JP2021070683A - Antitumor drug for fibrosarcoma, pharmaceutical composition for treating fibrosarcoma, antitumor effect potentiator, and pharmaceutical composition used for potentiating antitumor effect on fibrosarcoma - Google Patents

Abstract

To provide a novel antitumor drug and a novel pharmaceutical composition for treating tumor.SOLUTION: The present disclosure provides an antitumor drug or a pharmaceutical composition for treating tumor containing at least one component selected from the group consisting of (a) interleukin (IL)-36β or a modified body thereof; (b) a polynucleotide encoding IL-36β or a modified body thereof; (c) an IL-36β expression vector containing the (b) polynucleotide; and (d) a cell that secretes IL-36β or a modified body thereof.SELECTED DRAWING: None

Description

The present invention relates to antitumor agents and pharmaceutical compositions for treating tumors. It also relates to an antitumor effect enhancer and a pharmaceutical composition for enhancing the antitumor effect of IL-12.

Cancer is the leading cause of death in Japan, and the number of patients is large worldwide. Therefore, the development of better therapeutic agents is desired.

Interleukin (IL) -36 is a cytokine reported as a molecule homologous to IL-1 by analysis of a DNA database. IL-36, together with IL-1, IL-18, IL-33, IL-37, and IL-38, constitutes the IL-1 cytokine family. IL-36 is a secretory protein secreted from a specific producing cell, but the IL-36 gene does not have a sequence corresponding to the signal sequence, and the secretory mechanism has not been elucidated. The receptor for IL-36 consists of a heterodimer of the IL-36 receptor (IL-36R) and the IL-1 accessory protein (IL-1RAcP).

IL-36 has three isoforms as splicing variants, IL-36α, IL-36β and IL-36γ. IL-36β is mainly produced from epithelial cells, monocytes, B cells and the like. It has been reported that the truncated type IL-36β from which 30 amino acids at the N-terminal are removed has about 1000 times stronger activity than the natural type full-length IL-36β (Non-Patent Document 1). .. It has been reported that IL-36β acts on dendritic cells to enhance the production of IL-6, IL-12, GM-CSF, etc., and enhances the expression of MHC class II, CD80, CD86, etc. on the cell surface. (Non-Patent Document 2).

Towne JE, et al., Interleukin-36 (IL-36) ligands require processing for full agonist (IL-36α, IL-36β, and IL-36γ) or antagonist (IL-36Ra) activity. J Biol Chem. 2011 Dec 9; 286 (49): 42594-602. Vigne S, et al., IL-36R ligands are potent regulators of dendritic and T cells. Blood. 2011 Nov 24; 118 (22): 5813-23.

Although various antitumor agents have been developed so far, their effectiveness is often insufficient. In addition, cancer cells may acquire resistance to antitumor agents, making it difficult to continue treatment with the same antitumor agents. Therefore, there is a need for the development of a new antitumor agent capable of suppressing tumor growth by a mechanism different from that of conventional antitumor agents.

Therefore, an object of the present invention is to provide a novel antitumor agent and a novel pharmaceutical composition for treating a tumor. Another object of the present invention is to provide a novel drug and a pharmaceutical composition for enhancing the antitumor effect of IL-12.

The present invention includes the following aspects.
[1] (a) Interleukin (IL) -36β or a variant thereof; (b) A polynucleotide having a base sequence encoding IL-36β or a variant thereof; (c) Containing the polynucleotide of (b) above. An antitumor agent comprising at least one component selected from the group consisting of vectors; and (d) cells secreting IL-36β or variants thereof.
[2] The polynucleotide of (b) further has a base sequence encoding a signal peptide of a secretory protein, and the base sequence encoding the signal peptide is a base sequence encoding the IL-36β or a variant thereof. The antitumor agent according to [1], which is directly or indirectly linked in-frame to the 5'end of.
[3] The antitumor agent according to [1] or [2], which is administered in combination with IL-12.
[4] (a) IL-36β or a variant thereof; (b) a polynucleotide having a base sequence encoding IL-36β or a variant thereof; (c) a vector containing the polynucleotide of (b) above; and ( d) A pharmaceutical composition for treating a tumor, which comprises at least one component selected from the group consisting of cells secreting IL-36β or a variant thereof, and a pharmaceutically acceptable carrier.
[5] The polynucleotide of (b) further has a base sequence encoding a signal peptide of a secretory protein, and the base sequence encoding the signal peptide is a base sequence encoding the IL-36β or a variant thereof. The pharmaceutical composition according to [4], which is directly or indirectly linked in-frame to the 5'end of.
[6] The pharmaceutical composition according to [4] or [5], which is administered in combination with IL-12.
[7] The pharmaceutical composition according to [4] or [5], further comprising IL-12.
[8] (a) Interleukin (IL) -36β or a variant thereof; (b) A polynucleotide having a base sequence encoding IL-36β or a variant thereof; (c) Containing the polynucleotide of (b) above. An antitumor used to enhance the antitumor effect of IL-12, containing at least one component selected from the group consisting of a vector; and (d) a cell secreting IL-36β or a variant thereof. Effect enhancer.
[9] (a) Interleukin (IL) -36β or a variant thereof; (b) A polynucleotide having a base sequence encoding IL-36β or a variant thereof; (c) Containing the polynucleotide of (b) above. The antitumor effect of IL-12, containing at least one component selected from the group consisting of the vector; and (d) cells secreting IL-36β or variants thereof, and a pharmaceutically acceptable carrier. A pharmaceutical composition used to enhance.

According to the present invention, a novel antitumor agent and a novel pharmaceutical composition for treating a tumor are provided. Also provided are novel agents and pharmaceutical compositions for enhancing the antitumor effect of IL-12.

The outline of the construction method of pDFG-hPTH-truncated IL-36β-IRES-Neo is shown. The vector map of pDFG-hPTH-truncated IL-36β-IRES-Neo is shown. The outline of the construction method of pDFG-hGH-truncated IL-36β-IRES-Neo is shown. The vector map of pDFG-hGH-truncated IL-36β-IRES-Neo is shown. The vector map of pMFG-IRES-Neo is shown. The results of RT-PCR performed using the IL-36β amplification primer after introducing the IL-36β gene into mouse cancer cells (MCA205, B16-F10, MC38) are shown. The amount of IL-6 produced when the culture supernatant of IL-36β gene-introduced cells was allowed to act on mouse normal cell line lung fibroblasts (NMLF) is shown. In the figure, "IL-36beta" indicates IL-36β purchased from R & D Systems, Minneapolis, MN. The IL-36β was allowed to act on NMLF at the concentration in parentheses. "Full lens IL-36beta" is a mouse full-length IL-36β gene-introduced cell, and "hPTH truncated IL-36beta" is a polynucleotide in which a human parathyroid hormone signal sequence is added to a mouse IL-36β gene from which the 5'terminal side has been removed. The introduced cells, "hGH truncated IL-36beta", indicate the introduced cells of the polynucleotide in which the human growth hormone signal sequence is added to the mouse IL-36β gene from which the 5'terminal side has been removed. The results of comparing cell proliferation in IL-36β gene, truncated IL-36β gene-introduced cells and wild strain (WT) are shown. The tumor growth when the tumor cells into which the truncated IL-36β gene was introduced was administered to mice is shown. As the administration cells, cells in which the IL-36β gene was introduced into MCA205 were used. (A) As the administered cells, cells in which the truncated IL-36β gene was introduced into MCA205 were used. In (B), cells in which the truncated IL-36β gene was introduced into MC38 were used. The tumor growth when the tumor cells into which the truncated IL-36β gene was introduced was administered to mice is shown. As the administered cells, the IL-36β gene was introduced into B16-F10 and the cells were used. (A) shows the results in mice inoculated with ^{3 × 10 5 cells.} (B) shows the results in mice inoculated with ^{6 × 10 5 cells.} It shows a synergistic effect of suppressing tumor growth when a tumor cell into which the truncated IL-36β gene has been introduced and an IL-12 protein are administered to mice. As the administration cells, cells in which the hGH-truncated IL-36β gene was introduced into MCA205 were used. The liver weight on the 14th day (day 14) after the administration of the tumor cells into which the truncated IL-36β gene was introduced into the spleen of the mouse is shown. As the administration cells, cells in which the IL-36β gene was introduced into MC38 were used. The number of tumor nodules in the liver on the 14th day after administration (day 14) when tumor cells into which the truncated IL-36β gene was introduced into the spleen of a mouse is shown. As the administration cells, cells in which the truncated IL-36β gene was introduced into MC38 were used.

