Composition for treating cancer

Abstract

The present disclosure provides a composition that is for treating cancer and that contains at least one substance for breaking down a stem-loop structure in the 3'-untranslated region of Nfkbiz mRNA. Said stem-loop structure is at least one stem-loop structure selected from a first stem-loop structure formed in a region corresponding to positions 34-50 in SEQ ID NO: 9, a second stem-loop structure formed in a region corresponding to positions 100-116 in SEQ ID NO: 9, and a third stem-loop structure formed in a region corresponding to positions 129-143 in SEQ ID NO: 9.

Description

Compositions for treating cancer

[0001] This patent application claims priority to Japanese Patent Application No. 2022-088931, the entire contents of which are incorporated herein by reference. The present disclosure relates to compositions for treating cancer.

[0002] Cancer immunotherapy has made rapid progress with the development of immune checkpoint inhibitors, but only about 30% of patients respond to treatment. Furthermore, cancer immunotherapy using chimeric antigen receptor (CAR) T cells has proven highly effective against some blood cancers and is being used clinically, but its effectiveness against solid cancers is insufficient, and further improvement is needed.

[0003] IκBζ is a transcription factor that positively regulates immune responses. IκBζ belongs to the IκB family and is rapidly induced upon activation of TLR / IL-1R / IL-18R signaling. IκBζ binds to the NF-κB subunit p50, enhances transcription through chromatin remodeling, and positively regulates natural killer cell (NK cell) function, Th17 differentiation, and antibody production by B cells. Expression of Nfkbiz, which encodes IκBζ, is regulated by mRNA degradation.

[0004] Regnase-1 suppresses excessive immune responses in innate immune cells such as macrophages and dendritic cells by degrading mRNA encoding inflammatory cytokines such as interleukin (IL)-6 and molecules involved in inflammation induction. Regnase-1 also suppresses excessive T cell activation in adaptive immune T cells by degrading mRNA encoding molecules involved in T cell activation. Regnase-1 recognizes stem-loop structures present in the 3' untranslated region (3'UTR) of mRNA and degrades the mRNA (Patent Document 1, Non-Patent Document 1). Non-Patent Document 1 discloses that the stem-loop structure present in the 3'UTR of Nfkbiz mRNA is necessary for Regnase-1 to suppress Nfkbiz expression.

[0005] Regnase-3 is an RNase belonging to the same family as Regnase-1. Unlike Regnase-1, Regnase-3 is highly expressed in immune cells, particularly macrophages, and is transcriptionally regulated by TLR and IFN signals. Although its direct target in macrophages is unknown, Regnase-3 has been confirmed to bind to and degrade mRNAs such as Regnase-1 in vitro. Therefore, Regnase-3 is an RNase essential for immune homeostasis and is thought to be an important regulator of the TLR / IFN pathway in macrophages (Non-Patent Document 2).

[0006] International Publication No. 2019 / 182055

[0007] Mino, T. et al., Cell 161, 1058-1073 (2015)von Gamm M., et al., J Exp Med (2019) 216 (7): 1700-1723

[0008] The present application aims to provide a new method for treating cancer.

[0009] The inventors have found that disrupting a specific stem-loop structure out of five stem-loop structures conserved among species in the 3'UTR of Nfkbiz mRNA inhibits the degradation of Nfkbiz mRNA by Regnase-1 and Regnase-3, increases Nfkbiz expression, activates immune cells, and enhances their cytotoxic activity against cancer cells.

[0010] Thus, in one aspect, the present disclosure provides a composition for immunostimulation comprising at least one substance that disrupts a stem-loop structure in the 3' untranslated region of Nfkbiz mRNA.

[0011] In certain aspects, the present disclosure provides compositions for treating cancer comprising at least one agent that disrupts a stem-loop structure in the 3' untranslated region of Nfkbiz mRNA.

[0012] The present disclosure enables immune stimulation in a subject, and also enables cancer treatment through immune stimulation.

[0013] The genotype of the CreERT2+ mouse used in the examples and the experimental schedule are shown. A photograph of a spleen isolated from a mouse 14 days after gene deficiency is shown. CD62L in mouse spleen cells. ＋ CD44 － Naive T cells and CD62L － CD44 ＋ FACS plots showing effector CD4-positive T cells (top row) and the amount of CD69 expression on CD4-positive T cells (bottom row). ＋ CD44 － Naive T cells, central memory T cells and CD62L － CD44 ＋This is a FACS plot showing effector CD8-positive T cells. The interspecies alignment of nucleotides 29-148 in the 3'UTR of the Nfkbiz gene and the regions that can form secondary structures (SL2-5) are shown. A schematic representation of stem-loop structures (SL4 and 5) and the disruption of secondary structures by antisense nucleic acids against these structures is shown. This figure shows the luciferase activity of HeLa cells transfected with a reporter construct and antisense nucleic acid containing the 3'UTR of the mouse Nfkbiz gene shown in the figure. **p<0.01, ***p<0.001. This figure shows the luciferase activity of HeLa cells transfected with a reporter construct and antisense nucleic acid containing the 3'UTR of the human NFKBIZ gene shown in the figure. *p<0.05. This is a schematic representation of the changes in Nfkbiz gene expression induced by antisense nucleic acids. This is a schematic representation of the antitumor effect of cytotoxic T cells. Figure 1 shows the workflow of an in vitro T cell experiment using OT-I CD8-positive T cells. The percentage (upper) and mean fluorescence intensity (MFI) (lower) of IFNγ-, granzyme-b-, and TNF-producing CD8 T cells among OT-I CD8-positive T cells transfected with antisense nucleic acids are shown. ****p<0.0001. Figure 2 shows the antitumor activity of OT-I CD8 T cells transfected with antisense nucleic acids against the B16-OVA cell line. The vertical axis represents cytotoxicity, and the horizontal axis represents the ratio of OT-I CD8 T cells (effector) to B16-OVA cells (target). **p<0.01, ***p<0.001, ***p<0.0001. Figure 3 shows the time-dependent changes in Nfkbiz protein expression in mouse CD8-positive T cells transfected with antisense nucleic acids. Figure 1 shows the change in Nfkbiz expression over time in mouse bone marrow-derived macrophages transfected with antisense nucleic acid. **p<0.01, ***p<0.001, ****p<0.0001. Figure 2 shows the expression of Nfkbiz protein in mouse bone marrow-derived macrophages transfected with antisense nucleic acid. Figure 3 shows the change in Nfkbiz expression in mouse bone marrow-derived macrophages transfected with antisense nucleic acid in the presence of actinomycin D. *p<0.05, ***p<0.001. Figure 4 shows the IL-6 concentration in the culture supernatant of mouse bone marrow-derived macrophages transfected with antisense nucleic acid. ***p<0.001, ****p<0.0001.Figure 1 shows a schematic representation of antisense nucleic acids targeting the stem-loop structure (SL4, SL5, or both SL4 and SL5) in the 3'UTR of the Nfkbiz gene. Figure 2 shows the expression level of Nfkbiz in mouse bone marrow-derived macrophages transfected with antisense nucleic acids. *p<0.05, ***p<0.001. Figure 3 shows the expression of Nfkbiz protein in mouse bone marrow-derived macrophages transfected with antisense nucleic acids. Figure 4 shows the workflow of an in vivo tumor model using the B16 melanoma cell line. Figure 5 shows the change in tumor size over time in an in vivo tumor model transfected with mouse CD8-positive T cells transfected with antisense nucleic acids. Figure 6 shows FACS plots showing the expression of PD-1, LAG-3, TOX, and TCF-1 in mouse CD8-positive T cells transfected with antisense nucleic acids. Figure 7 shows the expression of NFKBIZ protein in human peripheral blood mononuclear cells (PBMCs) transfected with antisense nucleic acids. * indicates a non-specific band.

[0014] As used herein, when a numerical value is accompanied by the term "about," it is intended to encompass a range of ±10% of that value. For example, "about 20" is intended to include "18 to 22." A range of numerical values ​​includes all values ​​between and at the endpoints. "About" in reference to a range applies to both endpoints of the range. Thus, for example, "about 20 to 30" is intended to include "18 to 33."

[0015] Unless otherwise specified, terms used in this disclosure have the meanings commonly understood by those skilled in the art of organic chemistry, medicine, pharmacology, molecular biology, microbiology, etc. Below, definitions of some terms used in this disclosure are provided, but these definitions take precedence over common understandings in this disclosure.

[0016] Nfkbiz may be from any species, typically a mammal (e.g., human, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, etc.), particularly human or mouse Nfkbiz. Representative amino acid sequences of IκBζ encoded by Nfkbiz are registered under GenBank accession numbers NP_001005474.1 (human, SEQ ID NO: 1) and NP_001152866.1 (mouse, SEQ ID NO: 2). In the context of the present disclosure, IκBζ may be a protein having at least about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or more identity to the amino acid sequence of SEQ ID NO: 1 or 2, so long as the function of IκBζ is maintained.

[0017] The "3' untranslated region of Nfkbiz mRNA" or "3'UTR of Nfkbiz mRNA" refers to the 3' untranslated region of Nfkbiz mRNA. For example, the 3'UTR is the region from the nucleotide immediately following the stop codon of the mRNA to the 3' end. In the present disclosure, Nfkbiz mRNA encompasses mRNAs containing the nucleotide sequence encoding IκBζ as defined above and the nucleotide sequence of its 3'UTR. Examples of nucleotide sequences of the 3'UTR of Nfkbiz mRNA are described below. Representative nucleotide sequences of Nfkbiz mRNA are registered under GenBank accession numbers NM_001005474.3 (human, SEQ ID NO: 3) and NM_001159394.1 (mouse, SEQ ID NO: 4).

[0018] Examples of nucleotide sequences of the 3'UTR of Nfkbiz mRNA include sequences comprising at least the sequence set forth in SEQ ID NO:5 (human) or SEQ ID NO:6 (mouse). For example, the 3'UTR of Nfkbiz mRNA comprises the nucleotide sequence set forth in SEQ ID NO:7 (human) or SEQ ID NO:8 (mouse). For example, the 3'UTR of Nfkbiz mRNA is the nucleotide sequence set forth in SEQ ID NO:9 (human) or SEQ ID NO:10 (mouse). In some embodiments, the 3'UTR of Nfkbiz mRNA comprises a nucleotide sequence having at least about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or more identity to the nucleotide sequence of SEQ ID NO:5 or 6. In some embodiments, the 3'UTR of the Nfkbiz mRNA comprises a nucleotide sequence having at least about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or more identity to the nucleotide sequence of SEQ ID NO: 7 or 8. In some embodiments, the 3'UTR of the Nfkbiz mRNA consists of a nucleotide sequence having at least about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or more identity to the nucleotide sequence of SEQ ID NO: 9 or 10.

[0019] In the present disclosure, the identity of amino acid sequences or nucleotide sequences refers to the degree of similarity between two protein or nucleic acid sequences. Sequence identity is calculated by aligning the two sequences to be compared optimally (maximizing the amino acid or nucleotide identity) and determining the matching amino acids or nucleotides. If the two sequences are completely identical, the sequence identity is 100%. For example, sequence identity can be calculated using the following formula: Sequence identity (%) = [(number of identical residues) / (alignment length)] × 100. Algorithms for obtaining alignment and sequence identity include various algorithms commonly available to those skilled in the art (e.g., BLAST algorithm, FASTA algorithm). Sequence identity can be determined using sequence analysis software such as BLAST and FASTA.

