bases from the base sequence of (3).

Figure 1 shows the origin of the RNA consisting of the base sequence shown in SEQ ID NO: 1. As shown in Figure 1, the base sequence shown in SEQ ID NO: 1 is a part of the mRNA (accession number NM_000586, SEQ ID NO: 3) of human interleukin 2 (hIL-2). Furthermore, it is a part of the 3'-UTR.

As shown in Figure 1, the base sequence represented by SEQ ID NO:1 is 50 bases long and extends from base 971 to base 1020 of hIL-2 mRNA.

Figure 2 shows the origin of the RNA consisting of the base sequence shown in SEQ ID NO:2. As shown in Figure 2, the base sequence shown in SEQ ID NO:2 is a part of the mRNA (accession number NM_000572, SEQ ID NO:4) of human interleukin 10 (hIL-10). Furthermore, it is a part of the 3'-UTR.

As shown in FIG. 1, the base sequence represented by SEQ ID NO:1 is 50 bases long and extends from base 722 to base 771 of hIL-10 mRNA.

The RNA of the present disclosure may have the full length of the base sequence shown in SEQ ID NO: 1 or 2, or may have a base sequence that has 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 to the base sequence. Note that the identity of the base sequence can be determined, for example, by aligning two or more base sequences by blastn on the BLAST (BLAST: Basic Local Alignment Search Tool (nih.gov) page (https://blast.ncbi.nlm.nih.gov/Blast.cg) of NCBI under optimized conditions with highly similar sequence (megablast).

From the viewpoint of the present disclosure, a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 1 and 2 is considered to have 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. 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 is considered to have 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. Note that it may be preferable that the 5' end of a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 1 and 2 is U or is substituted with U.

The RNA of the present disclosure may be a part of the base sequence represented by SEQ ID NO: 1 or 2, or a base sequence having 90% or more identity to said base sequence (hereinafter, such sequences are collectively referred to as the base sequence represented by SEQ ID NO: 1, etc. and the base sequence represented by SEQ ID NO: 2, etc.). The base sequence represented by SEQ ID NO: 1, etc. and the base sequence represented by SEQ ID NO: 2, etc. each have a total length of 50 bases, and a part of the base sequences represented by SEQ ID NO: 1 and 2, etc. is, for example, a continuous sequence of 10 bases or more of the base sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2, etc., up to a maximum of 50 bases. The lower limit of the base length is, for example, 11 or more consecutive bases, 12 or more bases, 13 or more bases, 14 or more bases, 15 or more bases, 16 or more bases, 17 or more bases, 18 or more bases, 20 or more bases, 21 or more bases, 22 or more bases, 23 or more bases, 24 or more bases, 25 or more bases, 26 or more bases, 27 or more bases, 28 or more bases, 29 or more bases, 30 or more bases, 31 or more bases, 32 or more bases, 33 or more bases, 34 or more bases, 35 or more bases, 36 or more bases, 37 or more bases, 38 or more bases, 39 or more bases, 40 or more bases, 41 or more bases, 42 or more bases, 45 or more bases, 46 or more bases, 47 or more bases, 48 or more bases, or 49 or more bases. The upper limit of the base length is, for example, 49 bases or less, 48 bases or less, 47 bases or less, 46 bases or less, 45 bases or less, 44 bases or less, 43 bases or less, 42 bases or less, 41 bases or less, 40 bases or less, 39 bases or less, 38 bases or less, 37 bases or less, 36 bases or less, 35 bases or less, 34 bases or less, 33 bases or less, 32 bases or less, 31 bases or less, 30 bases or less, 29 bases or less, 28 bases or less, 27 bases or less, 26 bases or less, 25 bases or less, 23 bases or less, 22 bases or less, 21 bases or less, 20 bases or less, 19 bases or less, 18 bases or less, 17 bases or less, 16 bases or less, 15 bases or less, 14 bases or less, etc. A preferred base length can be set by arbitrarily combining these lower and upper limits. For example, it can be 10 to 50 bases, 15 to 50 bases, 20 to 50 bases, or 30 to 50 bases.