[Definition]
"Polynucleotide" refers to a nucleotide polymer in which nucleotides are attached by phosphodiester bonds. The "polynucleotide" may be DNA, RNA, or may be composed of a combination of DNA and RNA. A "polynucleotide" may be a polymer of natural nucleotides, a nucleotide in which at least one of a natural nucleotide and an unnatural nucleotide (an analog of a natural nucleotide, a base moiety, a sugar moiety and a phosphate moiety is modified). It may be a polymer with (for example, a phosphorothioate skeleton), etc., or it may be a polymer of unnatural nucleotides.
Unless otherwise specified, the base sequence of a "polynucleotide" is described by a generally accepted one-letter code. Unless otherwise specified, the base sequence is described from the 5'side to the 3'side.
Nucleotide residues constituting the "polynucleotide" may be simply described by adenine, thymine, cytosine, guanine, uracil, etc., or a single character code thereof.

"Gene" refers to a polynucleotide that contains at least one open reading frame that encodes a particular protein. The gene can include both exons and introns.

The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to polymers of amino acids linked by amide bonds. The "peptide", "polypeptide" and "protein" may be a polymer of natural amino acids, or may be a polymer of natural amino acids and unnatural amino acids (chemical analogs of natural amino acids, modified derivatives, etc.). It may be a polymer of unnatural amino acids.
Unless otherwise specified, the amino acid sequences of "peptide", "polypeptide" and "protein" are described by a generally accepted one-letter code or three-letter code. Unless otherwise specified, the amino acid sequence is described from the N-terminal side to the C-terminal side.
"Peptide" refers to a short amino acid polymer of about 50 amino acids or less, unless otherwise stated. "Polypeptide" and "protein" refer to amino acid polymers of about 50 amino acids or more, unless otherwise stated.

The term "operably linked" used with respect to a polynucleotide means that the first base sequence is located sufficiently close to the second base sequence and the first base sequence is the second base sequence or the second base sequence. It means that it can affect the area under the control of. For example, operably ligating a polynucleotide to a promoter means that the polynucleotide is ligated to be expressed under the control of the promoter.
The term "expressible state" refers to a state in which the gene of interest can be transcribed in the cell into which it has been introduced.
The term "expression vector" refers to a vector containing a target gene and comprising a system for making the target gene expressable in the cell into which the vector has been introduced.
"Promoter can function" means that a polynucleotide operably linked to the promoter can be expressed in the cells of the organism of interest.

"In-frame" means that the codon reading frame does not shift.

"IL-36β" refers to the protein of Il-36β. "IL-36β gene" refers to a gene encoding IL-36β.

Proteins, peptides, polynucleotides, vectors, and cells can be isolated. "Isolated" means a state isolated from the natural state. The proteins, peptides, polynucleotides, vectors, and cells described herein can be isolated polypeptides, isolated polynucleotides, isolated vectors, and isolated cells.

The "sequence identity" between amino acid sequences or base sequences is that two amino acid sequences or base sequences are juxtaposed with gaps in the parts corresponding to insertions and deletions so that the corresponding amino acids or bases match most. It is determined as the ratio of matching amino acids or bases to the entire amino acid sequence or base sequence excluding gaps in the obtained alignment. The sequence identity between amino acid sequences or base sequences can be determined by using various homology search software known in the art. For example, the amino acid sequence sequence identity value can be obtained by calculation based on the alignment obtained by the known homology search software BLASTP, and the base sequence sequence identity value can be obtained by the known homology search. It can be obtained by calculation based on the alignment obtained by the software BLASTN. Sequence identity is also referred to as homology.

Examples of the "stringent condition" include the method described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION (Sambrook et al., Cold Spring Harbor Laboratory Press). Stringent conditions include, for example, 6 × SSC (composition of 20 × SSC: 3M sodium chloride, 0.3M citric acid solution, pH 7.0), 5 × Denhardt solution (composition of 100 × Denhardt solution: 2% by mass). 42-70 ° C. in a hybridization buffer consisting of bovine serum albumin, 2% by mass ficol, 2% by mass polyvinylpyrrolidone), 0.5% by mass SDS, 0.1 mg / mL salmon sperm DNA, and 50% formamide. Conditions for hybridization can be mentioned by incubating for several hours to overnight. Examples of the washing buffer used for washing after incubation include a 1 × SSC solution containing 0.1% by mass SDS, and more preferably a 0.1 × SSC solution containing 0.1% by mass SDS.

[Anti-tumor agent]
In one embodiment, the present invention provides an antitumor agent. The antitumor agent is (a) interleukin (IL) -36β or a variant thereof; (b) a polynucleotide having a base sequence encoding IL-36β or a variant thereof; (c) the poly of (b) above. It contains at least one component selected from the group consisting of expression vectors containing nucleotides; and (d) cells secreting IL-36β or variants thereof.

As shown in Examples described later, tumor growth was suppressed in mice administered with tumor cells into which the IL-36β gene was introduced. This result indicates that the antitumor effect was exerted by the action of IL-36β secreted from the IL-36β-introduced cells. Therefore, the components (a) to (d) can be used as an antitumor agent.

The antitumor agent of the present embodiment can be administered to a subject having a tumor or a subject having a high risk of developing a tumor. The organism to which the antitumor agent is administered is not particularly limited, and examples thereof include humans and mammals other than humans. Examples of mammals other than humans include primates (monkeys, chimpanzees, gorillas, etc.), rodents (mouses, hamsters, rats, etc.), rabbits, dogs, cats, cows, goats, sheep, horses, pigs, and the like. However, it is not limited to these. Suitable administration targets include humans and mice.

<(A) IL-36β>
IL-36β is a cytokine belonging to the IL-1 cytokine family. IL-36β is mainly secreted from keratinocytes, nerve cells, tracheal epithelial cells, monocytes and the like. IL-36β binds to a receptor consisting of heterodimers of IL-36R and IL-1RAcp. IL-36β activates intracellular signal transduction by binding to the receptor and enhances the production of IL-6, IL-12, GM-CSF and the like.
Although the detailed mechanism of the antitumor effect of IL-36β is not clear, it is considered that cytokines induced by IL-36β or IL-36β act on various immune cells to enhance the antitumor immune response.

The organism from which IL-36β is derived is not particularly limited, but is preferably the same species as the organism to which the antitumor agent is administered. By using IL-36β of an organism of the same type as the subject to which the antitumor agent is administered, the immune response to IL-36β can be suppressed. For example, when the administration target of the antitumor agent is a human, it is preferable to use human IL-36β. When the administration target of the antitumor agent is a mouse, it is preferable to use mouse IL-36β.