[0020] The term "stem-loop structure" refers to a single-stranded nucleic acid structure in which complementary sequences present in two separate regions within the molecule are joined to form a stem portion, and the sequence sandwiched between the two regions forms a loop portion. The 3'UTR of Nfkbiz mRNA contains five stem-loop structures conserved across species. For ease of explanation, the five stem-loop structures will be referred to as SL1, SL2, SL3, SL4, and SL5, in order from the 5' end of the 3'UTR of Nfkbiz mRNA. In the present disclosure, it is preferred that at least one of SL2, SL4, and SL5 be disrupted. The nucleotide sequence of SEQ ID NO: 5 or 6 above includes SL4 and SL5. The nucleotide sequence of SEQ ID NO: 7 or 8 above includes SL2, SL3, SL4, and SL5. The nucleotide sequence of SEQ ID NO: 9 or 10 above is the entire length of the 3'UTR of Nfkbiz mRNA, and includes all stem-loop structures (SL1, SL2, SL3, SL4 and SL5).

[0021] In one embodiment, the stem-loop structure to be disrupted is a first stem-loop structure (SL2) formed in a region corresponding to positions 34 to 50 of SEQ ID NO:9 (human), a second stem-loop structure (SL4) formed in a region corresponding to positions 100 to 116 of SEQ ID NO:9, and / or a third stem-loop structure (SL5) formed in a region corresponding to positions 129 to 143 of SEQ ID NO:9 in the 3'UTR of any Nfkbiz mRNA. In one embodiment, the stem-loop structure to be disrupted is a first stem-loop structure (SL2) formed in a region corresponding to positions 35 to 51 of SEQ ID NO: 10 (mouse) in the 3'UTR of any Nfkbiz mRNA, a second stem-loop structure (SL4) formed in a region corresponding to positions 101 to 117 of SEQ ID NO: 10, and / or a third stem-loop structure (SL5) formed in a region corresponding to positions 130 to 144 of SEQ ID NO: 10.

[0022] In some embodiments, the stem-loop structure that is disrupted is at least one selected from the second stem-loop structure and the third stem-loop structure, hi some embodiments, the stem-loop structure that is disrupted is the second stem-loop structure and the third stem-loop structure.

[0023] A region in a second nucleotide sequence corresponding to a region in a first nucleotide sequence means a region in the second nucleotide sequence that matches the region in the first nucleotide sequence when the first nucleotide sequence and the second nucleotide sequence are optimally aligned.

[0024] The "substance that disrupts a stem-loop structure" is not particularly limited as long as it inhibits complementary binding within the stem-loop structure. It may be a substance that inhibits complementary binding between at least one, two, or three pairs of nucleotides. Examples of such a substance include an oligonucleotide (antisense nucleic acid) that binds to a nucleotide sequence that forms a stem-loop structure, or a substance for modifying a nucleotide sequence that forms a stem-loop structure using genome editing technology.

[0025] In one embodiment, the substance that disrupts the stem-loop structure is an antisense nucleic acid. The antisense nucleic acid can inhibit complementary binding within the stem-loop structure by binding to at least a portion of the region that forms the stem-loop structure in the 3'UTR of Nfkbiz mRNA, for example, at least one, two, or three nucleotides that form the stem portion of the stem-loop structure. For example, the antisense nucleic acid comprises a sequence complementary to a sequence comprising at least two or three, for example, three consecutive nucleotides that form the stem portion of the stem-loop structure. The consecutive nucleotides may be adjacent to the loop portion of the stem-loop structure, for example, positions 35 to 37, 47 to 49, 104 to 106, 110 to 112, 132 to 134, and 138 to 140 in SEQ ID NO: 9, and positions 36 to 38, 48 to 50, 105 to 107, 111 to 113, 133 to 135, and 139 to 141 in SEQ ID NO: 10. Antisense nucleic acids that do not themselves form stem-loop structures, hairpin structures, or multimers such as dimers are preferred. An antisense nucleic acid that disrupts one stem-loop structure consists of, for example, 10 to 30, 15 to 25, 18 to 22, or 19 to 21 nucleotides. An antisense nucleic acid that disrupts both the second stem-loop structure and the third stem-loop structure consists of, for example, 25 to 35, 25 to 30, or 25 to 27 nucleotides.

[0026] In one embodiment, the antisense nucleic acid is selected from: (a-1) an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 35 to 37 of SEQ ID NO:9, from the nucleotide sequence from positions 8 to 48 of SEQ ID NO:9; and (a-2) an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 47 to 49 of SEQ ID NO:9, from the nucleotide sequence from positions 36 to 76 of SEQ ID NO:9.

[0027] In one embodiment, the oligonucleotide (a-1) has at least about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or more sequence identity to SEQ ID NO: 11 (5'-taactgacagtgagtgttgc-3'). In another embodiment, the oligonucleotide (a-1) consists of a nucleotide sequence in which one or several, for example, two or three, nucleotides have been deleted, substituted, added, or inserted in the nucleotide sequence of SEQ ID NO: 11. In one embodiment, the oligonucleotide (a-1) binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 11. In one embodiment, the oligonucleotide (a-1) consists of a nucleotide sequence having about 90% or more identity to SEQ ID NO: 11. In one embodiment, the oligonucleotide (a-1) comprises the nucleotide sequence of SEQ ID NO: 11. In one embodiment, the oligonucleotide (a-1) consists of the nucleotide sequence of SEQ ID NO: 11.

[0028] In some embodiments, the oligonucleotide (a-2) has at least about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or more sequence identity to SEQ ID NO: 12 (5'-tacatcaggactgcctaact-3'). In another embodiment, the oligonucleotide (a-2) consists of a nucleotide sequence in which one or several, for example, two or three, nucleotides have been deleted, substituted, added, or inserted in the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the oligonucleotide (a-2) binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the oligonucleotide (a-2) consists of a nucleotide sequence having about 90% or more identity to SEQ ID NO: 12. In some embodiments, the oligonucleotide (a-2) comprises the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the oligonucleotide (a-2) consists of the nucleotide sequence of SEQ ID NO: 12.

[0029] In one embodiment, the antisense nucleic acid is selected from: (b-1) an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 104 to 106 of SEQ ID NO:9, from the nucleotide sequence from positions 77 to 111 of SEQ ID NO:9; and (b-2) an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 110 to 112 of SEQ ID NO:9, from the nucleotide sequence from positions 105 to 139 of SEQ ID NO:9.

[0030] In one embodiment, the oligonucleotide (b-1) has at least about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or more sequence identity to SEQ ID NO: 13 (5'-atagaaacaacttacatttg-3'). In another embodiment, the oligonucleotide (b-1) consists of a nucleotide sequence in which one or several, for example, two or three, nucleotides have been deleted, substituted, added, or inserted in the nucleotide sequence of SEQ ID NO: 13. In one embodiment, the oligonucleotide (b-1) binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 13. In one embodiment, the oligonucleotide (b-1) consists of a nucleotide sequence having about 90% or more identity to SEQ ID NO: 13. In one embodiment, the oligonucleotide (b-1) comprises the nucleotide sequence of SEQ ID NO: 13. In one embodiment, the oligonucleotide (b-1) consists of the nucleotide sequence of SEQ ID NO: 13.

[0031] In some embodiments, the oligonucleotide (b-2) has at least about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or more sequence identity to SEQ ID NO: 14 (5'-ctaaatatgtttgtttcata-3'). In another embodiment, the oligonucleotide (b-2) consists of a nucleotide sequence in which one or several, for example, two or three, nucleotides have been deleted, substituted, added, or inserted in the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the oligonucleotide (b-2) binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the oligonucleotide (b-2) consists of a nucleotide sequence having about 90% or more identity to SEQ ID NO: 14. In some embodiments, the oligonucleotide (b-2) comprises the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the oligonucleotide (b-2) consists of the nucleotide sequence of SEQ ID NO: 14.

[0032] In one embodiment, the oligonucleotide (b-2) has at least about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or more sequence identity to SEQ ID NO: 38 (5'-aactaaatatgtttgtttc-3'). In another embodiment, the oligonucleotide (b-2) consists of a nucleotide sequence in which one or several, for example, two or three, nucleotides have been deleted, substituted, added, or inserted in the nucleotide sequence of SEQ ID NO: 38. In one embodiment, the oligonucleotide (b-2) binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 38. In one embodiment, the oligonucleotide (b-2) consists of a nucleotide sequence having about 90% or more identity to SEQ ID NO: 38. In one embodiment, the oligonucleotide (b-2) comprises the nucleotide sequence of SEQ ID NO: 38. In one embodiment, the oligonucleotide (b-2) consists of the nucleotide sequence of SEQ ID NO: 38.

[0033] In one embodiment, the antisense nucleic acid is selected from: (c-1) an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 132 to 134, of SEQ ID NO:9, from the nucleotide sequence from positions 105 to 139 of SEQ ID NO:9; and (c-2) an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 138 to 140, of SEQ ID NO:9, from the nucleotide sequence from positions 133 to 167 of SEQ ID NO:9.

[0034] In one embodiment, the oligonucleotide (c-1) has at least about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or more sequence identity to SEQ ID NO: 15 (5'-ataatagtgaactaaatatg-3'). In another embodiment, the oligonucleotide (c-1) consists of a nucleotide sequence in which one or several, for example, two or three, nucleotides have been deleted, substituted, added, or inserted in the nucleotide sequence of SEQ ID NO: 15. In one embodiment, the oligonucleotide (c-1) binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 15. In one embodiment, the oligonucleotide (c-1) consists of a nucleotide sequence having about 90% or more identity to SEQ ID NO: 15. In one embodiment, the oligonucleotide (c-1) comprises the nucleotide sequence of SEQ ID NO: 15. In one embodiment, the oligonucleotide (c-1) consists of the nucleotide sequence of SEQ ID NO: 15.

[0035] In one embodiment, the oligonucleotide (c-1) has at least about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or more sequence identity to SEQ ID NO: 39 (5'-atagtgaactaaatatgt-3'). In another embodiment, the oligonucleotide (c-1) consists of a nucleotide sequence in which one or several, for example, two or three, nucleotides have been deleted, substituted, added, or inserted in the nucleotide sequence of SEQ ID NO: 39. In one embodiment, the oligonucleotide (c-1) binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 39. In one embodiment, the oligonucleotide (c-1) consists of a nucleotide sequence having about 90% or more identity to SEQ ID NO: 39. In one embodiment, the oligonucleotide (c-1) comprises the nucleotide sequence of SEQ ID NO: 39. In one embodiment, the oligonucleotide (c-1) consists of the nucleotide sequence of SEQ ID NO: 39.

[0036] In one embodiment, the oligonucleotide (c-2) has at least about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or more sequence identity to SEQ ID NO: 16 (5'-ttaatataacccactatata-3'). In another embodiment, the oligonucleotide (c-2) consists of a nucleotide sequence in which one or several, for example, two or three, nucleotides have been deleted, substituted, added, or inserted in the nucleotide sequence of SEQ ID NO: 16. In one embodiment, the oligonucleotide (c-2) binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 16. In one embodiment, the oligonucleotide (c-2) consists of a nucleotide sequence having about 90% or more identity to SEQ ID NO: 16. In one embodiment, the oligonucleotide (c-2) comprises the nucleotide sequence of SEQ ID NO: 16. In one embodiment, the oligonucleotide (c-2) consists of the nucleotide sequence of SEQ ID NO: 16.

[0037] In one embodiment, the antisense nucleic acid is (d) an oligonucleotide consisting of 25 to 35 nucleotides that binds to a sequence consisting of 25 to 35 consecutive nucleotides, including positions 110 to 134 of SEQ ID NO:9, within the nucleotide sequence from positions 105 to 139 of SEQ ID NO:9.

[0038] In one embodiment, the oligonucleotide (d) has at least about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or more sequence identity to SEQ ID NO: 17 (5'-atagtgaactaaatatgtttgtttc-3'). In another embodiment, the oligonucleotide (d) consists of a nucleotide sequence in which one or several, for example, two or three, nucleotides have been deleted, substituted, added, or inserted in the nucleotide sequence of SEQ ID NO: 17. In one embodiment, the oligonucleotide (d) binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 17. In one embodiment, the oligonucleotide (d) consists of a nucleotide sequence having about 90% or more identity to SEQ ID NO: 17. In one embodiment, the oligonucleotide (d) comprises the nucleotide sequence of SEQ ID NO: 17. In one embodiment, the oligonucleotide (d) consists of the nucleotide sequence of SEQ ID NO: 17.