The RNA of the present disclosure may contain a base sequence derived from the base sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2, and may have any sequence at the 3' end and 5' end of these base sequences. The base sequence selected based on the base sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 can be appropriately selected, for example, based on the NK cell activation ability disclosed in the Examples described below.

Chemical Modifications in RNA
The RNA of the present disclosure may be appropriately chemically modified in consideration of stability, delivery, and the like in the body.

For example, the RNA of the present disclosure may be modified with a nucleotide unit represented by the following formula (1):

In the nucleotide unit represented by formula (1), R ¹ represents a hydroxyl group, a hydroxyl group in which the hydrogen atom is substituted with an alkyl group or an alkenyl group, or a halogen atom.

The alkyl group may be a saturated hydrocarbon group that is linear, branched, cyclic, or a combination thereof. In general, a lower alkyl group is 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 for example, methyl groups and ethyl groups are preferred, and 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 groups are particularly preferred. Examples of cyclic alkyl groups having 1 to 4 carbon atoms include cyclopropyl, cyclobutyl, and cyclopropylmethyl groups.

Alkenyl groups include saturated hydrocarbon groups that 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.

The halogen atom is not particularly limited, but may be, for example, a fluorine atom.

^R2 can represent ^NHR3 having a linking group, i.e., ^NHR3 is bonded to the 4' carbon atom via a linking group bonded to the nitrogen atom.

The linking group can be, for example, a divalent hydrocarbon group having one or more carbon atoms. In other words, examples of divalent hydrocarbon groups include alkylene groups having 1 to 8 carbon atoms and alkenylene groups having 2 to 8 carbon atoms.

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 or 2 to 3 carbon atoms. 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. Other examples include butane-1,2-diyl. Particularly preferred examples include ethylene, propane-1,3-diyl, and n-butane-1,1-diyl.

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

In the nucleotide unit represented by formula (1), 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, is preferred from the viewpoint of the NK cell activation ability of the RNA of the present disclosure.

Examples of R ³ include a hydrogen atom, an alkyl group, or an alkenyl group. Examples of the alkyl group include the alkyl groups already described, as well as lower alkyl groups, such as alkyl groups having 1 to 3 carbon atoms, alkyl groups having 1 or 2 carbon atoms, and methyl groups. Examples of the alkenyl group include the alkenyl groups already described, as well as lower alkenyl groups. When R ³ is a group such as a hydrogen atom, the linking group is preferably an alkylene group having 2 or more carbon atoms, such as 3 or more, or such as 4 or more, and for example 6 or less, such as 5 or less, or for example 4 or less.

When ^R3 is a hydrogen atom, ^R2 is _NH2 (amino group) having a linking group, that is, when the linking group is an alkylene group, it is an aminoalkyl group. When ^R2 in formula (1) is an aminoalkyl group such as an aminoethyl group, it is considered to be suitable as the RNA of the present disclosure.

n represents 0 or 1. When n is 0, the 3' position of the nucleotide unit is ^O- , and may be, for example, OH. When n is 1, ^X1 represents an oxygen atom or a sulfur atom, and ^X2 represents OH (or ^O- ) or SH (or ^S- ). When ^X1 or ^X2 is a sulfur atom or SH ( ^S- ), the nucleotide unit has the next nucleotide unit on 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 or the like, it may be a salt with an appropriate base.

B represents a purine base or a pyrimidine base. Purine bases are not particularly limited, but examples include purine-9-yl groups, as well as various substituted purine-9-yl groups such as 2,6-dimethoxypurine-9-yl and 2,6-dichloropurine-9-yl. Pyrimidine bases include 2-oxo-pyrimidin-1-yl and 2-oxo-pyrimidin-1-yl. Further examples include 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.

B may also be a modified base as described below.

The RNA of the present disclosure has at least one nucleotide unit represented by formula (1) in the 5'-terminal region within 10 bases from the 5'-terminal and/or in the 3'-terminal region within 10 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.

The 5'-terminal region is, for example, within 9 bases, 8 bases, 7 bases, 6 bases, 5 bases, 4 bases, 3 bases, 2 bases, or 1 base from the 5'-terminal. The 3'-terminal region is within 9 bases, 8 bases, 7 bases, 6 bases, 5 bases, 4 bases, 3 bases, 2 bases, or 1 base from the 3'-terminal.