IL-36β may be a natural type or a modified type. Natural IL-36β is IL-36β produced in nature. Natural IL-36β is, for example, IL-36β produced in the body of mammals as described above. Preferred examples of the natural IL-36β include human IL-36β and mouse IL-36β. An example of the amino acid sequence of human IL-36β (NCBI Gene ID: 27177) is SEQ ID NO: 2 (SEQ ID NO: 1 is the base sequence encoding the amino acid sequence), and SEQ ID NO: 4 (SEQ ID NO: 3 encodes the amino acid sequence). Base sequence). An example of the amino acid sequence of mouse IL-36β (NCBI Gene ID: 69677) is shown in SEQ ID NO: 6 (SEQ ID NO: 5 is the base sequence encoding the amino acid sequence). The natural IL-36β is not limited to those having the amino acid sequence, and may be homologs (orthologs, paralogs) thereof. The amino acid sequence of native IL-36β can be obtained from a sequence database such as GenBank. In the present specification, the natural IL-36β may be described as “full length IL-36β” or “full lens IL-36β”.

Modified IL-36β is a modified protein of natural IL-36β. The modified IL-36β has an amino acid sequence in which one or more amino acids have been modified (substitution, deletion, addition, insertion, or a combination thereof) as compared with the amino acid sequence of the natural IL-36β. The number of the modified amino acids is not particularly limited as long as it functions as IL-36β, and is, for example, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 1. 10 or 1 to 5 or the like can be mentioned. The modified IL-36β may have an amino acid sequence having 80% or more sequence identity with the amino acid sequence of the natural IL-36β. The sequence identity may be, for example, 85% or more, 90% or more, or 95% or more. The modified IL-36β may be referred to as a variant of IL-36β. The term "IL-36β or a variant thereof" includes both natural Il-36β and modified Il-36β.

The modified IL-36β preferably has an activity equal to or higher than that of the natural IL-36β. Examples of the activity of natural IL-36β (hereinafter, also referred to as “IL-36β activity”) include binding activity to a receptor consisting of a heterodimer of IL-36R and IL-1RAcp, and a cell having the receptor. Examples thereof include an activity of causing (for example, lung fibroblasts) to produce IL-6, an antitumor activity, and the like. The modified IL-36β having the same or higher activity as the natural IL-36β has, for example, 80 or more, 85 or more, 90 or more, or 95 or more activity when the activity exhibited by the natural IL-36β is 100. Shown.

In mouse IL-36β, it has been reported that the modified IL-36β from which 30 amino acids at the N-terminal have been removed exhibits about 1000 times stronger activity than the natural IL-36β (Towne JE, et al., J Biol Chem. 2011 Dec 9; 286 (49): 42594-602.). It has also been reported that in human IL-36β, the modified IL-36β from which the four N-terminal amino acids have been removed exhibits stronger activity than the natural IL-36β (Towne JE, et. al., J Biol Chem. 2011 Dec 9; 286 (49): 42594-602.). Therefore, a preferable example of the modified IL-36β is one in which the amino acid in the N-terminal region of the natural IL-36β is removed (hereinafter, also referred to as “truncated IL-36β”). The number of N-terminal amino acids removed is, for example, 40 or less, 35 or less, or 30 or less. The number of N-terminal amino acids removed is, for example, 1-40, 5-35, or 10-30 in the case of mouse IL-36β. The number of N-terminal amino acids removed is, for example, 1-40, 2-30, 3-20, or 4-10 in the case of human IL-36β. An example of the amino acid sequence of human truncated IL-36β is shown in SEQ ID NO: 8 (SEQ ID NO: 7 is the base sequence encoding the amino acid sequence) and SEQ ID NO: 10 (SEQ ID NO: 9 is the base sequence encoding the amino acid sequence). .. An example of the amino acid sequence of mouse truncated type IL-36β is shown in SEQ ID NO: 12 (SEQ ID NO: 11 is the base sequence encoding the amino acid sequence).

<(B) Polynucleotide>
The polynucleotide (b) has a base sequence encoding IL-36β or a variant thereof (hereinafter, referred to as “IL-36β coding sequence”). The IL-36β coding sequence is the base sequence of the IL-36β gene. Therefore, it can be said that the polynucleotide (b) is a polynucleotide containing the IL-36β gene.

≪IL-36β coding sequence≫
The IL-36β coding sequence may encode native IL-36β or may encode modified IL-36β. Examples of the polynucleotide (b) include the following polynucleotides.
(1) A polynucleotide encoding a polypeptide having the amino acid sequence of natural IL-36β (eg, SEQ ID NO: 2, 4 or 6).
(2) An amino acid sequence in which one or more amino acids are modified (deletion, substitution, addition, insertion, or a combination thereof) in the amino acid sequence of natural IL-36β (eg, SEQ ID NO: 2, 4 or 6). A polynucleotide encoding a polypeptide having and having IL-36β activity.
(3) A poly comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence of natural IL-36β (eg, SEQ ID NO: 2, 4 or 6) and encoding a polypeptide having IL-36β activity. nucleotide.
(4) A polynucleotide having the base sequence of the natural IL-36β gene (eg, SEQ ID NO: 1, 3 or 5).
(5) Nucleotide sequence in which one or more bases are modified (deletion, substitution, addition, insertion, or a combination thereof) in the base sequence of the natural IL-36β gene (eg, SEQ ID NO: 1, 3 or 5). A polynucleotide encoding a polypeptide having IL-36β activity.
(6) A polypeptide having a base sequence having 80% or more sequence identity with the base sequence of the natural IL-36β gene (eg, SEQ ID NO: 1, 3 or 5) and having IL-36β activity is encoded. Polynucleotide to be.
(7) A poly that hybridizes with a polynucleotide having the base sequence of the natural IL-36β gene (eg, SEQ ID NO: 1, 3 or 5) under stringent conditions and encodes a polypeptide having IL-36β activity. nucleotide.

The number of modified amino acids in (2) and the value of sequence identity in (3) are as exemplified in <(a) IL-36β>.
The number of modified bases in (5) is, for example, 1 to 100, 1 to 80 or less, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 10 or 1 to 5 or the like can be mentioned.
The sequence identity in (6) may be, for example, 85% or more, 90% or more, or 95% or more.

The IL-36β coding sequence may encode a truncated type IL-36β. Examples of the polynucleotide having the base sequence encoding the truncated type IL-36β include the following polynucleotides.
(8) A polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 8, 10 or 12.
(9) The amino acid sequence shown in SEQ ID NO: 8, 10 or 12 has an amino acid sequence in which one or more amino acids have been modified (deletion, substitution, addition, insertion, or a combination thereof) and has IL-36β activity. A polynucleotide encoding a polypeptide having.
(10) A polynucleotide comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8, 10 or 12 and encoding a polypeptide having IL-36β activity.
(11) A polynucleotide having the nucleotide sequence shown in SEQ ID NO: 7, 9 or 11.
(12) The base sequence shown in SEQ ID NO: 7, 9 or 11 has a base sequence in which one or more bases are modified (deletion, substitution, addition, insertion, or a combination thereof) and has IL-36β activity. A polynucleotide encoding a polypeptide having.
(13) A polynucleotide encoding a polypeptide having a base sequence having 80% or more sequence identity with the base sequence shown in SEQ ID NO: 7, 9 or 11 and having IL-36β activity.
(14) A polynucleotide that hybridizes with a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 7, 9 or 11 under stringent conditions and encodes a polypeptide having IL-36β activity.