[0039] One or more antisense nucleic acids can be used. When two or more antisense nucleic acids are used, a composition containing all of the antisense nucleic acids may be used, or two or more compositions each containing one or more antisense nucleic acids may be used in combination. When two or more antisense nucleic acids are contained in one composition, or when two or more antisense nucleic acids are administered simultaneously, it is preferable that the antisense nucleic acids do not form complementary bonds.

[0040] In one embodiment, a combination of at least two antisense nucleic acids is used, including one antisense nucleic acid selected from (a-1) and (a-2), one antisense nucleic acid selected from (b-1) and (b-2), and one antisense nucleic acid selected from (c-1) and (c-2). For example, (a-1) / (b-1), (a-1) / (b-2), (a-2) / (b-1), (a-2) / (b-2), (a-1) / (c-1), (a-1) / (c-2), (a-2) / (c-1), (a-2) / (c-2), (b-1) / (c-1), (b-1) / (c-2), (b-2) / (c-1), (b-2) / (c-2), (a-1) / (b- Antisense nucleic acid combinations of (a-1) / (c-1), (a-1) / (b-1) / (c-2), (a-1) / (b-2) / (c-1), (a-1) / (b-2) / (c-2), (a-2) / (b-1) / (c-1), (a-2) / (b-1) / (c-2), (a-2) / (b-2) / (c-1), or (a-2) / (b-2) / (c-2) may be used.

[0041] In one embodiment, a combination of one antisense nucleic acid selected from the above (a-1) and (a-2) and the above (d) is used. For example, the combination of antisense nucleic acids (a-1) / (d) or (a-2) / (d) can be used.

[0042] In one embodiment, the antisense nucleic acid is selected from: (a-1') an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 36 to 38, of SEQ ID NO: 10, from the nucleotide sequence from positions 9 to 49 of SEQ ID NO: 10; and (a-2') an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 48 to 50, of SEQ ID NO: 10, from the nucleotide sequence from positions 37 to 77 of SEQ ID NO: 10.

[0043] In one embodiment, the antisense nucleic acid is selected from: (b-1') an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 105 to 107 of SEQ ID NO: 10, from the nucleotide sequence from positions 78 to 112 of SEQ ID NO: 10; and (b-2') an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 111 to 113 of SEQ ID NO: 10, from the nucleotide sequence from positions 106 to 140 of SEQ ID NO: 10.

[0044] In one embodiment, the antisense nucleic acid is selected from: (c-1') an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 133 to 135 of SEQ ID NO: 10, from the nucleotide sequence from positions 106 to 140 of SEQ ID NO: 10; and (c-2') an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 139 to 141 of SEQ ID NO: 10, from the nucleotide sequence from positions 134 to 168 of SEQ ID NO: 10.

[0045] In one embodiment, the antisense nucleic acid is (d') an oligonucleotide consisting of 25 to 35 nucleotides that binds to a sequence consisting of 25 to 35 consecutive nucleotides, including positions 111 to 135 of SEQ ID NO: 10, within the nucleotide sequence from positions 106 to 140 of SEQ ID NO: 10.

[0046] The above (a-1'), (a-2'), (b-1'), (b-2'), (c-1'), (c-2') and (d') are defined in accordance with the above (a-1), (a-2), (b-1), (b-2), (c-1), (c-2) and (d), respectively. In some embodiments, oligonucleotides (a-1'), (a-2'), (b-1'), (b-2'), (c-1'), (c-2') and (d') have at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO:18 (5'-taactgacagtgagtgtcgc-3'), SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:19 (5'-ctaaatgggtttgtttcata-3'), SEQ ID NO:20 (5'-ataatagtgaactaaatggg-3'), SEQ ID NO:21 (5'-ttaatataatccactatata-3') and SEQ ID NO:22 (5'-atagtgaactaaatgggtttgtttc-3'), respectively. In another embodiment, the oligonucleotides (a-1'), (a-2'), (b-1'), (b-2'), (c-1'), (c-2') and (d') each consist of a nucleotide sequence in which one or several, for example, two or three nucleotides have been deleted, substituted, added or inserted in the nucleotide sequence of SEQ ID NOs: 18, 12, 13, 19, 20, 21 and 22. In one embodiment, the oligonucleotides (a-1'), (a-2'), (b-1'), (b-2'), (c-1'), (c-2') and (d') bind to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NOs: 18, 12, 13, 19, 20, 21 and 22, respectively. In some embodiments, oligonucleotides (a-1'), (a-2'), (b-1'), (b-2'), (c-1'), (c-2'), and (d') consist of nucleotide sequences having about 90% or greater identity to SEQ ID NOs: 18, 12, 13, 19, 20, 21, and 22, respectively. In some embodiments, oligonucleotides (a-1'), (a-2'), (b-1'), (b-2'), (c-1'), (c-2'), and (d') comprise the nucleotide sequences of SEQ ID NOs: 18, 12, 13, 19, 20, 21, and 22, respectively.In some embodiments, the oligonucleotides (a-1'), (a-2'), (b-1'), (b-2'), (c-1'), (c-2') and (d') consist of the nucleotide sequences of SEQ ID NOs: 18, 12, 13, 19, 20, 21 and 22, respectively.

[0047] In one embodiment, the oligonucleotides (b-2') and (c-1') have at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 40 (5'-aactaaatgggtttgtttc-3') and SEQ ID NO: 41 (5'-atagtgaactaaatgggt-3'), respectively. In another embodiment, the oligonucleotides (b-2') and (c-1') consist of the nucleotide sequences of SEQ ID NOs: 40 and 41, respectively, in which one or several, for example, two or three, nucleotides have been deleted, substituted, added, or inserted. In one embodiment, the oligonucleotides (b-2') and (c-1') bind to nucleotide sequences complementary to the nucleotide sequences of SEQ ID NOs: 40 and 41, respectively. In some embodiments, oligonucleotides (b-2') and (c-1') consist of nucleotide sequences having about 90% or more identity to SEQ ID NOs: 40 and 41, respectively. In some embodiments, oligonucleotides (b-2') and (c-1') comprise the nucleotide sequences of SEQ ID NOs: 40 and 41, respectively. In some embodiments, oligonucleotides (b-2') and (c-1') consist of the nucleotide sequences of SEQ ID NOs: 40 and 41, respectively.

[0048] Antisense nucleic acids may be composed of natural nucleotides, artificial nucleotides, or one or more natural nucleotides and one or more artificial nucleotides. Natural nucleotides include, for example, deoxyribonucleotides or ribonucleotides. Artificial nucleotides may be selected that have a structure different from that of natural nucleotides and that enhance nuclease resistance or binding affinity to the target sequence. For example, the artificial nucleotides described in Deleavey, GF, & Damha, MJ (2012). Designing chemically modified oligonucleotides for targeted gene silencing. Chemistry & biology, 19(8), 937-954, which is incorporated herein by reference, may be used. Examples of artificial nucleotides include abasic nucleosides, arabinonucleosides, 2'-deoxyuridine, α-deoxyribonucleosides, β-L-deoxyribonucleosides, nucleosides with other sugar modifications, peptide nucleic acids (PNAs), phosphate-linked peptide nucleic acids (PHONAs), bridged artificial nucleic acids (LNAs), 2'-O,4'-C-ethylene-bridged nucleic acids (ENAs), constrained ethyl (cEt), morpholino nucleic acids, etc. Artificial nucleotides with sugar modifications include substituted pentasaccharides such as 2'-O-methylribose, 2'-O-methoxyethylribose, 2'-deoxy-2'-fluororibose, and 3'-O-methylribose; 1',2'-deoxyribose; arabinose; substituted arabinose sugars; hexasaccharides; and alpha-anomeric sugar modifications. Examples of artificial nucleotides having modified bases include those having pyrimidines such as 5-hydroxycytosine, 5-methylcytosine, 5-fluorouracil, and 4-thiouracil; purines such as 6-methyladenine and 6-thioguanosine; and other heterocyclic bases. The artificial nucleotides in the antisense nucleic acid may all be of the same type, or may be two or more different types of artificial nucleotides.

[0049] In one embodiment, the antisense nucleic acid is a morpholino oligonucleotide, which is an oligonucleotide having a morpholine ring instead of the ribose or deoxyribose of RNA or DNA, and has a structure in which the oligonucleotides shown below are linked together repeatedly: (wherein B is a base, e.g., adenine, cytosine, guanine, thymine, or uracil, and the dashed line indicates the point of attachment to the adjacent structure.) In some embodiments, the antisense nucleic acid is single-stranded DNA.

[0050] Antisense nucleic acids may be linked to one or more moieties or conjugates that enhance the activity or cellular uptake of the antisense nucleic acid. Such moieties include, but are not limited to, cholesterol moieties, cholic acid, thioethers (e.g., hexyl-S-tritylthiol), thiocholesterol, aliphatic chains (e.g., dodecanediol or undecyl residues), phospholipids (e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate), polyamine or polyethylene glycol chains, adamantane acetic acid, palmityl moieties, or octadecylamine, hexylamino-carbonyl-t-oxycholesterol, or octaguanidine dendrimer moieties. Oligonucleotides containing such moieties and methods for preparing such oligonucleotides are known in the art.

[0051] The present composition can stimulate immunity and therefore can be used to treat cancer through cancer immunotherapy. In the present disclosure, the cancer may be any cancer, whether primary or metastatic. Examples of cancer include malignant melanoma, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, classical Hodgkin's lymphoma, head and neck cancer, gastric cancer, malignant pleural mesothelioma, colorectal cancer, esophageal cancer, cancer of unknown primary site, urothelial carcinoma, hepatocellular carcinoma, solid tumors, breast cancer, endometrial cancer, malignant pleural mesothelioma, Merkel cell carcinoma, brain tumor, cholangiocarcinoma (including intrahepatic cholangiocarcinoma), kidney cancer, and colon cancer. In some embodiments, the cancer is malignant melanoma, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, classical Hodgkin's lymphoma, head and neck cancer, gastric cancer, malignant pleural mesothelioma, colorectal cancer, esophageal cancer, carcinoma of unknown primary, urothelial carcinoma, hepatocellular carcinoma, solid tumor, breast cancer, endometrial cancer, malignant pleural mesothelioma, or Merkel cell carcinoma. In some embodiments, the cancer is brain cancer or bile duct cancer. In some embodiments, the cancer is malignant melanoma, kidney cancer, or colon cancer. In some embodiments, the cancer is malignant melanoma.

[0052] As used herein, "treat" or "treatment" means reducing or eliminating cancer cells, slowing or stopping the progression, recurrence or metastasis of cancer, and / or reducing, alleviating, ameliorating or eliminating the symptoms of cancer in a cancer patient.

[0053] Subjects for disease treatment or prevention include animals, typically mammals (eg, humans, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, etc.), and particularly humans.

[0054] Cancer immunotherapy can be immune cell therapy, such as CAR-T cell therapy, and immune cells can be treated with the present composition. Immune cells may be any cells involved in immune responses, including lymphoid cells such as T cells, NK cells, and B cells; antigen-presenting cells such as monocytes, macrophages, and dendritic cells; and granulocytes such as neutrophils, eosinophils, basophils, and mast cells. Isolated or purified immune cells may be treated with the present composition, or cell populations containing immune cells (e.g., peripheral blood mononuclear cells, umbilical cord blood cells) may be treated. T cells can be obtained by isolation and purification from immune cells infiltrating body fluids such as blood and bone marrow fluid, tissues such as the spleen, thymus, and lymph nodes, or cancer tissues such as primary tumors, metastatic tumors, and cancerous ascites. They may also be prepared from pluripotent stem cells such as ES cells and iPS cells. Such T cells may include alpha-beta T cells, gamma-delta T cells, CD8-positive T cells, CD4-positive T cells, tumor-infiltrating T cells, memory T cells, naive T cells, NKT cells, and naive CD8-positive T cells. The immune cells may be isolated from a subject.