The RNA of the present disclosure has at least one nucleotide unit represented by the nucleotide unit represented by formula (1) in the 5'-terminal region. The number of nucleotide units represented by the nucleotide unit represented by formula (1) 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 unit represented by the nucleotide unit represented by formula (1) is provided, for example, in a region within 6 bases from the 5'-terminal. It is believed that this makes it possible to effectively protect the 5'-terminal side. It may be effective to provide the nucleotide unit represented by formula (1) at least at the base position of the 5'-terminal or two bases from the 5'-terminal, for example.

The RNA of the present disclosure has at least one nucleotide unit represented by the nucleotide unit represented by formula (1) in the 3'-terminal region. The number of nucleotide units represented by formula (1) 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 unit represented by formula (1) is provided, for example, in a region within 6 bases from the 3'-terminal. It is believed that this makes it possible to effectively protect the 3'-terminal side. It may be effective to provide the nucleotide unit represented by formula (1) at least at the base position of the 3'-terminal or 2 bases from the 5'-terminal, for example.

The RNA of the present disclosure can have at least one nucleotide unit represented by formula (1) in both the 3'-terminal region and the 5'-terminal region. The number of nucleotide units represented by formula (1) in each of these terminal regions is, for example, 1 to 5, or, for example, 1 to 4, or, for example, 1 to 3, or, for example, 1 to 2, or, for example, 1. In each of these terminal regions, it may be preferable to have one or two nucleotide units represented by the nucleotide unit represented by formula (1) within 3 bases or within 2 bases from each end. Furthermore, it may be preferable that at least one or all of the nucleotide units represented by formula (1) in each of these terminal regions have a fluorine atom at the 2'-position of ribose and an aminoethyl group at the 4'-position. 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, for example, cytosine or uracil.

In view of the above, for example, the following aspects may be preferable for the RNA of the present disclosure.
[1] A nucleotide unit represented by the formula (1) is a nucleotide unit in which n is 1, R ¹ is a fluorine atom, and R ² is an aminoethyl group;
[2] [1], which has at least one nucleotide unit represented by formula (1) within 2 bases from the 3'-terminus of the 3'-terminus region and at least one nucleotide unit represented by formula (1) within 2 bases from the 5'-terminus of the 5'-terminus region, wherein the base sequence has 90% or more identity with a base sequence of 10 to 50 consecutive bases in the base sequence represented by SEQ ID NO: 1 or 2. [3] [1], which has 90% or more identity with a base sequence of 20 to 50 consecutive bases in the base sequence represented by SEQ ID NO: 1 or 2. [4] [1], which has at least one nucleotide unit represented by formula (1) within 2 bases from the 3'-terminus of the 3'-terminus region and at least one nucleotide unit represented by formula (1) within 2 bases from the 5'-terminus of the 5'-terminus region. In the above-mentioned base sequence [5] [1], the base sequence has 90% or more identity with a base sequence of 30 to 50 consecutive bases in the base sequence represented by SEQ ID NO: 1 or 2. In the above-mentioned base sequence [6] [1], the base sequence has 90% or more identity with a base sequence of 40 to 50 consecutive bases in the base sequence represented by SEQ ID NO: 1 or 2. In the above-mentioned base sequence [7] [1] to [6], the base of the nucleotide unit is cytosine or uracil.

The 5'-terminal region and 3'-terminal region of the RNA of the present disclosure may have a structure in which ribose is linked by phosphorothioate bonds, in addition to having a nucleotide unit represented by formula (1). Linkage by phosphorothioate bonds may also be provided in the nucleotide unit represented by formula (1), but it may be advantageous for nucleotides corresponding to other bases that are not substituted by the nucleotide unit to also be linked by phosphorothioate bonds.

The number and the position of phosphorothioate bonds in the 5'-terminal region and the 3'-terminal region are not particularly limited. For example, the 5'-terminal region and the 3'-terminal region may have 1 to 8, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 consecutive phosphorothioate bonds, including the first linking portion from the 5'-terminal and the 3'-terminal, respectively, or may have 1 to 4, for example, 1 to 2 bases apart from the first linking portion from the terminal.