Examples of the number of modified amino acids in (9) include 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10 or 1 to 5. Be done. The value of sequence identity in (10) may be, for example, 85% or more, 90% or more, or 95% or more. The number of modified bases in (11) is, for example, 1 to 100, 1 to 80 or less, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 10 or 1 to 5 or the like can be mentioned. The sequence identity in (12) may be, for example, 85% or more, 90% or more, or 95% or more.

≪Other base sequences≫
The polynucleotide (b) may contain another base sequence in addition to the IL-36β coding sequence. Examples of other base sequences include a base sequence encoding a signal peptide (hereinafter referred to as “signal peptide coding sequence”), a sequence controlling the expression of the IL-36β gene (hereinafter referred to as “expression control sequence”), and the like. Can be mentioned.

(Signal peptide coding sequence)
The polynucleotide (b) preferably has a signal peptide coding sequence of a secretory protein in addition to the IL-36β coding sequence. The signal peptide coding sequence is preferably directly or indirectly linked in-frame to the 5'end of the IL-36β coding sequence. When the polynucleotide of (b) is introduced into a cell by linking the signal peptide coding sequence to the 5'end of the IL-36β coding sequence in-frame, the IL-36β expressed in the cell is released. It can be secreted extracellularly.

The signal peptide coding sequence is not particularly limited as long as it is a base sequence encoding a signal peptide that can function as a secretory signal in the cells of the organism to which the antitumor agent is administered. From the viewpoint that IL-36β can be stably secreted extracellularly, it is preferable to use a signal peptide coding sequence of a secretory protein derived from an organism of the same type as the subject to which the antitumor agent is administered. For example, when the administration target of the antitumor agent is a human, it is preferable to use the signal peptide coding sequence of a human secreted protein. When the administration target of the antitumor agent is a mouse, it is preferable to use the signal peptide coding sequence of the secretory protein of the mouse.

Examples of secretory proteins include, but are not limited to, peptide hormones such as parathyroid hormone and growth hormone; cytokines such as IFN-γ, GM-CSF and IL-6; immunoglobulin heavy chains or light chains and the like. ..
As an example of the signal peptide coding sequence, the signal peptide coding sequence of human parathyroid hormone (hPTH) is shown in SEQ ID NO: 14 (SEQ ID NO: 13 is the amino acid sequence of the signal peptide encoded by the coding sequence). The signal peptide coding sequence of human growth hormone (hGH) is shown in SEQ ID NO: 16 (SEQ ID NO: 15 is the amino acid sequence of the signal peptide encoded by the coding sequence).

The signal peptide coding sequence may be directly linked to the 5'end of the IL-36β coding sequence, or may be indirectly linked via a linker. The linker is not particularly limited as long as it does not impair the activity of IL-36β when translated. Examples of the length of the linker include 3 base lengths, 6 base base lengths, 9 base base lengths, 12 base base lengths, and the like.
The signal peptide coding sequence is preferably linked directly to the 5'end of the IL-36β coding sequence.

(Expression control sequence)
The polynucleotide (b) preferably has an expression control sequence in addition to the IL-36β coding sequence. Expression control sequences include, for example, promoters, enhancers, poly-A addition signals, terminators and the like.
The promoter is not particularly limited as long as it can function in the cells of the organism to which the antitumor agent is administered. Examples of promoters include CMV (cytomegalovirus) promoter, SRα promoter, SV40 early promoter, retrovirus LTR, RSV (rous sarcoma virus) promoter, HSV-TK (herpes simplex virus thymidine kinase) promoter, EF1α promoter, and metallothioneine. Promoters, heat shock promoters and the like can be mentioned, but the present invention is not limited thereto.
The IL-36β coding sequence is preferably operably linked to the appropriate promoter. When having the signal peptide coding sequence of the polynucleotide of (b), it is preferable that the signal peptide coding sequence and the IL-36β coding sequence are operably linked to a suitable promoter.

<(C) Vector>
The vector of (c) contains the polynucleotide of (b) above. The type of vector is not particularly limited, and an expression vector or the like generally used for gene expression can be used. The vector may be linear or cyclic. The vector may be a non-viral vector such as a plasmid, a viral vector, or a transposon vector. Further, the vector may be an episomal vector, an artificial chromosome vector, or the like.

Examples of the viral vector include Sendai virus vector, retrovirus (including lentivirus) vector, adenovirus vector, adeno-associated virus vector, herpesvirus vector, vaccinia virus vector, poxvirus vector, poliovirus vector, and sylvis virus vector. , Loved virus vector, paramix virus vector, orthomix virus vector and the like.

Examples of the plasmid vector include plasmid vectors for animal cell expression such as pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo.

An episomal vector is a vector that can autonomously replicate extrachromosomally. Examples of the episomal vector include a vector containing a sequence required for autonomous replication derived from EBV, SV40, etc. as a vector element. Specific examples of the vector element required for autonomous replication include a replication origin and a gene encoding a protein that binds to the origin of replication and controls replication. For example, in EBV, the replication origin oriP and the EBNA-1 gene can be mentioned, and in SV40, the replication origin ori and the SV40LT gene can be mentioned.

Examples of the artificial chromosome vector include a YAC (Yearst artificial chromosome) vector, a BAC (Bacterial artificial chromosome) vector, and a PAC (P1-derivated artificial chromosome) vector.

Among them, the vector is preferably a viral vector. As the viral vector, a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a Sendai virus vector, or a vaccinia virus vector is preferable.
The vector can be prepared by using a commercially available one. For example, a vector containing the polynucleotide of (b) can be prepared by inserting the polynucleotide of (b) into a multicloning site of a commercially available vector. The insertion of the polynucleotide of (b) into a commercially available vector can be performed, for example, by combining restriction enzyme treatment and ligase treatment.

When the vector (c) is a targeting vector that is integrated into the genome of a target cell using genome editing technology or the like, the vector preferably contains a homology arm for homologous recombination. The homology arm has a sequence homologous to any genomic region of the cell of the organism to which the antitumor agent is administered. The genomic region homologous to the homology arm is the region into which the polynucleotide of (b) above is inserted. The homology arm is arranged adjacent to the 5'side and the 3'side of the polynucleotide of (b).
The sequence of the homology arm can be appropriately designed by selecting the target region into which the polynucleotide of (b) is inserted, depending on the type of the target cell. For example, when the target cell is a tumor cell, the tumor gene contained in the tumor cell may be selected as a target region to design a homology arm.

<(D) cell>
The cell (d) is a cell that secretes IL-36 or a variant thereof (hereinafter, referred to as "IL-36 secreting cell"). The IL-36 secretory cell may be a natural IL-36 secretory cell, and is a cell into which the polynucleotide of (b) has been introduced using a gene recombination technique (hereinafter, referred to as “IL-36β gene transfer cell”). It may be. The IL-36 secretory cells are preferably cells of the organism to which the antitumor agent is administered.

Natural IL-36 secretory cells include, for example, epithelial cells, monocytes, B cells and the like.
Examples of the IL-36β gene-introduced cell include a cell into which the polynucleotide of (b) has been introduced using the vector of (c). The host cell for gene transfer is not particularly limited, and examples thereof include tumor cells collected from a subject to which an antitumor agent is administered; immune cells such as dendritic cells, T cells and natural killer cells; and fibroblasts; and the like. .. The host cells are preferably obtained from the subject to which the antitumor agent is administered. Alternatively, the host cell is preferably a cell having the same HLA type as the subject to which the antitumor agent is administered.
IL-36β gene transfer cells can be produced by using a known gene transfer method depending on the type of vector. Examples of the gene transfer method include a virus infection method, a lipofection method, a microinjection method, a calcium phosphoric acid method, a DEAE-dextran method, an electroporation method, a method using a transposon, and a particle gun method.
The IL-36β gene-introduced cell may be prepared by using a targeting vector among the vectors of the above (c) and using a known gene editing technique or the like. Examples of gene editing techniques include techniques using endonucleases such as zinc finger nucleases, TALENs (transcriptional activation-like effector nucleases), and CRISPR (Crustered Regularly Interspaced Short Palindromic Repeat) -Cas systems.