[0055] The composition is used to introduce a substance that disrupts a stem-loop structure into immune cells. The introduction method is not limited, and methods commonly used in immune cell therapy, such as CAR-T cell therapy, can be used. For example, nucleic acids can be introduced by known methods such as the calcium phosphate method, lipofection, microinjection, and electroporation. Therefore, the composition may contain a substance that disrupts the stem-loop structure in the 3'UTR of Nfkbiz mRNA together with a substance that assists gene introduction. Alternatively, the composition may be a solution or lyophilized product of the substance that disrupts the stem-loop structure.

[0056] Immune cells treated with the composition can be administered to a subject. The administration method is not limited, and methods commonly used in immune cell therapy can be used. For example, administration can be into the carotid artery, subcutaneously, intradermally, intratumorally, intralymph node, intraspinal, intramuscularly, intravenously, or intraperitoneally. A composition containing the immune cells can also be administered.

[0057] Cancer immunotherapy may involve administering the composition to a subject. The route of administration of the composition includes, but is not limited to, oral or parenteral administration. Various known administration forms can be employed depending on the application site and the target disease. For example, parenteral administration may be systemic or local, and more specifically, examples include intratracheal administration, intrathecal administration, subarachnoid administration, intracranial administration, intravenous administration, intraarterial administration, intraportal vein administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, intranasal administration, and oral administration. In one embodiment, the composition is administered intravenously.

[0058] Dosage forms include granules, fine granules, powders, coated tablets, tablets, suppositories, powders, capsules, microcapsules, chewable tablets, liquids, suspensions, emulsions, etc. Dosage forms that extend the release of the active substance may also be used. Dosage forms for injection or infusion include aqueous and non-aqueous injection solutions (which may contain antioxidants, buffers, bacteriostats, isotonic agents, etc.) and aqueous and non-aqueous injection suspensions (which may contain suspending agents, thickeners, etc.). These dosage forms may be provided as liquids in sealed ampoules or vials, or as lyophilized products that can be prepared by adding sterile liquids (e.g., water for injection) immediately before use. Injection solutions or suspensions may be prepared from powders, granules, or tablets.

[0059] These dosage forms are prepared by conventional formulation methods. Furthermore, various pharmaceutically acceptable pharmaceutical substances can be blended as needed for the formulation. The pharmaceutical substances can be appropriately selected depending on the dosage form of the preparation, and examples thereof include buffering agents, surfactants, stabilizers, preservatives, excipients, diluents, additives, disintegrants, binders, coating agents, lubricants, glidants, flavoring agents, sweeteners, solubilizers, etc.

[0060] The dosage and frequency of administration of the composition can be appropriately determined by those skilled in the art depending on the species of animal to be administered, the health condition, age, body weight, route of administration, dosage form, etc., of the subject, so that an effective amount of the active ingredient is administered to the subject. The effective amount in a given situation can be easily determined by routine experimentation and is within the skill and judgment of an ordinary clinician. For example, when the active ingredient is an antisense nucleic acid, the antisense nucleic acid can be administered in an amount of about 0.01 to about 100 mg / kg body weight, about 0.05 to about 10 mg / kg body weight, or about 0.1 to about 5 mg / kg body weight.

[0061] The present composition can be used alone or in combination with one or more additional active ingredients, particularly active ingredients for the treatment of cancer. "Combined use" of ingredients means not only the use of a dosage form containing all ingredients or the use of a combination of dosage forms containing each ingredient separately, but also the simultaneous administration, sequential administration, or delayed administration of each ingredient, as long as they are used for the treatment of cancer. Two or more additional active ingredients can also be used in combination. Active ingredients suitable for combination include, for example, known anticancer agents.

[0062] Examples of known anticancer agents include immune checkpoint inhibitors, alkylating agents, antimetabolites, anticancer antibiotics, plant alkaloids, antihormones, platinum compounds, cytokine preparations, molecularly targeted drugs, tumor immunotherapeutic agents, and cancer vaccines. In one embodiment, the active ingredient used in combination with the present composition is an immune checkpoint inhibitor. Immune checkpoint inhibitors are substances that inhibit the function of immune checkpoint molecules (e.g., CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM3, BTLA, B7H3, B7H4, 2B4, CD160, A2aR, KIR, VISTA, TIGIT, etc.) that suppress immune responses against self and / or excessive immune responses.

[0063] Other cancer treatments can also be administered in combination with cancer immunotherapy. Suitable treatments include, for example, chemotherapy, radiation therapy, hematopoietic stem cell transplantation, endoscopic resection, surgery, etc.

[0064] It is known that tumor-infiltrating CD8 T cells, despite expressing IL-18R, do not transmit signals via cancer cell-derived decoys. Without being limited by theory, it is believed that because IκB-ζ acts downstream of IL-18R signaling, for example, the present composition can activate T cells independently of the binding of IL-18 to IL-18R, and can activate exhausted T cells that have lost IL-18R expression. It is also expected to boost T cells resistant to PD-1 therapy, suppress the differentiation of tumor-infiltrating T lymphocytes into exhausted T cells, and promote their differentiation into effector T cells. NK cells also exert anti-tumor immunity through IL-18R signaling, and anti-tumor effects targeting these cells are also expected. Increased expression of Nfkbiz can also activate the immune response of macrophages and dendritic cells, potentially enhancing cancer immunity.

[0065] In one aspect, a method of treating cancer is provided, comprising administering to a subject in need thereof an effective amount of at least one substance that disrupts a stem-loop structure in the 3' untranslated region of Nfkbiz mRNA. In one aspect, at least one substance that disrupts a stem-loop structure in the 3' untranslated region of Nfkbiz mRNA is provided for treating cancer. In one aspect, a use of at least one substance that disrupts a stem-loop structure in the 3' untranslated region of Nfkbiz mRNA for treating cancer is provided. In one aspect, a use of at least one substance that disrupts a stem-loop structure in the 3' untranslated region of Nfkbiz mRNA in the manufacture of a composition for treating cancer is provided.

[0066] In another aspect, the present disclosure provides a method for screening for cancer treatment drugs, comprising the following steps: (a) introducing a candidate substance into cells expressing a reporter gene linked to the 3' untranslated region of Nfkbiz mRNA and Regnase-1 and / or Regnase-3; (b) measuring the expression level of the reporter gene in the cells; and (c) selecting, as a cancer treatment drug, a candidate substance that increases the expression level compared to the expression level measured in the absence of the candidate substance.

[0067] In step (a) of the above method, the 3' untranslated region of Nfkbiz mRNA is as described above, and for example, contains at least the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6. Any reporter gene can be used as long as it directly or indirectly generates a detectable label, and examples include the chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein (GFP) gene, β-glucuronidase (GUS) gene, luciferase gene, and other marker genes. The 3' untranslated region of Nfkbiz mRNA is ligated downstream of the reporter gene.

[0068] Regnase-1 and Regnase-3 may be from any species, typically mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, etc.), particularly humans and mice. Representative amino acid sequences of Regnase-1 are registered under GenBank accession numbers NP_001310479.1 (human, SEQ ID NO: 23) and NP_694799.1 (mouse, SEQ ID NO: 24), and representative nucleotide sequences are registered under GenBank accession numbers NM_001323550 (human, SEQ ID NO: 25) and NM_153159.2 (mouse, SEQ ID NO: 26). Representative amino acid sequences of Regnase-3 are registered under GenBank accession numbers NP_203748.1 (human, SEQ ID NO: 27) and NP_001156393.1 (mouse, SEQ ID NO: 28), and representative nucleotide sequences are registered under GenBank accession numbers NM_033390.2 (human, SEQ ID NO: 29) and NM_001162921.2 (mouse, SEQ ID NO: 30).

[0069] For purposes of the present disclosure, "Regnase-1" and "Regnase-3" include proteins having at least about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or more identity to the amino acid sequences of SEQ ID NO: 23 or 24 and SEQ ID NO: 27 or 28, respectively, so long as the functions of Regnase-1 and Regnase-3 are maintained. Regnase-1 and Regnase-3 may be expressed natively by cells used in the above methods, or may be expressed from genes introduced into cells by genetic recombinant technology.

[0070] The cells used in the above method are not limited as long as they are capable of expressing a reporter gene linked to the 3' untranslated region of Nfkbiz mRNA and, if necessary, Regnase-1 and / or Regnase-3. Examples include HeLa cells, HEK293 cells, HepG2 cells, and COS-7 cells. Construction of a vector for introducing a nucleic acid, transformation, etc. can be carried out by known methods (Molecular Cloning: A Laboratory Manual 2nd edition (1989), Cold Spring Harbor Laboratory Press).

[0071] "Candidate substances" include all substances, such as proteins, amino acids, nucleic acids, lipids, carbohydrates, and low-molecular-weight compounds. Typically, purified and isolated candidate substances can be used, but unpurified and unisolated crude products are also acceptable. Candidate substances may be provided in the form of a compound library, a nucleic acid library, a random peptide library, or the like, or may be provided as natural products. Candidate substances may be designed based on the stem-loop structure present in the 3' untranslated region of Nfkbiz mRNA. Candidate substances can be introduced into cells by known methods depending on the type of candidate substance.

[0072] In step (b) of the above method, the expression level of the reporter gene may be measured by a known method depending on the reporter gene. In step (c) of the above method, a candidate substance that increases the expression of the reporter gene by, for example, about 10% or more, preferably about 20% or more, more preferably about 30% or more, and even more preferably about 50% or more, compared to the expression level measured in the absence of the candidate substance, may be selected as a cancer treatment drug.

[0073] In one aspect, a composition for stimulating immunity is provided, comprising at least one substance that disrupts a stem-loop structure in the 3' untranslated region of Nfkbiz mRNA. In the present disclosure, "stimulating immunity" refers to activating immune cells, enhancing immunity, and / or relieving immune suppression. The immunity may be innate or adaptive immunity. Details of the substance that disrupts the stem-loop structure and the method of use of the composition are similar to those of the composition for treating cancer described above.

[0074] In one aspect, a method of immune stimulation is provided, comprising administering to a subject in need thereof an effective amount of at least one substance that disrupts a stem-loop structure in the 3' untranslated region of Nfkbiz mRNA. In one aspect, at least one substance that disrupts a stem-loop structure in the 3' untranslated region of Nfkbiz mRNA is provided for immune stimulation. In one aspect, a use of at least one substance that disrupts a stem-loop structure in the 3' untranslated region of Nfkbiz mRNA for immune stimulation is provided. In one aspect, a use of at least one substance that disrupts a stem-loop structure in the 3' untranslated region of Nfkbiz mRNA in the manufacture of a composition for immune stimulation is provided.

[0075] In one embodiment, a substance that disrupts the stem-loop structure in the 3' untranslated region of Nfkbiz mRNA is provided. Details of this substance are as described above. Two or more substances may be used in combination.

[0076] In one aspect, a method for stimulating immune cells is provided, comprising introducing into immune cells in vitro at least one substance that disrupts the stem-loop structure in the 3' untranslated region of Nfkbiz mRNA. The method may also comprise isolating the immune cells from a subject. In one aspect, an immune cell is provided that comprises at least one substance that disrupts the stem-loop structure in the 3' untranslated region of Nfkbiz mRNA. In one aspect, a composition for treating cancer is provided, comprising an immune cell that comprises at least one substance that disrupts the stem-loop structure in the 3' untranslated region of Nfkbiz mRNA. In these aspects, the immune cell is as described above, e.g., a naive CD8-positive T cell.