The RNA of the present disclosure has at least one nucleotide unit represented by formula (1) in the 5'-terminal region and/or 3'-terminal region, and the other nucleotide units may be natural nucleotides, i.e., nucleotide units having natural ribose, or 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 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 its 5'-terminal region and/or 3'-terminal region have chemically modified ribose having a methoxy group at the 2'-position of ribose.

In view of the above, for example, in the RNA of the present disclosure, in any of the above aspects [1] to [6], 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.

The RNA of the present disclosure may further be chemically modified to convert uridine in the RNA to a uridine analog. The chemical modification to convert uridine to a uridine analog is not particularly limited in terms of 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 may be uridine analogs, for example, 90% or more, or for example, 100% or more.

Nucleobases and nucleosides containing uridine analogs include, but are not limited to, uridine analogs selected from the group consisting of uridine 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. For example, the uridine analog is 1-methyl-pseudouridine.

The RNA of the present disclosure may further comprise a chemical modification that converts cytidine in the RNA into a cytidine analog. The chemical modification that converts cytidine into a cytidine analog is not particularly limited in terms of the percentage of the total number of cytidines in the RNA of the present disclosure. For example, 80% or more of the total number of cytidines may be cytidine analogs, for example, 90% or more, or for example, 100% or more.

Nucleobases and nucleosides containing cytidine analogues 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 ... These include za-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.

The RNA of the present disclosure may further include a chemical modification that converts adenine in the RNA to an adenine analog. The chemical modification that converts adenine to an adenine analog is not particularly limited in terms of the percentage of the total number of adenines in the RNA of the present disclosure. For example, 80% or more of the total number of adenines may be adenine analogs, for example, 90% or more, or for example, 100% or more.

Nucleobases and nucleosides containing adenine analogues 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-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- These include 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.

The RNA of the present disclosure may further be chemically modified to convert guanine in the RNA to a guanine analogue. The chemical modification to convert guanine to a guanine analogue is not particularly limited in terms of the percentage of the total number of guanines in the RNA of the present disclosure. For example, 80% or more of the total number of guanines may be guanine analogues, for example, 90% or more, or for example, 100% or more.

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-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 Anosine, 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 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.

The RNA of the present disclosure may optionally include known modifications in RNA pharmaceuticals, such as at least one translation initiation sequence, such as a Kozak sequence, an internal ribosome entry site (IRES), and/or a fragment thereof.

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

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' terminus via a 5'-5'-triphosphate bond. The 5' cap may be methylated, e.g. m7GpppN, where N is the terminal 5' nucleotide of the 5' capped nucleic acid.

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

The RNA of the present disclosure may be an mRNA or a portion thereof. If it is an mRNA or a portion thereof, it may include a polyA tail.

The RNA of the present disclosure may be an RNAi inducer, an RNAi agent, an siRNA, an shRNA, an miRNA, an antisense RNA, an RNA, an aptamer, a vector, etc.

The RNA disclosed herein may have a labeling molecule, such as a fluorescent substance or a chromogenic substance, attached to a portion of it, as necessary.

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

The RNA of the present disclosure can be used as an activator of NK cells and T cells or an agent for activating cell migration. When the RNA of the present disclosure is administered to cells that overexpress the ZC3H12D protein (e.g., 786-O cells in FIG. 4), it can activate the migration ability of NK cells, induce the anti-metastatic activity of the cells, and, in the case of natural killer (NK) cells, increase the tumor-destructive activity. In the case of T cells, it can also increase the tumor-destructive activity. Thus, the RNA of the present disclosure can be used in a method for activating NK cells and CTL cells (cytotoxic T cells) and a method for activating cell migration, etc., by administering it to in vitro cells, humans, or non-human animals.

The various embodiments of the RNA disclosed herein can be applied, either alone or in combination, to each of the following aspects of the RNA disclosed herein:

(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, thereby exerting an antitumor effect.

The RNA disclosed herein as an active ingredient of an antitumor agent is 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.