When producing IL-36β gene-introduced cells, the vector of (c) above may contain a marker gene. The marker gene can be used to select IL-36β transgenic cells. As the marker gene, a commonly used marker gene can be used without particular limitation. Examples of the marker gene include a drug resistance gene. Examples of the drug resistance gene include, but are not limited to, a neomycin resistance gene, an ampicillin resistance gene, a hyglomycin resistance gene, a tetracycline resistance gene, and a chloramphenicol resistance gene.
The marker gene may be linked to the polynucleotide of (b) above by interposing a base sequence encoding a self-cleaving end peptide such as a 2A peptide, an IRES (internal ribozyme entry site) sequence, or the like. By interposing these sequences, the IL-36β gene and the marker gene can be expressed independently from one promoter.

The secretion of IL-36β in IL-36β transgenic cells can be confirmed by a known method. For example, by measuring the IL-36β concentration in the culture supernatant of the IL-36β gene-introduced cells by Western blotting or ELISA, it is confirmed whether the IL-36β gene-introduced cells secrete IL-36β. can do. Alternatively, IL-6β-producing cells such as fibroblasts are allowed to act on the culture supernatant of IL-36β gene-introduced cells, and the amount of IL-6 secreted from the fibroblasts is measured to measure the IL-6β. It can be confirmed whether the transgenic cells secrete IL-36β.

The antitumor agent of the present embodiment may contain any one of the components (a) to (d) alone, or may contain two or more in combination.
The antitumor agent can contain at least one component selected from the group consisting of the above (a) to (d) as an active ingredient of the antitumor effect. The "anti-tumor effect" refers to an effect of suppressing tumor growth as compared with the case where an anti-tumor agent is not used. Antitumor effects include tumor metastasis suppression, tumor regression, and tumor eradication.
The antitumor agent can contain a therapeutically effective amount of at least one component selected from the group consisting of the above (a) to (d). "Therapeutically effective amount" means the amount of an ingredient effective for the treatment or prevention of a disease. The therapeutically effective amount may vary depending on the condition, age, sex, body weight, etc. of the disease to be administered. In the antitumor agent, the therapeutically effective amount may be an amount in which at least one component selected from the group consisting of the above (a) to (d) can suppress tumor growth.
By administering an antitumor agent to a subject having a tumor, the growth of the tumor in the subject can be suppressed. By administering an antitumor agent to a subject at high risk of developing a tumor, the development of a tumor in the subject can be suppressed.

The tumor targeted by the antitumor agent of the present embodiment is not particularly limited. Examples of the tumor include melanoma, glioma, malignant mesoderma, lung cancer, pancreatic cancer, head and neck cancer, liver cancer, uterine cancer, bladder cancer, biliary tract cancer, esophageal cancer, and testis. Tumors, thyroid cancers, brain tumors, prostate cancers, colon cancers, kidney cancers, ovarian cancers, breast cancers, adenocarcinomas, squamous cell carcinoma, glandular squamous cell carcinoma, vaginal cancer, cervical cancer, Cancers such as spleen cancer, tracheal cancer, bronchial cancer, small intestine cancer, gastric cancer, bile sac cancer, testicular cancer; osteosarcoma, chondrosarcoma, Ewing sarcoma, malignant vascular endothelial tumor, malignant Schwan tumor, soft sarcoma , Fibrosarcoma; blastoma, hepatoblastoma, myeloma, renal blastoma, pancreatic blastoma, pleural lung blastoma, retinal blastoma, etc .; germ cell tumor; lymphoma, leukemia, myeloma Hematological cancers such as, but are not limited to these.

The antitumor agent of the present embodiment may be administered to a subject as a pharmaceutical composition described later in combination with a pharmaceutically acceptable carrier or the like.

[Pharmaceutical composition]
In one embodiment, the invention provides a pharmaceutical composition for treating a tumor. The pharmaceutical composition comprises (a) IL-36β or a variant thereof; (b) a polynucleotide having a nucleotide sequence encoding IL-36β or a variant thereof; (c) an expression comprising the polynucleotide of (b) above. It contains at least one component selected from the group consisting of a vector; and (d) a cell secreting IL-36β or a variant thereof, and a pharmaceutically acceptable carrier.

The pharmaceutical composition of this embodiment is used to treat a tumor. Tumor treatment includes suppressing tumor growth in subjects with tumors, regressing tumors in subjects with tumors, eradicating tumors in subjects with tumors, and metastasis of tumors in subjects with tumors. It is a concept that includes suppression, suppression of tumor development in a subject having a high risk of tumor development, suppression of tumor recurrence in a subject whose tumor has been removed by surgical operation or radiotherapy, and the like. Therefore, the pharmaceutical composition of the present embodiment is at least one selected from the group consisting of suppression of tumor growth, tumor regression, tumor eradication, tumor metastasis suppression, tumor development suppression, and tumor recurrence suppression. It can also be said that it is a pharmaceutical composition used to exert an effect. Examples of the tumor to be treated include those similar to those mentioned in the above section [Anti-tumor agent].

The components (a) to (d) are the same as those described in the above section [Anti-tumor agent].
The "pharmaceutically acceptable carrier" means a carrier that does not inhibit the physiological activity of the active ingredient and does not show substantial toxicity to the administration subject. By "not showing substantial toxicity" is meant that the ingredient is not toxic to the subject at commonly used doses. In the pharmaceutical composition of the present embodiment, the pharmaceutically acceptable carrier does not inhibit the antitumor activity of the components (a) to (d) and exhibits substantial toxicity to the administration subject thereof. Not a carrier. The pharmaceutically acceptable carrier includes any known pharmaceutically acceptable ingredient, which is typically considered an inactive ingredient. The pharmaceutically acceptable carrier is not particularly limited, and is, for example, a solvent, a diluent, a vehicle, an excipient, a flow accelerator, a binder, a granulator, a dispersant, a suspending agent, a wetting agent, and the like. Lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, fillers, preservatives (eg antioxidants), chelating agents, flavoring agents, sweeteners, thickeners, buffers, colorants, etc. Can be mentioned. As the pharmaceutically acceptable carrier, one type may be used alone, or two or more types may be used in combination. When the pharmaceutical composition contains the above (b) or (c), the pharmaceutically acceptable carrier may be a solvent, a diluent, a vehicle, a buffer solution or the like. When the pharmaceutical composition contains the above (d), the pharmaceutically acceptable carrier may be a cell culture solution, a buffer solution (physiological saline, PBS, citric acid buffer solution, etc.) or the like.
In the pharmaceutical composition of the present embodiment, in addition to those listed as the pharmaceutically acceptable carriers, additives and the like commonly used in the pharmaceutical field can be used without particular limitation. When the pharmaceutical composition contains the above (b) or (c), the pharmaceutical composition may contain a transfection accelerator, a genome editing reagent (CRISPR / Cas9 system, etc.) and the like.