[0077] The present application provides, for example, the following embodiments: [1] A composition for stimulating immunity, comprising at least one substance that disrupts a stem-loop structure in the 3' untranslated region of Nfkbiz mRNA. [2] A composition for treating cancer, comprising at least one substance that disrupts a stem-loop structure in the 3' untranslated region of Nfkbiz mRNA. [3] The composition according to paragraph 1 or 2, wherein the stem-loop structure is at least one stem-loop structure selected from the following stem-loop structures: a first stem-loop structure formed in a region corresponding to positions 34 to 50 of SEQ ID NO:9, a second stem-loop structure formed in a region corresponding to positions 100 to 116 of SEQ ID NO:9, and a third stem-loop structure formed in a region corresponding to positions 129 to 143 of SEQ ID NO:9. [4] The composition according to any one of items 1 to 3, comprising at least one substance that disrupts at least one stem-loop structure selected from the second stem-loop structure and the third stem-loop structure. [5] The composition according to any one of items 1 to 4, comprising at least one substance that disrupts the second stem-loop structure and the third stem-loop structure. [6] The composition according to any one of items 1 to 5, wherein the 3' untranslated region of Nfkbiz mRNA comprises a nucleotide sequence having about 90% or more identity to the nucleotide sequence of SEQ ID NO: 5. [7] The composition according to any one of items 1 to 6, wherein the 3' untranslated region of Nfkbiz mRNA comprises the nucleotide sequence of SEQ ID NO: 5. [8] The composition according to any one of items 1 to 7, wherein the 3' untranslated region of Nfkbiz mRNA comprises a nucleotide sequence having about 90% or more identity to the nucleotide sequence of SEQ ID NO: 7. [9] The composition according to any one of items 1 to 8, wherein the 3' untranslated region of Nfkbiz mRNA comprises the nucleotide sequence of SEQ ID NO: 7.

[10] The composition according to any one of items 1 to 9, wherein the 3' untranslated region of Nfkbiz mRNA consists of a nucleotide sequence having about 90% or more identity to the nucleotide sequence of SEQ ID NO: 9.

[11] The composition according to any one of items 1 to 10, wherein the 3' untranslated region of Nfkbiz mRNA consists of the nucleotide sequence of SEQ ID NO: 9.

[12] The composition according to any one of items 1 to 11, wherein at least one substance that disrupts the stem-loop structure is an oligonucleotide that binds to a nucleotide sequence that forms a stem-loop structure.

[0078]

[13] At least one substance that disrupts a stem-loop structure is an oligonucleotide selected from: (a) a first oligonucleotide, (a-1) an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 35 to 37, of SEQ ID NO: 9, among the nucleotide sequence from positions 8 to 48 of SEQ ID NO: 9; and (a-2) an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 47 to 49, of SEQ ID NO: 9, among the nucleotide sequence from positions 36 to 76 of SEQ ID NO: 9; (b) a second oligonucleotide, (b-1) an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 104 to 106, of SEQ ID NO: 9, among the nucleotide sequence from positions 77 to 111 of SEQ ID NO: 9; and (b-2) an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 110 to 112, of SEQ ID NO: 9, from the nucleotide sequence from positions 105 to 139 of SEQ ID NO: 9; (c) a third oligonucleotide selected from: (c-1) an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 132 to 134, of SEQ ID NO: 9, from the nucleotide sequence from positions 105 to 139 of SEQ ID NO: 9; and (c-2) an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 138 to 140, of SEQ ID NO: 9, from the nucleotide sequence from positions 133 to 167 of SEQ ID NO: 9; (d) a fourth oligonucleotide consisting of 25 to 35 nucleotides that binds to a sequence consisting of 25 to 35 consecutive nucleotides including positions 110 to 134 of SEQ ID NO: 9, within the nucleotide sequence from positions 105 to 139 of SEQ ID NO: 9;(e) a combination of at least two oligonucleotides selected from a first oligonucleotide, a second oligonucleotide, and a third oligonucleotide; or (f) a combination of a first oligonucleotide and a fourth oligonucleotide.

[0079]

[14] At least one substance that disrupts a stem-loop structure is an oligonucleotide selected from: (a) a first oligonucleotide, (a-1) an oligonucleotide consisting of a nucleotide sequence having 90% or more identity with SEQ ID NO: 11, which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 35 to 37, of SEQ ID NO: 9, among the nucleotide sequence from positions 8 to 48 of SEQ ID NO: 9; and (a-2) an oligonucleotide consisting of a nucleotide sequence having 90% or more identity with SEQ ID NO: 12, which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 47 to 49, of SEQ ID NO: 9, among the nucleotide sequence from positions 36 to 76 of SEQ ID NO: 9; (b) a second oligonucleotide, (b-1) an oligonucleotide consisting of a nucleotide sequence having 90% or more identity with SEQ ID NO: 13, which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 104 to 106, of SEQ ID NO: 9, among the nucleotide sequence from positions 77 to 111 of SEQ ID NO: 9; and (b-2) an oligonucleotide selected from the group consisting of: (b-1) an oligonucleotide having a nucleotide sequence having 90% or more identity with SEQ ID NO: 15, which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 110 to 112, of SEQ ID NO: 9, among the nucleotide sequence from positions 105 to 139 of SEQ ID NO: 9; (c-2) an oligonucleotide selected from the group consisting of: (b-2) an oligonucleotide having 90% or more identity with SEQ ID NO: 14, which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 110 to 112, of SEQ ID NO: 9, among the nucleotide sequence from positions 105 to 139 of SEQ ID NO: 9; (c-1) an oligonucleotide having a nucleotide sequence having 90% or more identity with SEQ ID NO: 15, which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 132 to 134, of SEQ ID NO: 9, among the nucleotide sequence from positions 105 to 139 of SEQ ID NO: 9; and (c-2) an oligonucleotide having a nucleotide sequence having 90% or more identity with SEQ ID NO: 16, which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 138 to 140, of SEQ ID NO: 9, among the nucleotide sequence from positions 133 to 167 of SEQ ID NO: 9;(d) a fourth oligonucleotide, which is an oligonucleotide consisting of a nucleotide sequence having 90% or more identity to SEQ ID NO: 17, and which binds to a sequence consisting of 25 to 35 consecutive nucleotides, including positions 110 to 134 of SEQ ID NO: 9, within the nucleotide sequence of positions 105 to 139 of SEQ ID NO: 9; (e) a combination of at least two oligonucleotides selected from the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide; or (f) a combination of the first oligonucleotide and the fourth oligonucleotide.

[0080]

[15] At least one substance that disrupts a stem-loop structure is an oligonucleotide selected from: (a) a first oligonucleotide, (a-1) an oligonucleotide consisting of a nucleotide sequence of SEQ ID NO: 11 in which one, two, or three nucleotides have been deleted, substituted, added, or inserted, and which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 35 to 37, of SEQ ID NO: 9, among the nucleotide sequence of positions 8 to 48 of SEQ ID NO: 9; and (a-2) an oligonucleotide consisting of a nucleotide sequence of SEQ ID NO: 12 in which one, two, or three nucleotides have been deleted, substituted, added, or inserted, and which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 47 to 49, of SEQ ID NO: 9, among the nucleotide sequence of positions 36 to 76 of SEQ ID NO: 9; (b) a second oligonucleotide, (b-1) an oligonucleotide consisting of a nucleotide sequence of SEQ ID NO: 13 in which one, two or three nucleotides have been deleted, substituted, added or inserted, and which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 104 to 106, of SEQ ID NO: 9, among the nucleotide sequence of positions 77 to 111, and (b-2) an oligonucleotide consisting of a nucleotide sequence of SEQ ID NO: 14 in which one, two or three nucleotides have been deleted, substituted, added or inserted, and which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 110 to 112, of SEQ ID NO: 9, among the nucleotide sequence of positions 105 to 139, and (c) a third oligonucleotide, (c-1) an oligonucleotide consisting of a nucleotide sequence of SEQ ID NO: 15 in which one, two, or three nucleotides have been deleted, substituted, added, or inserted, and which binds to a sequence consisting of 10 to 30 consecutive nucleotides including positions 132 to 134 of SEQ ID NO: 9, within the nucleotide sequence of positions 105 to 139 of SEQ ID NO: 9; and(c-2) an oligonucleotide selected from the group consisting of SEQ ID NO: 16, wherein one, two, or three nucleotides have been deleted, substituted, added, or inserted, and which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 138 to 140, of SEQ ID NO: 9, within the nucleotide sequence from positions 133 to 167 of SEQ ID NO: 9; (d) a fourth oligonucleotide, wherein one, two, or three nucleotides have been deleted, substituted, added, or inserted, and which binds to a sequence consisting of 25 to 35 consecutive nucleotides, including positions 110 to 134, of SEQ ID NO: 9, within the nucleotide sequence from positions 105 to 139 of SEQ ID NO: 9; (e) a combination of at least two oligonucleotides selected from the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide; or (f) a combination of the first oligonucleotide and the fourth oligonucleotide.

[0081]

[16] At least one substance that disrupts a stem-loop structure is selected from: (a) a first oligonucleotide, the oligonucleotide being selected from: (a-1) an oligonucleotide that binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 11, and (a-2) an oligonucleotide that binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 12; (b) a second oligonucleotide, the oligonucleotide being selected from: (b-1) an oligonucleotide that binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 13, and (b-2) an oligonucleotide that binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 14; (c) a third oligonucleotide, the oligonucleotide being selected from: (c-1) an oligonucleotide that binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 15, and (c-2) an oligonucleotide that binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 16; (d) a fourth oligonucleotide, the oligonucleotide that binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 17; (e) a combination of at least two oligonucleotides selected from a first oligonucleotide, a second oligonucleotide, and a third oligonucleotide; or (f) a combination of a first oligonucleotide and a fourth oligonucleotide.

[0082]

[17] The composition according to any one of Items 1 to 16, wherein the at least one substance that disrupts a stem-loop structure is: (a) a first oligonucleotide selected from (a-1) an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 11, and (a-2) an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 12; (b) a second oligonucleotide selected from (b-1) an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 13, and (b-2) an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 14; (c) a third oligonucleotide selected from (c-1) an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 15, and (c-2) an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 16; (d) a fourth oligonucleotide selected from the nucleotide sequence of SEQ ID NO: 17; (e) a combination of at least two oligonucleotides selected from the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide; or (f) a combination of the first oligonucleotide and the fourth oligonucleotide.

[0083]

[18] The composition according to any one of Items 1 to 17, wherein the at least one substance that disrupts a stem-loop structure is: (a) a first oligonucleotide selected from (a-1) an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 11, and (a-2) an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 12; (b) a second oligonucleotide selected from (b-1) an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 13, and (b-2) an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 14; (c) a third oligonucleotide selected from (c-1) an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 15, and (c-2) an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 16; (d) a fourth oligonucleotide selected from the nucleotide sequence of SEQ ID NO: 17; (e) a combination of at least two oligonucleotides selected from the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide; or (f) a combination of the first oligonucleotide and the fourth oligonucleotide.