Tumors include head and neck cancer, esophageal cancer, stomach 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, mesothelioma, etc.

Immune cell activation can be evaluated by IFNγ production in immune cells, or improved migration ability of immune cells or tumor cells expressing ZC3H12D protein. Antitumor activity can be evaluated by known methods for evaluating antitumor activity.

(Activator (stimulator) for 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 a human or non-human animal in vitro. The above-described embodiments of the antitumor agent are applicable to the cells, immune cells, and immune cell activation.

(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 anti-tumor activity of the cells and inhibit the metastasis of tumor cells. The cells, immune cells, activation of immune cells, and anti-tumor activity are the same as those described above for the anti-tumor agent.

Inhibition of tumor cell metastasis can be evaluated by measuring the number of metastatic tumor cells, etc.

Therapy intended to inhibit the metastasis of primary cancers using metastasis inhibitors is performed in combination with or independently of treatment for the primary cancer. For example, when metastasis inhibitors are used in combination, they can be used before, after, or simultaneously with one or more of the following: surgical treatment for the primary cancer, treatment with anticancer drugs, radiation therapy (X-rays, proton beams, heavy particle beams), and electromagnetic wave therapy (radio waves). Combination with surgical treatment is one of the preferred embodiments.

Typically, the following applications are mentioned:
(a) When the 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, electromagnetic therapy, etc. (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 is 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.

(Enhancer for enhancing the immunostimulating effect of an immunostimulant and a 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 RNA can be used 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. The above-mentioned embodiments of the antitumor agent and metastasis inhibitor are applicable to the activation of cells, immune cells, and the suppression of immune cell activation, antitumor activity, and metastasis.

The immunostimulant used in combination may be a known immunostimulant. Also, the metastasis inhibitor used in combination may be a known metastasis inhibitor.

The antitumor agents used in combination include 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, and also include salts of the compounds. These antitumor agents are well known and commercially available products can be used.

The administration method of the above-mentioned concomitant drug and the enhancer of the present disclosure is not particularly limited, and they may be administered simultaneously, sequentially, or at intervals, so long as the desired enhancing effect of the enhancer of the present disclosure is achieved. The order of administration of these compositions is also not particularly limited, and the antitumor agent may be administered before, simultaneously with, or after the RNA.

The drug to be used in combination with the enhancer of the present disclosure may be used in combination as an individual drug, or may be provided as a combination drug or drug kit with a prescribed usage method.

(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, as well as 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.

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

When using the RNA of the present disclosure as an active ingredient of a medicine, a pharmaceutical carrier can be added as necessary, and various administration forms can be adopted depending on the preventive or therapeutic purpose. The form of the antitumor agent containing the above-mentioned RNA may be, for example, any of injections, suppositories, oral preparations, ointments, eye drops, etc., and preferably, an injection (intravenous injection, etc.) is adopted. Each of these administration forms can be produced by a formulation method known and commonly used by those skilled in the art.

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

The agents and pharmaceutical compositions of the present invention are administered to mammals, preferably humans.

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 compound is preferably administered in a dose of 10 ng to 1 mg, more preferably 100 ng to 100 μg, and even more preferably 1 μg to 10 μg per day for an adult (body weight 50 kg).

The amount of RNA to be incorporated into each of the above dosage unit forms is set appropriately depending on the properties of the RNA used, the patient's symptoms, the dosage form, etc.

The disclosures of all patent applications and publications cited herein are hereby incorporated by reference in their entirety.

The present disclosure will be explained in more detail below with reference to examples, but the present disclosure is not limited to these.

Unless otherwise stated, the following method was followed:

(1) 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.

(2) 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: 8) and 5′-CTCGAGTTAAGGATCCCCCAACGGAGCACC-3′ (SEQ ID NO: 9), 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.

(3) 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. An aliquot (50 μL) of the cell suspension (2 × 10 ⁵ to 2 × 10 ⁶ cells/mL) was inoculated into each of the upper wells and incubated for 3.5 h.