The dosage form of the pharmaceutical composition is not particularly limited, and can be a dosage form generally used as a pharmaceutical preparation. The pharmaceutical composition may be an oral preparation or a parenteral preparation. Examples of the oral preparation include tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, liquids, drop loves, emulsions and the like. Examples of parenteral preparations include injections, suppositories, ointments, sprays, nasal drops, inhalants and the like. The pharmaceutical compositions of these dosage forms can be formulated according to a conventional method (for example, the method described in the Japanese Pharmacopoeia).
The pharmaceutical composition of the present embodiment is preferably a parenteral preparation, more preferably an injection.

The pharmaceutical composition of the present embodiment may contain an active ingredient other than the above (a) to (d). The active ingredient is not particularly limited, and for example, vitamins and derivatives thereof, anti-inflammatory agents, anti-inflammatory agents, blood circulation promoters, stimulants, hormones, stimulants, analgesics, cell activators, plants and. Animal / microbial extracts, antipruritics, anti-inflammatory analgesics, antifungal agents, antihistamines, hypnotic sedatives, tranquilizers, antihypertensive agents, antihypertensive diuretics, antibiotics, anesthetics, antibacterial agents, antiepileptic agents, coronary vessels Examples include, but are not limited to, dilators, diuretics, antipruritics, keratin softening and stripping agents. Moreover, the active ingredient may be another antitumor agent. Other antitumor agents may be appropriately selected according to the type of cancer to be applied. As the other active ingredient, one kind may be used alone, or two or more kinds may be used in combination. The pharmaceutical composition of the present embodiment may contain IL-12. When the components (a) to (d) are used in combination with IL-12, the antitumor effect is synergistically improved.

The pharmaceutical composition of the present embodiment can contain at least one selected from the group consisting of the above (a) to (d) as an active ingredient. The pharmaceutical composition can contain a therapeutically effective amount of at least one component selected from the group consisting of the above (a) to (d). When the pharmaceutical composition contains the above (a) to (c), the therapeutically effective amount may be, for example, 0.01 μg to 100 mg, 0.1 μg to 10 mg, 0.5 μg to 5 mg, or 0.5 μg to 5 mg per administration unit form. Examples thereof include 1 μg to 1 mg. When the pharmaceutical composition contains the above (d), the therapeutically effective amount is, for example, 10 ³ to 10 ¹⁰ cells, 10 ⁴ to 10 ⁹ cells, or 10 ⁵ to 10 cells per administration unit form. ^Eight and the like can be mentioned.

[Administration method]
The antitumor agent or pharmaceutical composition can be administered to a subject in need of these administrations by a known method. The administration method may be oral administration or parenteral administration, but parenteral administration is preferable. Parenteral routes include all non-oral routes of administration, such as intravenous, intramuscular, subcutaneous, intranasal, intradermal, eye drops, intracerebral, rectal, intravaginal and intraperitoneal administration. The administration method may be topical administration or systemic administration. Among them, the administration method is preferably administration by injection. Preferred administration routes include intravenous administration, subcutaneous administration, intratumoral administration and the like.

The dose and interval of administration of the antitumor agent or pharmaceutical composition can be appropriately selected depending on the age, sex, body weight, etc. of the subject to be administered, the type of disease, the degree of progression, symptoms, etc., the administration method, and the like. The dose can be a therapeutically effective amount of at least one component selected from the group consisting of (a) to (d). When the antitumor agent or the pharmaceutical composition contains the above (a) to (c), the therapeutically effective amount is, for example, 0.01 μg to 100 mg, 0.1 μg to 10 mg, 0.5 μg per administration. Examples thereof include ~ 5 mg, 1 μg ~ 1 mg, and the like. When the pharmaceutical composition contains the above (d), the therapeutically effective amount is, for example, 10 ³ to 10 ¹⁰ cells, 10 ⁴ to 10 ⁹ cells, or 10 ⁵ to 10 cells per administration. ^Eight and the like can be mentioned.
it can.

The antitumor agent or pharmaceutical composition may be administered in a single dose or in multiple doses. In the case of multiple administrations, the administration interval may be, for example, daily, weekly, 10 to 30 days, monthly, 3 to 6 months, annually, or the like.

The antitumor agent or pharmaceutical composition is preferably administered in combination with IL-12. As shown in Examples described later, when administered in combination with IL-12, the antitumor effect of the antitumor agent or pharmaceutical composition is synergistically improved. The antitumor agent or pharmaceutical composition may be administered at the same time as IL-12, or may be administered at a different time from IL-12. Examples of the dose of IL-12 include 0.01 μg to 100 mg, 0.1 μg to 10 mg, 0.5 μg to 5 mg, 1 μg to 1 mg, and the like.

[Anti-tumor effect enhancer, pharmaceutical composition used to enhance anti-tumor effect]
In one embodiment, the present invention provides an antitumor effect enhancer used to enhance the antitumor effect of IL-12. The antitumor effect enhancer comprises (a) IL-36β or a variant thereof; (b) a polynucleotide having a base sequence encoding IL-36β or a variant thereof; (c) the polynucleotide of (b) above. It contains at least one component selected from the group consisting of an expression vector containing; and (d) a cell secreting IL-36β or a variant thereof.
Also, in one embodiment, the present invention provides a pharmaceutical composition used to enhance the antitumor effect of IL-12. The pharmaceutical composition contains at least one component selected from the group consisting of (a) to (d) and a pharmaceutically acceptable carrier.

The components (a) to (d) are the same as those described in the above section [Anti-tumor agent]. As shown in Examples described later, the components (a) to (d) have an effect of enhancing the antitumor effect of IL-12. Therefore, a drug or pharmaceutical composition containing at least one component selected from the group consisting of (a) to (d) can be used to enhance the antitumor effect of IL-12. The component that may be contained in the antitumor effect enhancer or pharmaceutical composition of the present embodiment in addition to at least one component selected from the group consisting of (a) to (d) is the above-mentioned [antitumor]. Agents] or similar to those listed in the section [Pharmaceutical Compositions].

The present invention may also include the following embodiments:
In one embodiment, the present invention provides at least one component selected from the group consisting of (a) to (d) above, which is used to treat a tumor.
In one embodiment, the present invention provides at least one component selected from the group consisting of (a)-(d) above, which is used in combination with IL-12 to treat a tumor.

In one embodiment, the present invention provides a method of treating a tumor, which comprises administering to a subject at least one component selected from the group consisting of (a) to (d). The subject is a subject in need of treatment of the tumor. The subject is, for example, a subject having a tumor or a subject at high risk of developing a tumor.
In one embodiment, the present invention comprises administering to the subject at least one component selected from the group consisting of (a) to (d), and administering IL-12 to the subject. Provide a method of treating a tumor. The components (a) to (d) and IL-12 may be administered at the same time or separately.
In one embodiment, the present invention provides at least one component selected from the group consisting of (a) to (d) above, which is used in the production of an antitumor agent.
In one embodiment, the present invention provides at least one component selected from the group consisting of (a) to (d) above, which is used in the production of a pharmaceutical composition for treating a tumor.
In one embodiment, the present invention provides at least one component selected from the group consisting of (a) to (d) above, which is used to enhance the antitumor effect of IL-12.
In one embodiment, the present invention is at least one selected from the group consisting of (a) to (d) above, which is used in the production of an antitumor agent used to enhance the antitumor effect of IL-12. Ingredients are provided.
In one embodiment, the present invention provides a combination of IL-12 with at least one component selected from the group consisting of (a)-(d) above, which is used to treat a tumor.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to the following Examples.

[Cloning of mouse IL-36β- cDNA]
Reagents were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) unless otherwise specified.