[0084]

[19] At least one substance that disrupts a stem-loop structure is an oligonucleotide selected from: (a) a first oligonucleotide, (a-1) an oligonucleotide consisting of a nucleotide sequence having 90% or more identity with SEQ ID NO: 11, which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 35 to 37, of SEQ ID NO: 9, among the nucleotide sequence from positions 8 to 48 of SEQ ID NO: 9; and (a-2) an oligonucleotide consisting of a nucleotide sequence having 90% or more identity with SEQ ID NO: 12, which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 47 to 49, of SEQ ID NO: 9, among the nucleotide sequence from positions 36 to 76 of SEQ ID NO: 9; (b) a second oligonucleotide, (b-1) an oligonucleotide consisting of a nucleotide sequence having 90% or more identity with SEQ ID NO: 13, which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 104 to 106, of SEQ ID NO: 9, among the nucleotide sequence from positions 77 to 111 of SEQ ID NO: 9; and (b-2) an oligonucleotide selected from the group consisting of a nucleotide sequence having 90% or more identity to SEQ ID NO: 38, which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 110 to 112, of SEQ ID NO: 9, from the nucleotide sequence from positions 105 to 139 of SEQ ID NO: 9; (c) a third oligonucleotide selected from the group consisting of: (c-1) an oligonucleotide having 90% or more identity to SEQ ID NO: 39, which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 132 to 134, of SEQ ID NO: 9, from the nucleotide sequence from positions 105 to 139 of SEQ ID NO: 9; and (c-2) an oligonucleotide having 90% or more identity to SEQ ID NO: 16, which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 138 to 140, of SEQ ID NO: 9, from the nucleotide sequence from positions 133 to 167 of SEQ ID NO: 9;(d) a fourth oligonucleotide, which is an oligonucleotide consisting of a nucleotide sequence having 90% or more identity to SEQ ID NO: 17, and which binds to a sequence consisting of 25 to 35 consecutive nucleotides, including positions 110 to 134 of SEQ ID NO: 9, within the nucleotide sequence of positions 105 to 139 of SEQ ID NO: 9; (e) a combination of at least two oligonucleotides selected from the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide; or (f) a combination of the first oligonucleotide and the fourth oligonucleotide.

[0085]

[20] At least one substance that disrupts a stem-loop structure is an oligonucleotide selected from: (a) a first oligonucleotide, (a-1) an oligonucleotide consisting of a nucleotide sequence of SEQ ID NO: 11 in which one, two, or three nucleotides have been deleted, substituted, added, or inserted, and which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 35 to 37, of SEQ ID NO: 9, among the nucleotide sequence of positions 8 to 48 of SEQ ID NO: 9; and (a-2) an oligonucleotide consisting of a nucleotide sequence of SEQ ID NO: 12 in which one, two, or three nucleotides have been deleted, substituted, added, or inserted, and which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 47 to 49, of SEQ ID NO: 9, among the nucleotide sequence of positions 36 to 76 of SEQ ID NO: 9; (b) a second oligonucleotide, (b-1) an oligonucleotide consisting of a nucleotide sequence of SEQ ID NO: 13 in which one, two or three nucleotides have been deleted, substituted, added or inserted, and which binds to a sequence consisting of 10 to 30 consecutive nucleotides including positions 104 to 106 of SEQ ID NO: 9 in the nucleotide sequence from positions 77 to 111 of SEQ ID NO: 9, and (b-2) an oligonucleotide consisting of a nucleotide sequence of SEQ ID NO: 38 in which one, two or three nucleotides have been deleted, substituted, added or inserted, and which binds to a sequence consisting of 10 to 30 consecutive nucleotides including positions 110 to 112 of SEQ ID NO: 9 in the nucleotide sequence from positions 105 to 139 of SEQ ID NO: 9, (c) a third oligonucleotide, (c-1) an oligonucleotide consisting of a nucleotide sequence of SEQ ID NO: 39 in which one, two, or three nucleotides have been deleted, substituted, added, or inserted, and which binds to a sequence consisting of 10 to 30 consecutive nucleotides including positions 132 to 134 of SEQ ID NO: 9, within the nucleotide sequence of positions 105 to 139 of SEQ ID NO: 9; and(c-2) an oligonucleotide selected from the group consisting of SEQ ID NO: 16, wherein one, two, or three nucleotides have been deleted, substituted, added, or inserted, and which binds to a sequence consisting of 10 to 30 consecutive nucleotides, including positions 138 to 140, of SEQ ID NO: 9, within the nucleotide sequence from positions 133 to 167 of SEQ ID NO: 9; (d) a fourth oligonucleotide, wherein one, two, or three nucleotides have been deleted, substituted, added, or inserted, and which binds to a sequence consisting of 25 to 35 consecutive nucleotides, including positions 110 to 134, of SEQ ID NO: 9, within the nucleotide sequence from positions 105 to 139 of SEQ ID NO: 9; (e) a combination of at least two oligonucleotides selected from the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide; or (f) a combination of the first oligonucleotide and the fourth oligonucleotide.

[0086]

[21] At least one substance that disrupts a stem-loop structure is selected from: (a) a first oligonucleotide, the oligonucleotide being selected from: (a-1) an oligonucleotide that binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 11, and (a-2) an oligonucleotide that binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 12; (b) a second oligonucleotide, the oligonucleotide being selected from: (b-1) an oligonucleotide that binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 13, and (b-2) an oligonucleotide that binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 38; (c) a third oligonucleotide, the oligonucleotide being selected from: (c-1) an oligonucleotide that binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 39, and (c-2) an oligonucleotide that binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 16; (d) a fourth oligonucleotide, the oligonucleotide that binds to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 17; (e) a combination of at least two oligonucleotides selected from a first oligonucleotide, a second oligonucleotide, and a third oligonucleotide; or (f) a combination of a first oligonucleotide and a fourth oligonucleotide.

[0087]

[22] At least one substance that disrupts a stem-loop structure is selected from: (a) a first oligonucleotide, which is selected from: (a-1) an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 11, and (a-2) an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 12; (b) a second oligonucleotide, which is selected from: (b-1) an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 13, and (b-2) an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 38; (c) a third oligonucleotide, which is selected from: (c-1) an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 39, and (c-2) an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 16; (d) a fourth oligonucleotide, which is selected from: (e) a combination of at least two oligonucleotides selected from the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide; or (f) a combination of the first oligonucleotide and the fourth oligonucleotide. 22. The composition according to any one of items 1 to 13 and items 19 to 21,

[0088]

[23] At least one substance that disrupts a stem-loop structure is selected from: (a) a first oligonucleotide, which is selected from: (a-1) an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 11, and (a-2) an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 12; (b) a second oligonucleotide, which is selected from: (b-1) an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 13, and (b-2) an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 38; (c) a third oligonucleotide, which is selected from: (c-1) an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 39, and (c-2) an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 16; (d) a fourth oligonucleotide, which is an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 17; (e) a combination of at least two oligonucleotides selected from the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide; or (f) a combination of the first oligonucleotide and the fourth oligonucleotide. 23. The composition according to any one of items 1 to 13 and items 19 to 22, wherein

[0089]

[24] The composition according to any of items 13 to 23, wherein any one of the first to third oligonucleotides consists of 15 to 25, preferably 18 to 22, and more preferably 19 to 21 nucleotides.

[25] The composition according to any of items 13 to 24, wherein the fourth oligonucleotide consists of 25 to 30, preferably 25 to 27 nucleotides.

[26] The composition according to any of items 13 to 25, wherein at least one substance that disrupts a stem-loop structure is a combination of a second oligonucleotide and a third oligonucleotide.

[27] The composition according to any of items 13 to 25, wherein at least one substance that disrupts a stem-loop structure is a fourth oligonucleotide.

[28] The composition according to any one of items 2 to 27, wherein the cancer is malignant melanoma, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, classical Hodgkin's lymphoma, head and neck cancer, gastric cancer, malignant pleural mesothelioma, colorectal cancer, esophageal cancer, cancer of unknown primary origin, urothelial carcinoma, hepatocellular carcinoma, solid tumor, breast cancer, endometrial cancer, malignant pleural mesothelioma, Merkel cell carcinoma, brain tumor, bile duct cancer, kidney cancer, or colon cancer.

[29] The composition according to any one of items 2 to 28, wherein the cancer is malignant melanoma, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, classical Hodgkin's lymphoma, head and neck cancer, gastric cancer, malignant pleural mesothelioma, colorectal cancer, esophageal cancer, cancer of unknown primary origin, urothelial carcinoma, hepatocellular carcinoma, solid tumor, breast cancer, endometrial cancer, malignant pleural mesothelioma, or Merkel cell carcinoma.

[30] The composition according to any one of items 2 to 28, wherein the cancer is brain tumor or bile duct cancer.

[31] The composition according to any one of items 2 to 28, wherein the cancer is malignant melanoma, kidney cancer, or colorectal cancer.

[32] The composition according to any one of items 2 to 28 and 31, wherein the cancer is malignant melanoma.

[0090]

[33] A method for screening for a cancer therapeutic agent, comprising the following steps: (a) introducing a candidate substance into cells expressing a reporter gene linked to the 3' untranslated region of Nfkbiz mRNA and Regnase-1 and / or Regnase-3, (b) measuring the expression level of the reporter gene in the cells, and (c) selecting, as a cancer therapeutic agent, a candidate substance that increases the expression level compared to the expression level measured in the absence of the candidate substance.

[34] The method of item 33, wherein the 3' untranslated region of Nfkbiz mRNA comprises a nucleotide sequence having about 90% or more identity to the nucleotide sequence of SEQ ID NO: 5.

[35] The method of item 33 or 34, wherein the 3' untranslated region of Nfkbiz mRNA comprises the nucleotide sequence of SEQ ID NO: 5.

[36] The method of any of items 33 to 35, wherein the 3' untranslated region of Nfkbiz mRNA comprises a nucleotide sequence having about 90% or more identity to the nucleotide sequence of SEQ ID NO: 7.

[37] The method of any of items 33 to 36, wherein the 3' untranslated region of Nfkbiz mRNA comprises the nucleotide sequence of SEQ ID NO: 7.

[38] The method of any of items 33 to 37, wherein the 3' untranslated region of Nfkbiz mRNA consists of a nucleotide sequence having about 90% or more identity to the nucleotide sequence of SEQ ID NO: 9.

[39] The method of any of items 33 to 38, wherein the 3' untranslated region of Nfkbiz mRNA consists of the nucleotide sequence of SEQ ID NO: 9.

[40] The method of any of items 33 to 39, wherein the candidate substance is designed based on a stem-loop structure present in the 3' untranslated region of Nfkbiz mRNA.

[41] A substance that disrupts the stem-loop structure in the 3' untranslated region of Nfkbiz mRNA.

[42] A method for activating immune cells, comprising a step of introducing at least one substance that disrupts the stem-loop structure in the 3' untranslated region of Nfkbiz mRNA into immune cells in vitro.

[43] Immune cells comprising at least one substance that disrupts the stem-loop structure in the 3' untranslated region of Nfkbiz mRNA.

[44] A composition for treating cancer, comprising immune cells containing at least one substance that disrupts a stem-loop structure in the 3' untranslated region of Nfkbiz mRNA.

[0091] All documents cited in this specification are incorporated herein by reference. The present invention will be described in more detail in the following examples, but the present invention is not limited to these examples. The above descriptions are all non-limiting, and the present invention is defined in the appended claims, and various modifications are possible within the scope of the technical idea thereof.

[0092] [Materials and Methods] Oligonucleotide Design Morpholino oligonucleotides with the following sequences were produced by GeneTools, LLC. Mouse Nfkbiz SL2-targeting morpholino oligo (SL2-mNfkbiz-MO); 5'-taactgacagtgagtgtcgc-3' (SEQ ID NO: 18) Mouse Nfkbiz SL3-targeting morpholino oligo (SL3-mNfkbiz-MO); 5'-aatacagggcaaatggtcta-3' (SEQ ID NO: 31) Mouse Nfkbiz SL4-targeting morpholino oligo (SL4-ASO); 5'-aactaaatgggtttgtttc-3' (SEQ ID NO: 40) Mouse Nfkbiz SL5-targeting morpholino oligo (SL5-ASO); 5'-atagtgaactaaatgggt-3' (SEQ ID NO: 41) Mouse Nfkbiz SL45-targeting morpholino oligo (SL45-mNfkbiz-MO, Nfkbiz-ASO, SL4 / 5-ASO); 5'-atagtgaactaaatgggtttgtttc-3' (SEQ ID NO: 22) Human NFKBIZ SL45-targeting morpholino oligo (SL45-hNFKBIZ-MO); 5'-atagtgaactaaatatgtttgtttc-3' (SEQ ID NO: 17)

[0093] Luciferase reporter assay: 1x10 HeLa cells ４Cells were seeded at 1000 cells / well in a 24-well plate. 24 hours later, control oligonucleotides (Standard Control, GeneTools, LLC) or Nfkbiz-targeting oligonucleotides were transfected using Endo-Porter (GeneTools, LLC) at a final concentration of 3 μM. Immediately afterward, pGL3-luciferase (no insert) or a vector containing the mouse Nfkbiz 3'UTR sequence downstream of the luciferase gene was transfected using Lipofectamine LTX Reagent (Thermo Fisher Scientific). Cells were harvested 24 hours later. Luciferase activity in the cell lysates was measured using the Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was normalized using the Renilla luciferase gene.