(Synthesis of chemically modified RNA)
In the base sequence shown in FIG. 3 (a modified sequence of a partial sequence of human IL-2 (a base sequence represented by SEQ ID NO: 1 in which A at the 5' end is replaced with U) (SEQ ID NO: 5), a partial sequence of human IL-10 (SEQ ID NO: 2), and a partial sequence of human IL-1β (SEQ ID NO: 6)), three types of RNAs (HumanIL-2-5_OMe_AE, HumanIL-10-1_OMe_AE, and HumanIL1β_OMe_AE) having two types of chemical modifications 2'-OMe and 2'-F_4'-AE shown in FIG. 3 were synthesized according to a standard method. Note that the nucleotide unit having a fluorine atom at the 2' position and an aminoethyl group at the 4' position was synthesized in its entirety according to the phosphoramidite method in accordance with JP 2019-10035 A and the like. Note that, as a control, a 50mer PolyA (PolyA_50mer_OMe_AE) (SEQ ID NO: 7) chemically modified as shown in formula (1) was also synthesized.

(Confirmation of cell migration activation effect of chemically modified RNA in ZC3H12D overexpressing cells)
The three types of chemically modified RNAs synthesized in Example 1 and 50mer PolyA_50mer_OMe_AE as a control were administered to the medium containing 786-O cells overexpressing ZC3H12D protein, and the cell migration ability was measured by the average number of migrated cells. 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. An aliquot (50 μL) of the cell suspension (1 × 10 ⁶ /ml) was placed in the upper well, and the medium containing each of the above RNAs at a concentration of 10 ng/ml was placed in the lower well. The wells were incubated for 3.5 hours, and the number of cells that migrated to the lower well was counted. The results are shown in Figure 4.

As shown in Figure 4, HumanIL-2-5_OMe_AE and HumanIL-10-1_OMe_AE showed significantly improved migration activity compared to no additive, control and HumanIL1β_OMe_AE. From the above, since HumanIL-2-5_OMe_AE and HumanIL-10-1_OMe_AE have 1.2 to 1.5 times higher migration activation activity than HumanIL1β_OMe_AE with similar modification, it was found that RNA containing the base sequence in HumanIL-2-5_OMe_AE and HumanIL-10-1_OMe_AE strongly improves the migration activation activity of NK cells. It was found that this improvement in migration activation activity contributes to antitumor activity, tumor metastasis activity, immunostimulatory activity, and enhanced activity of antitumor activity and immunostimulatory activity.

(Confirmation of cell migration activation effect of chemically modified RNA in ZC3H12D overexpressing cells 2)
The two types of RNAs having chemical modifications 2'-OMe and 2'-F_4'-AE (HumanIL1β_OMe_AE and HumanIL-2-5_OMe_AE) synthesized in Example 1 and the control were examined for their migration activation effect in accordance with the migration assay of Example 2, with the HumanIL1β_OMe_AE concentration being fixed at 10 ng/ml, while the HumanIL-2-5_OMe_AE concentration was varied from 0.1 to 10 ng/ml. The results are shown in Figure 5.

As shown in Figure 5, HumanIL-2-5_OMe-AE showed high migration activation ability even at low concentrations (1ng/ml to 5ng/ml) compared to no additive (none), the control, and HumanIL1β_OMe_AE (10ng/ml). These results indicate a significant improvement in migration activity. These results indicate that RNA containing a base sequence with chemical modifications in HumanIL-2-5_OMe_AE strongly improves the migration activation ability of NK cells.

(Killing assay of human colon cancer cell line DLD-1 by CD56+ ^CD3- NK cells in human peripheral blood)
Human colon cancer cell line DLD-1 was seeded on a culture dish, and then CD56 ⁺ CD3 ^- NK cells isolated from human peripheral blood using a cell sorter in the same number as the DLD-1 cells were added and cultured for 24 hours. After culture, the DLD-1 cells were stained with Zombie Green dye. Cells stained with Zombie Green dye are dead cells. The more cells stained with Zombie Green dye, the more dead cells there are.