1. 1. Preparation of Bone Marrow Cells The femur and tibia of a 7-week-old male C57BL / 6J purchased from Tokyo, Japan, while maintaining the specific muscular free (SPF) conditions, were sterilized with scissors. It was collected and the attached muscle tissue was carefully removed. The femur and tibia were sterilized by immersing them in 70% ethanol for 1 to 2 minutes, and then washed with 1xphosphate buffered saline (PBS, Life Technologies, Carlsbad, CA, USA; Table 1). Cut off both ends of the femur and tibia, extrude bone marrow fluid using a 1 mL syringe with a 26 G needle, and in RPMI park memorial instance (RPMI) -1640 medium (SIGMA-ALDRICH, St. Louis, MO, MO, USA; collected in Table 2, hereinafter referred to as "RPMI-1640 medium"). Bone marrow fluid was extruded from the bone marrow several more times using RPMI-1640 medium in the dish. The removed bone marrow fluid was filtered using a cell strainer and centrifuged (4 ° C., 1200 rpm, 5 minutes). After removing the supernatant, 20 mL of PBS was added to wash the cells thoroughly, and the cells were centrifuged (4 ° C., 1200 rpm, 5 minutes). After removing the supernatant, an appropriate amount of ACK lysis buffer (Table 3) was added according to the obtained cells to hemolyze the erythrocytes contained in the cells, and the cells were centrifuged (4 ° C., 1200 rpm, 5 minutes). After centrifugation, the supernatant was quickly removed and suspended in PBS. Cells were filtered using a cell strainer and centrifuged (4 ° C., 1200 rpm, 5 minutes). After removing the supernatant, the cells were suspended in RPMI-1640 medium, and the number of cells was calculated using a hemocytometer.

2. Extraction of Total RNA Bone marrow cells (6.4x10 ⁶ cells) were dispensed into 1.5 mL microtubes, centrifuged (4 ° C., 12,000 g, 5 minutes), the supernatant was removed, and 300 μL of PBS was added. Stir well. After performing centrifugation twice with PBS, add 500 μL of denaturing (D) -solution (Table 4) 500 μL, 2M sodium acetate 50 μL, and PhOH saturated with RNase free water 500 μL to each tube except PBS, and stir well. , Flushed. Further, _{150 μL of CHCl 3} was added, the mixture was stirred using a vortex mixer, flushed, and then allowed to stand on ice for 15 minutes. Then, the mixture was centrifuged (4 ° C., 12,000 g, 5 minutes), and the separated upper layers (aqueous layers) were collected in new 1.5 mL microtubes. The same amount of 2-propanol as the collected upper layer was added, mixed by inversion, flushed, and then allowed to stand at −20 ° C. for 1 hour. Then, the mixture was centrifuged (4 ° C., 12,000 g, 20 minutes) to remove the supernatant. To each tube, 150 μL of D-solution and 150 μL of 2-propanol were added, stirred using a vortex mixer, flushed, and then allowed to stand at −20 ° C. for 1 hour. Then, the mixture was centrifuged (4 ° C., 12,000 g, 10 minutes) to remove the supernatant. 1 mL of 70% Ethanol was added to each tube, and the mixture was stirred until the pellets were peeled off from the bottom of the tube, and then centrifuged (4 ° C., 12,000 g, 5 minutes) to remove the supernatant. The washing operation with 70% Ethanol was performed again to remove the supernatant, and then the pellets were dried. After drying, 20 μL of RNase free water was added and stirred well, and the concentration and purity of total RNA were measured using Nano Drop 1000 (Thermo Scientific, Waltham, MA, USA).

3. 3. Synthesis of Complementary DNA (CDNA) A PrimeScript RT-PCR Kit from TakaRa (Shiga, Japan) was used. The reagents shown in Table 5 were mixed in PCR tubes. It ^{was set in a Veriti TM} Thermal cycler (Applied Biosystems, Tokyo, Japan) and annealed (65 ° C., 5 minutes). Then, each tube was allowed to stand on ice, and the reagents shown in Table 6 were added. The cDNA was synthesized again by setting it in the Veriti ^TM Thermal cycler and performing a reverse transcription reaction (42 ° C., 30 minutes) and an extension reaction (95 ° C., 5 minutes). The resulting cDNA was stored at 4 ° C.

4. Primer design The sequence of the known mouse IL-36β gene (SEQ ID NO: 5) was searched by NCBI (www.ncbi.nlm.nih.gov/pubmed/), and Primer3 (frodo.wi.mit.edu/) and CLUSTLAW. (Www.genome.jp/tools/clustalw/) was used to design a primer (long primer) that amplifies all the ORFs of the mouse IL-36β gene. Mouse β-actin was used as a control. The designed primer sequences are shown in Table 7.

5. Examination of expression of mouse IL-36β gene by RT-PCR method Reactions shown in Table 8 using the cDNA obtained in 3 above and mouse β-actin primer or short primer (Operon Biotechnology, Tokyo, Japan; Table 7). The solution was prepared. It ^{was set in the Veriti TM} Thermal cycler and RT-PCR was performed (94 ° C. 5 minutes, 94 ° C. 45 seconds, 60 ° C. 45 seconds, 72 ° C. 45 seconds, 72 ° C. 1 minute, 30 cycles; or 95 ° C. 5 minutes, 95 ° C. 30 seconds, 58 ° C. 30 seconds, 72 ° C. 30 seconds, 72 ° C. 1 minute, 40 cycles). A sample obtained by adding 2 μL of loading buffer (Table 9) to 8 μL of the obtained PCR product was used as a sample, and 100 bp DNA ladder (Bio-Regenerations, Kanagawa, Japan) was used as a DNA size marker. Samples and DNA size markers were electrophoresed on Mupid-2 plus (Advance, Tokyo, Japan) using 1xTris Acetate ETDA (TAE; Table 10) and agarose gels. The agarose gel used was diluted with 1xTAE so that Agarose S (NIPPON GENE, Tokyo, Japan) had a concentration of 2%. After electrophoresis, the gel was stained with ethidium bromide (NIPPON GENE), and the PCR product of the target region was confirmed using FAS-III (TOYOBO, Osaka, Japan). As a result, bands matching the size of the mouse IL-36β gene and the mouse β-actin were confirmed, respectively.

6. Amplification of mouse IL-36β gene by RT-PCR method New England BioLabs Japan (Tokyo, Japan)'s Phusion ^TM High-Fidelity PCR Kit was used.
The reagents shown in Table 11 were prepared using the cDNA obtained in 3 and the longprimer (Operon Biotechnology; Table 7). It ^{was set in the Veriti TM} Thermal cycler, and RT-PCR was performed under PCR conditions (98 ° C. 30 seconds, 98 ° C. 10 seconds, 57 ° C. 30 seconds, 72 ° C. 30 seconds, 72 ° C. 1 minute, 33 cycles). A sample obtained by adding 4 μL of loading buffer to 20 μL of the obtained PCR product was used as a sample, and 1 kbp DNA ladder (Bio-Regenerations) was used as a DNA size marker. Samples and DNA size markers were electrophoresed on Mupid-2 plus using 1xTAE and agarose gels. The agarose gel used was diluted with 1xTAE so that the agarose S had a concentration of 1%. After electrophoresis, the gel was stained with AtlasSight DNA stain (Funakoshi, Tokyo, Japan), and the PCR product of the target region was confirmed using TRANSILLUMINATOR (Advance). As a result, a band matching the size of the mouse IL-36β gene was confirmed.