[0094] Mice Wild-type C57BL / 6JJcl mice were purchased from CLEA Japan, Inc. Regnase-1 flox mice (Uehata, T., et al. (2013). Cell 153, 1036-1049), Nfkbiz flox mice (Okuma A. et al., Immunity 2013 Mar 21;38(3):450-60), CreERT2 mice (Ventura A., et al., Nature 2007 Feb 8;445(7128):661-5), and OT-I mice (Hogquist KA, et al., Cell 1994 Jan 14;76(1):17-27) have been reported in previous studies. Regnase-3 knockout mice were generated by genome editing using the CRISPR-Cas9 method. Mice were raised under SPF conditions, and 8-12 week old mice were used for analysis.

[0095] Isolation of naive CD8+ T cells. Spleens and lymph nodes from C57BL / 6JJcl or OT-I mice were collected and crushed in staining buffer (PBS (pH 7.2) containing 0.5% BSA and 2 mM EDTA) to obtain a cell suspension. Erythrocytes were disrupted using ACK lysis buffer (Gibco). CD8+ T cells were isolated by negative selection using a Pan T Cell Isolation Kit II (Miltenyi Biotec) supplemented with biotin-anti-CD4 antibody. Subsequently, cells were stained with PE-anti-CD44 antibody, followed by negative selection using Anti-PE MicroBeads (Miltenyi Biotec), and the resulting cells were isolated as naive CD8+ T cells.

[0096] Cellular transfection of antisense nucleic acids. 2x10 mouse naive CD8+ T cells were placed in a 24-well plate coated with hamster IgG (MP Biochemicals). ５ Cells were plated at 1000 cells / well and stimulated with anti-CD3ε antibody (final concentration 0.25 μg / mL) and anti-CD28 antibody (final concentration 1.0 μg / mL) for 24 hours. Activated T cells were harvested, washed with PBS, and then electroporated with control or Nfkbiz target nucleic acids using the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza). Cells were then transferred to T-flasks and cultured for 3 days in RPMI medium (2-mercaptoethanol, penicillin-streptomycin, 10% FCS) containing IL-2 (10 ng / mL), IL-7 (2.5 ng / mL), and IL-15 (25 ng / mL).

[0097] Isolation of bone marrow-derived macrophages and oligonucleotide transfection. Femurs, tibias, and sacrum were collected from wild-type C57BL / 6JJcl mice and mashed in RPMI (2-mercaptoethanol, penicillin-streptomycin, 10% FCS) solution. Cells were collected by filtration through a cell strainer (100 μM). After centrifugation, the cell pellet was subjected to erythrocyte disruption using ACK lysis buffer (Gibco). After washing once with RPMI, bone marrow cells were cultured in the presence of M-CSF (20 ng / mL) for 6 days to isolate bone marrow-derived macrophages. After 6 days of culture, cells were harvested from the plate with 10 mM EDTA-containing PBS and further washed with PBS. Control oligonucleotides or Nfkbiz-targeted oligonucleotides were then electroporated using a P2 Primary Cell 4D-Nucleofector™ X Kit (Lonza). Thereafter, the cells were transferred to a 24-well plate and cultured in RPMI containing M-CSF (20 ng / mL) for 24 hours.

[0098] Measurement of cytokines and cytotoxic substances. After nucleic acid transfection, T cells were harvested, washed with PBS, and plated onto a 96-well U-bottom plate. These cells were stimulated with IL-12 (10 ng / mL) and IL-18 (1.0 ng / mL) for 4 hours in the presence of Golgi stop (BD Biosciences). After stimulation, cells were harvested and stained for cell surface antigens with anti-CD8a and anti-TCRβ. Subsequently, cells were fixed using fixation / permeabilization buffer and intracellularly stained for IFNγ, granzyme-b, and TNF. Cytokines were measured using a FACS (BD LSR Fortessa).

[0099] Cancer killing assay: Ovalbumin-expressing B16 cancer cell line (B16-OVA) was cultured in DMEM (supplemented with penicillin-streptomycin and 10% FCS), and then replaced with RPMI (supplemented with 2-mercaptoethanol, penicillin-streptomycin and 10% FCS) culture medium. ４The T cells were seeded into a 96-well flat-bottom plate at 100 cells / well. Subsequently, the transfected T cells were harvested and washed with PBS. After co-culture, the T cells were seeded onto the 96-well plate seeded with B16-OVA at a ratio of 1:1 to 1:16 relative to the number of B16-OVA cells. After 4 hours, the culture supernatant was collected. The OD490 of this culture was measured using the CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega). The OD490 measured in a control group without T cells was used as background and subtracted from each OD490 value. Antitumor activity was calculated as the % cytotoxicity, with the OD490 at which all cancer cells were killed being the maximum value.

[0100] [Results] Study 1: The Reg1 / 3-Nfkbiz regulatory axis plays a crucial role in regulating T cell activation. Nfkbiz mRNA is an important target gene for Regnase 1 and Regnase 3. To clarify the role of Nfkbiz as a Regnase target gene in vivo, we performed the following analysis using a tamoxifen-inducible gene deletion system in CreERT2 mice. Gene deletion was performed by intraperitoneal administration of 2 mg tamoxifen in 100 μL of corn oil for 5 consecutive days, followed by analysis on day 14. The genotypes used in the analysis and the experimental schedule are shown in Figure 1. As a result, mice doubly deficient in Regnase-1 and Regnase-3 (DKO) exhibited significant splenomegaly. However, mice deficient in Regnase 1 and Regnase 3 as well as Nfkbiz (TKO) did not exhibit splenomegaly, as did wild-type mice (Figure 2). Next, flow cytometry (FACS) analysis was performed using spleen cells from the mice. Figure 3 shows the CD62L expression in CD4-positive T cells. ＋ CD44 － Naive T cells and CD62L － CD44 ＋The figures show the expression levels of effector T cells and the activation marker CD69. Compared to wild-type mice, DKO spleen cells showed an increase in effector T cells and higher CD69 expression levels. However, these phenotypes were not different between TKO mice and wild-type mice, and no splenomegaly was observed. Similarly, CD8-positive T cells (Figure 4) were also expressed in DKO spleen cells, suggesting that CD62L － CD44 ＋ Although effector T cells were significantly increased in TKO mice, no difference was observed between the control and TKO mice. These results suggest that Regnase-1 and Regnase-3 regulate T cell activation by suppressing Nfkbiz expression.

[0101] Test 2: Immunostimulatory Strategy Targeting Nfkbiz mRNA Instability Elements The nucleotide sequences of the 3' untranslated regions of the Nfkbiz genes of various species were aligned. Figure 5 shows the alignment of nucleotides 29-148 and the regions that may form secondary structures (SL2-5). Figure 6 is a schematic diagram showing the stem-loop structure (SL4 and 5) present in the 3' untranslated region of mouse Nfkbiz mRNA and the disruption of this secondary structure by antisense nucleic acids. As shown in the right diagram of Figure 6, the antisense nucleic acid that binds complementarily to the region from a portion of SL4 (the 3' stem portion of SL4) to a portion of SL5 (the 5' stem portion of SL5) in the 3' untranslated region of mouse Nfkbiz mRNA is referred to as SL45-mNfkbiz-MO.

[0102] Luciferase reporter assays were performed using a vector containing the mouse Nfkbiz 3'UTR sequence inserted downstream of the luciferase gene of pGL3-luciferase. The method and results are shown in Figure 7. HeLa cells transfected with Nfkbiz-targeting morpholino oligos against SL2 or SL4 and 5 (SL2-mNfkbiz-MO or SL45-mNfkbiz-MO) showed significantly higher luciferase activity than HeLa cells transfected with a control morpholino oligo (control-MO). HeLa cells transfected with Nfkbiz-MO against SL3 (SL3-Nfkbiz-MO) showed no difference in luciferase activity compared to HeLa cells transfected with control-MO.

[0103] A similar luciferase reporter assay was performed using a vector in which the human NFKBIZ 3'UTR sequence was inserted downstream of the luciferase gene of pGL3-luciferase. The method and results are shown in Figure 8. Luciferase activity was significantly higher in HeLa cells transfected with NFKBIZ-targeting morpholino oligos against SL4 and 5 (SL45-hNFKBIZ-MO) than in HeLa cells transfected with a control morpholino oligo (control-MO).

[0104] These results suggest that disruption of a specific stem-loop structure in the 3' untranslated region of the Nfkbiz gene increases Nfkbiz expression. Figure 9 shows a schematic diagram of changes in Nfkbiz gene expression caused by antisense nucleic acids.

[0105] Test 3: Nfkbiz-targeted nucleic acid medicine exerts a cancer-suppressing effect. Figure 10 is a schematic diagram showing the antitumor effect of cytotoxic T cells. As shown in Figure 11, OT-I CD8-positive T cells were transfected with SL45-mNfkbiz-MO or control-MO and cultured for 72 hours before undergoing cytokine measurement and cancer-killing assay.

[0106] The expression levels of IFNγ, granzyme-b, and TNF were assessed by FACS. Figure 12 shows the percentage (upper panel) and mean fluorescence intensity (MFI) (lower panel) of IFNγ-, granzyme-b-, and TNF-producing CD8 T cells. The expression levels of IFNγ and granzyme-b were significantly higher in SL45-mNfkbiz-MO-transfected cells than in the control group.

[0107] The results of the cancer killing assay are shown in Figure 13. The antitumor activity of OT-ICD8 T cells against the B16-OVA cell line (melanoma cell line) was compared between the SL45-mNfkbiz-MO-transfected group and the control-MO-transfected group. The cytotoxicity of the SL45-mNfkbiz-MO-transfected group was higher than that of the control-MO-transfected group, and increased in a dose-dependent manner.

[0108] Test 4: Nfkbiz targeting nucleic acid exhibits sustained enhancement of Nfkbiz protein expression in CD8+ T cells. CD62L isolated from spleens and lymph nodes of wild-type C57BL / 6JJcl mice ＋ CD44 － Naive CD8+ T cells were stimulated with anti-CD3ε antibody (final concentration 0.25 μg / mL) and anti-CD28 antibody (final concentration 1.0 μg / mL) for 24 hours, and then Nfkbiz-ASO or control oligonucleotide (Control-oligo) was introduced by electroporation at 10 μM or 50 μM. Changes in Nfkbiz expression over time after ASO introduction were evaluated by Western blot. The results are shown in Figure 14. Nfkbiz-ASO demonstrated a sustained enhancement of Nfkbiz protein expression in CD8+ T cells.

[0109] Test 5: Nfkbiz-targeting nucleic acids increase Nfkbiz expression in macrophages and enhance inflammatory responses. Bone marrow-derived macrophages from wild-type C57BL / 6JJcl mice were transfected with 50 μM Nfkbiz-ASO or control oligonucleotide, followed by stimulation with LPS (100 ng / mL). Nfkbiz expression after stimulation was quantified over time by qPCR. Furthermore, Nfkbiz expression levels at 24 hours were evaluated by Western blot. The results are shown in Figures 15 and 16. Nfkbiz-ASO sustainedly enhanced Nfkbiz expression in bone marrow-derived macrophages.