The Zombie Green staining rate (%) was measured when NK cells were activated with 10 ng/ml of various chemically modified RNAs for 24 hours in the presence of 200 IU/ml of IL2 before adding the NK cells to the culture medium. The results are shown in Figure 6. In Figure 6, "NK (-)" indicates the case where no RNA was added and no NK cells were added, "No stimulation" indicates the case where no RNA was added and no activated NK cells were added, and "IL-12" indicates the case where NK cells were activated with IL-12 protein instead of RNA.

As shown in Figure 6, HumanIL-2-5_OMe_AE was found to have a higher killing ability than the control, HumanIL1β_OMe_AE, and no additive (none).

SEQ ID NO: 8, 9: Primers

Claims (10)

A cell activator comprising, as an active ingredient, RNA containing any one of the base sequences selected from the group consisting of (1), (2), (3) and (4) below.
(1) A base sequence represented by SEQ ID NO: 1 or a base sequence having 90% or more identity thereto. (2) A base sequence of 10 or more consecutive bases from the base sequence of (1). (3) A base sequence represented by SEQ ID NO: 2 or a base sequence having 90% or more identity thereto. (4) A base sequence of 10 or more consecutive bases from the base sequence of (3). An antitumor agent comprising, as an active ingredient, RNA comprising any one of the base sequences selected from the group consisting of (1), (2), (3) and (4) below:
(1) A base sequence represented by SEQ ID NO: 1 or a base sequence having 90% or more identity thereto. (2) A base sequence of 10 or more consecutive bases from the base sequence of (1). (3) A base sequence represented by SEQ ID NO: 2 or a base sequence having 90% or more identity thereto. (4) A base sequence of 10 or more consecutive bases from the base sequence of (3). The antitumor agent according to claim 2, comprising as an active ingredient an RNA having the base sequence and at least one nucleotide unit represented by the following formula (1) in its 3'-terminal region within 10 bases from the 3'-terminus and/or in its 5'-terminal region within 10 bases from the 5'-terminus.

(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 represents NHR ³ having a linking group, the linking group being 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 represents OH (or O ⁻ ) or SH (or S ⁻ ); and B represents a purine base or a pyrimidine base.) The antitumor agent according to claim 3, wherein the RNA has 1 to 5 nucleotide units in the 3'-terminal region and/or the 5'-terminal region. The antitumor agent according to claim 3 or 4, wherein the nucleotide unit is a nucleotide unit represented by formula (1), in which R ¹ is a methoxy group or a fluorine atom, and R ² is an aminoethyl group. The antitumor agent according to claim 3 or 4, wherein, in the nucleotide unit, n is 1, and the RNA has a nucleotide unit in which either ^X1 or ^X2 in the formula (1) is S or SH ( ^S- ) in each of the 3'-terminal region and/or the 5'-terminal region. The antitumor agent according to claim 3 or 4, 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. An immunostimulant comprising, as an active ingredient, any one of base sequences selected from the group consisting of (1), (2), (3) and (4) below.
(1) A base sequence represented by SEQ ID NO: 1 or a base sequence having 90% or more identity thereto. (2) A base sequence of 10 or more consecutive bases from the base sequence of (1). (3) A base sequence represented by SEQ ID NO: 2 or a base sequence having 90% or more identity thereto. (4) A base sequence of 10 or more consecutive bases from the base sequence of (3). A tumor metastasis inhibitor comprising, as an active ingredient, RNA comprising any one of the base sequences selected from the group consisting of (1), (2), (3) and (4) below:
(1) A base sequence represented by SEQ ID NO: 1 or a base sequence having 90% or more identity thereto. (2) A base sequence of 10 or more consecutive bases from the base sequence of (1). (3) A base sequence represented by SEQ ID NO: 2 or a base sequence having 90% or more identity thereto. (4) A base sequence of 10 or more consecutive bases from the base sequence of (3). 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 any one of the base sequences selected from the group consisting of (1), (2), (3) and (4) below.
(1) A base sequence represented by SEQ ID NO: 1 or a base sequence having 90% or more identity thereto. (2) A base sequence of 10 or more consecutive bases from the base sequence of (1). (3) A base sequence represented by SEQ ID NO: 2 or a base sequence having 90% or more identity thereto. (4) A base sequence of 10 or more consecutive bases from the base sequence of (3).