[0110] As described above, mouse bone marrow-derived macrophages were transfected with Nfkbiz-ASO or control oligonucleotide and then stimulated with LPS (100 ng / mL). Two hours after LPS stimulation, actinomycin D (5 μg / mL) was added, and Nfkbiz expression was then assessed by qPCR. The results are shown in Figure 17. Nfkbiz-ASO suppressed the attenuation of Nfkbiz expression in the presence of a transcription inhibitor, suggesting that the increase in Nfkbiz expression by Nfkbiz-ASO was due to the inhibition of Nfkbiz degradation.

[0111] As described above, mouse bone marrow-derived macrophages were transfected with Nfkbiz-ASO or control oligonucleotide, and then stimulated with LPS (100 ng / mL) for 24 hours. IL-6 concentrations in the culture supernatants were assessed by ELISA. The results are shown in Figure 18. These results suggest that Nfkbiz-ASO increases IL-6 production and enhances the inflammatory response.

[0112] Mouse bone marrow-derived macrophages were transfected with 50 μM SL4-mNfkbiz-MO (SL4-ASO), SL5-mNfkbiz-MO (SL5-ASO), SL45-mNfkbiz-MO (SL4 / 5-ASO), or control oligos by electroporation. A schematic diagram of each MO is shown in Figure 19. Twenty-four hours after transfection, Nfkbiz expression levels were assessed by qPCR and Western blot. The results are shown in Figures 20 and 21. Nfkbiz degradation tended to be suppressed by the use of morpholino oligos targeting SL4 and SL5 separately, whereas Nfkbiz degradation was strongly suppressed by morpholino oligos simultaneously targeting SL4 and SL5.

[0113] Study 6: Nfkbiz-ASO-transduced cytotoxic T cells exert tumor regression effects in vivo. 5x10 ５ The B16-OVA cell line was inoculated subcutaneously into the flank. Meanwhile, ovalbumin (OVA)-specific naive CD8-positive T cells were inoculated into the OT-1 ＋ Rosa － E2a-Td-tomato ＋ RAG2 －／－ Tumors were isolated from the spleen and lymph nodes of mice and stimulated with anti-CD3ε antibody / anti-CD28 antibody. 24 hours later, Nfkbiz-ASO or control-oligo was introduced by electroporation and cultured for 4 days in RPMI medium containing IL-2 / IL-7 / IL-15. On the 7th day after tumor inoculation, 5x10 cells were injected into mice matched for tumor size. ６ The aforementioned T cells or 200 μL of PBS (control) were injected via the orbital vein (N=3). Tumor size was measured over time. The workflow of this study is shown in Figure 22, and the results are shown in Figure 23. Tumor growth was suppressed in mice injected with cytotoxic T cells transfected with Nfkbiz-ASO.

[0114] Test 7: Enhancement of Nfkbiz expression by Nfkbiz-ASO suppresses T cell exhaustion. Nfkbiz-ASO was introduced into C57BL / 6JJcl mouse CD8-positive T cells by electroporation and cultured for 4 days in the presence of IL-2 / IL-7 / IL-15. After culture, the cells were collected, and the expression of PD-1, LAG-3, TOX, and TCF-1 was evaluated by flow cytometry. PD-1, LAG-3, and TOX are markers that indicate T cell exhaustion, and TCF-1 is a stem cell-like memory T cell marker. The results are shown in Figure 24. In T cells transduced with Nfkbiz-ASO (upper right and lower right), compared to T cells transduced with control-oligo (upper left and lower left), both the PD-1-positive / LAG-3-positive cell population (cells within the box in the upper diagram) and the TOX-positive / TCF1-positive cell population (cells within the box in the lower diagram) were reduced (numbers in the diagram indicate the percentage of cells within the box). These results indicate that T cell exhaustion can be suppressed by enhancing Nfkbiz expression by Nfkbiz-ASO.

[0115] Test 8: Increased NFKBIZ expression in PBMCs after transfection with human NFKBIZ-ASO After transfection with Nfkbiz-ASO into human peripheral blood mononuclear cells (PBMCs) by electroporation, the expression level of NFKBIZ protein in the PBMCs was analyzed by Western blot. The results are shown in Figure 25. NFKBIZ protein expression was increased in PBMCs transfected with Nfkbiz-ASO compared to PBMCs transfected with control oligonucleotide.

[0116] These results suggest that disruption of a specific stem-loop structure in the 3' untranslated region of the Nfkbiz gene enhances the antitumor activity of cytotoxic T cells.

[0117] The present disclosure provides a method for stimulating immunity and a method for treating cancer, which are useful in the medical field.

Claims

1. A composition for treating cancer, comprising at least one substance that disrupts a stem-loop structure in the 3' untranslated region of Nfkbiz mRNA, A composition, wherein the stem loop structure is at least one stem loop structure selected from the following stem loop structures: a first stem-loop structure formed in a region corresponding to positions 34 to 50 of SEQ ID NO:9; A second stem-loop structure formed in a region corresponding to positions 100 to 116 of SEQ ID NO:9; and A third stem-loop structure formed in a region corresponding to positions 129 to 143 of SEQ ID NO:

9.

2. The composition of claim 1 , wherein at least one stem-loop structure selected from the second stem-loop structure and the third stem-loop structure is disrupted.

3. The composition of claim 1 , wherein the second stem-loop structure and the third stem-loop structure are disrupted.

4. The composition of claim 1 , wherein the 3′ untranslated region of the Nfkbiz mRNA comprises a nucleotide sequence having about 90% or greater identity to the nucleotide sequence of SEQ ID NO:

5.

5. The composition of claim 1 , wherein the 3′ untranslated region of Nfkbiz mRNA comprises a nucleotide sequence having about 90% or greater identity to the nucleotide sequence of SEQ ID NO:

7.

6. At least one substance that disrupts the stem-loop structure, (a) a first oligonucleotide, (a-1) an oligonucleotide consisting of 10 to 30 nucleotides, which binds to a sequence consisting of 10 to 30 consecutive nucleotides including positions 35 to 37 of SEQ ID NO: 9, in the nucleotide sequence consisting of positions 8 to 48 of SEQ ID NO: 9; and (a-2) an oligonucleotide consisting of 10 to 30 nucleotides, which binds to a sequence consisting of 10 to 30 consecutive nucleotides including positions 47 to 49 of SEQ ID NO: 9, in the nucleotide sequence consisting of positions 36 to 76 of SEQ ID NO: 9; An oligonucleotide selected from (b) a second oligonucleotide, (b-1) an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides including positions 104 to 106 of SEQ ID NO: 9 in the nucleotide sequence consisting of positions 77 to 111 of SEQ ID NO: 9; and (b-2) an oligonucleotide consisting of 10 to 30 nucleotides, which binds to a sequence consisting of 10 to 30 consecutive nucleotides including positions 110 to 112 of SEQ ID NO: 9, in the nucleotide sequence consisting of positions 105 to 139 of SEQ ID NO: 9; An oligonucleotide selected from (c) a third oligonucleotide, (c-1) an oligonucleotide consisting of 10 to 30 nucleotides that binds to a sequence consisting of 10 to 30 consecutive nucleotides including positions 132 to 134 of SEQ ID NO: 9 in the nucleotide sequence consisting of positions 105 to 139 of SEQ ID NO: 9; and (c-2) an oligonucleotide consisting of 10 to 30 nucleotides, which binds to a sequence consisting of 10 to 30 consecutive nucleotides including positions 138 to 140 of SEQ ID NO:9, in the nucleotide sequence consisting of positions 133 to 167 of SEQ ID NO:9; An oligonucleotide selected from (d) a fourth oligonucleotide, the fourth oligonucleotide consisting of 25 to 35 nucleotides, which binds to a sequence consisting of 25 to 35 consecutive nucleotides including positions 110 to 134 of SEQ ID NO:9, among the nucleotide sequence of positions 105 to 139 of SEQ ID NO:9; (e) a combination of at least two oligonucleotides selected from a first oligonucleotide, a second oligonucleotide, and a third oligonucleotide; or (f) a combination of the first oligonucleotide and the fourth oligonucleotide; 2. The composition of claim 1 ,

7. At least one substance that disrupts the stem-loop structure, (a) a first oligonucleotide, (a-1) an oligonucleotide consisting of a nucleotide sequence having 90% or more identity with SEQ ID NO: 11, which binds to a sequence consisting of 10 to 30 consecutive nucleotides including positions 35 to 37 of SEQ ID NO: 9, among the nucleotide sequence of positions 8 to 48 of SEQ ID NO: 9; and (a-2) an oligonucleotide consisting of a nucleotide sequence having 90% or more identity to SEQ ID NO: 12, which binds to a sequence consisting of 10 to 30 consecutive nucleotides including positions 47 to 49 of SEQ ID NO: 9, among the nucleotide sequence of positions 36 to 76 of SEQ ID NO: 9; An oligonucleotide selected from (b) a second oligonucleotide, (b-1) an oligonucleotide consisting of a nucleotide sequence having 90% or more identity to SEQ ID NO: 13, which binds to a sequence consisting of 10 to 30 consecutive nucleotides including positions 104 to 106 of SEQ ID NO: 9, among the nucleotide sequence of positions 77 to 111 of SEQ ID NO: 9; and (b-2) an oligonucleotide consisting of a nucleotide sequence having 90% or more identity to SEQ ID NO: 38, which binds to a sequence consisting of 10 to 30 consecutive nucleotides including positions 110 to 112 of SEQ ID NO: 9, among the nucleotide sequence of positions 105 to 139 of SEQ ID NO: 9; An oligonucleotide selected from (c) a third oligonucleotide, (c-1) an oligonucleotide consisting of a nucleotide sequence having 90% or more identity to SEQ ID NO: 39, which binds to a sequence consisting of 10 to 30 consecutive nucleotides including positions 132 to 134 of SEQ ID NO: 9, among the nucleotide sequence of positions 105 to 139 of SEQ ID NO: 9; and (c-2) an oligonucleotide consisting of a nucleotide sequence having 90% or more identity to SEQ ID NO: 16, which binds to a sequence consisting of 10 to 30 consecutive nucleotides including positions 138 to 140 of SEQ ID NO: 9, among the nucleotide sequence of positions 133 to 167 of SEQ ID NO: 9; An oligonucleotide selected from (d) a fourth oligonucleotide, which binds to a sequence consisting of 25 to 35 consecutive nucleotides including positions 110 to 134 of SEQ ID NO:9, among the nucleotide sequence of positions 105 to 139 of SEQ ID NO:9, and which has a nucleotide sequence having 90% or more identity to SEQ ID NO:17; (e) a combination of at least two oligonucleotides selected from a first oligonucleotide, a second oligonucleotide, and a third oligonucleotide; or (f) a combination of the first oligonucleotide and the fourth oligonucleotide; 2. The composition of claim 1 ,

8. The composition of claim 6 , wherein the at least one substance that disrupts the stem-loop structure is a combination of a second oligonucleotide and a third oligonucleotide.

9. The composition of claim 6 , wherein the at least one substance that disrupts the stem-loop structure is a fourth oligonucleotide.

10. The composition according to any one of claims 1 to 9, wherein the cancer is malignant melanoma, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, classical Hodgkin's lymphoma, head and neck cancer, gastric cancer, malignant pleural mesothelioma, colorectal cancer, esophageal cancer, cancer of unknown primary site, urothelial carcinoma, hepatocellular carcinoma, solid cancer, breast cancer, endometrial cancer, Merkel cell carcinoma, brain tumor, bile duct cancer, kidney cancer or colon cancer.

11. A composition for stimulating immunity, comprising at least one substance that disrupts a stem-loop structure in the 3' untranslated region of Nfkbiz mRNA, A composition, wherein the stem loop structure is at least one stem loop structure selected from the following stem loop structures: a first stem-loop structure formed in a region corresponding to positions 34 to 50 of SEQ ID NO:9; A second stem-loop structure formed in a region corresponding to positions 100 to 116 of SEQ ID NO:9; and A third stem-loop structure formed in a region corresponding to positions 129 to 143 of SEQ ID NO:9.

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