7. Extraction of DNA from an electrophoresis gel A DNA Fragment Purification manufactured by TOYOBO was used.
Under LED irradiation, the PCR band of interest was excised from the gel using a scalpel and transferred to a 1.5 mL microtube. 400 μL of the adsorbent was added and allowed to stand at room temperature until the gel was completely dissolved. After thawing, 30 μL of magnetic beads were added, and the mixture was allowed to stand for 2 minutes with occasional stirring and then flushed. Bound / Free (B / F) separation was performed, 600 μL of the cleaning solution was added, and the mixture was stirred with a vortex mixer for 10 seconds and flushed. B / F separation was performed again, and 1 mL of 75% Ethanol was added for washing. After stirring with a vortex mixer for 10 seconds and flushing, B / F separation was performed. The magnetic beads were dried at 55 ° C., 10 μL of RNase free water was added, and the beads were stirred and flushed using a vortex mixer for 10 seconds. After allowing to stand at room temperature for 2 minutes, B / F separation was performed, and the supernatant was collected in a new 1.5 mL microtube. The extracted DNA was measured for concentration and purity using Nano Drop 1000.

8. Deoxyadenosine (dA) Addition Reaction New England BioLabs Japan's NEBNext® dA-Tailing Module was used.
The reagents shown in Table 12 were mixed in 1.5 mL microtubes. Incubation was carried out at 37 ° C. for 30 minutes, and A was added to the 3'end of the extracted DNA.

9. Ligase reaction Promega (San Francisco, CA, USA) pGEM ^TM- T Easy Vector Systems was used.
The reagents shown in Table 13 were mixed in PCR tubes, incubated overnight at 4 ° C., and the gene of interest was inserted into the pGEM-T Easy Vector.

10. Transformation and Escherichia coli selection 1 μL of the ligase reaction product obtained in 9 and 10 μL of JM109 competent cell 10 μL (Promega) were gently mixed with a 1.5 mL microtube and allowed to stand on ice for 20 minutes. Then, the tube was placed in a water bath at 42 ° C. for 40 to 50 seconds, heat shock was applied to the JM109 competent cells, and the cells were returned to the ice and allowed to stand for 2 minutes. 90 μL of Super Optimal vibration with Catabolite repression (SOC) medium (Table 14) returned to room temperature was added, and the cells were cultured with shaking at 37 ° C. and 50 rpm for 1.5 hours. Then, X-Gal IPG Spray (Funakoshi) was sown in a Lysogeny Broth (LB) agar plate medium (Table 15) so as to spread over a petri dish, and 30 to 33 μL of the transformed solution was added. Then, it was shake-cultured at 37 ° C. for 18 hours. Positive colonies were selected by a blue-white assay with a two-clone sterile toothpick and placed in 3 mL of LB medium (Table 16) in a 15 mL tube. This was shake-cultured overnight at 37 ° C. and 150 rpm.

11. Plasmid extraction and restriction enzyme treatment (Alw NI)
The cultured Escherichia coli was dispensed into a 1.5 mL microtube, centrifuged (4 ° C., 1580 g, 10 minutes), the supernatant was removed, 150 μL of glucose solution (Table 17) was added, and the mixture was stirred using a vortex mixer. .. Alkaline solution (Table 18) 200 μL was added, and the mixture was lightly inverted and mixed, and allowed to stand on ice for 5 minutes. Then, 150 μL of potassium acetate (Table 19) was added, mixed by inversion, allowed to stand on ice for 3 minutes, and centrifuged (4 ° C., 20,400 g, 5 minutes). The supernatant was transferred to a new 1.5 mL microtube. An equal amount of PhOH saturated with RNase free water and CHCl ₃ were added, stirred using a vortex mixer, centrifuged (4 ° C., 20,400 g, 5 minutes), and then the separated upper layer (aqueous layer) was recovered. After performing this operation again, 2.5 times the amount of the collected supernatant, 100% Ethanol, was added, mixed well, and allowed to stand at room temperature for 10 minutes. Centrifugation (4 ° C., 20,400 g, 10 minutes) was performed to remove the supernatant, and then the pellet was dried. Then 100 μL of RNase free water was added. The extracted plasmid was measured for concentration and purity using Nano Drop 1000. The extracted plasmid was treated with the restriction enzyme Alw NI (New England BioLabs Japan) (37 ° C., 16 hours).
A mixture of 10 μL of restriction enzyme reaction solution and 2 μL of loading buffer was used as a sample, and 1 kbp DNA ladder was used as a DNA size marker. Samples and DNA size markers were run on a Mupid-2 plus using a 1xTAE and an agarose gel. The agarose gel used was diluted with 1xTAE so that the agarose S had a concentration of 1%. After electrophoresis, the gel was stained with ethidium bromide, and the PCR product of the target region was confirmed using FAS-III. Since a band was confirmed in the target region, it was confirmed that the mouse IL-36β gene was inserted into the pGEM-T Easy Vector.

12. The plasmid obtained in Sequence Analysis 11 was prepared with the compositions shown in SP6 primer (Operon Biotechnology; Table 7) and Table 20, and the sequence analysis was performed by requesting the Operon Biotechnology. The sequence analysis results were examined for homology with the reported mouse IL-36β gene sequence (SEQ ID NO: 5) using NCBI and CLUSTLAW. The sequence analysis results were consistent with the reported mouse IL-36β gene sequence, confirming that the mouse IL-36β cDNA could be cloned.

[Introduction of mouse IL-36β gene into tumor cells by retroviral vector]
1. 1. Cells used Fibrosarcoma cells (MCA205), melanoma cells (B16-F10), colon cancer cells (MC38) and packaging cells (ΦCRIP, BOSC23) of mouse cell lines are available from the Medical Cell Resources Center of the Institute of Aging Medicine, Tohoku University. offered. Packaging cells (EcoPack 2-293) were purchased from Clontech (CA, USA).

2. Cell culture Cells are cultured in RPMI-1640 medium or RPMI-1640 medium for Ecopack (Table 21) (hereinafter, may be collectively referred to simply as "medium") under the conditions of _{5% CO 2, 37 ° C.} did. When the cells were 80-90% confluent ^{, the medium in a 75 cm 2} flask (TPP ^TM , trasadingen, CH) was removed and PBS was added to wash the cells. After removing the PBS, 0.05% trypsin-EDTA (Biological, Beit HaEmek, Israel) was added, and the mixture _{was incubated at 5% CO 2} , 37 ° C. for 3 to 5 minutes. After confirming that the cells were peeled off, a medium was added to stop the trypsin reaction, and the cells were collected. The collected cells were centrifuged at 4 ° C., 1,300 rpm, and 5 minutes to precipitate the cells. The supernatant was removed and fresh medium was added to prepare a cell suspension. 50 μL of cell suspension and 50 μL of trypan blue staining solution were placed in a 1.5 mL microtube, and the number of cells was counted by the dye exclusion method using a hemocytometer. The total number of cells was calculated from the counted number of cells, and the required number of cells was seeded in a new flask and subcultured.

3. 3. Construction of recombinant retroviral vector (pMEI-5-Neo-full-length IL-36β)
A DNA fragment containing IL-36β cDNA was cut out from the pGEM-Teasy prepared above with restriction enzymes NotI and SalI (TaKaRa, Shiga, Japan) and inserted into pMEI-5 Neo DNA (TaKaRa Bio) (pMEI-5). -Neo-full-length IL-36β). It was confirmed that the IL-36β cDNA was inserted into the pMEI-5 Neo DNA from the size of the DNA fragment cleaved with the restriction enzyme EcoRI (TaKaRa).

(Construction of pDFG-hPTH-truncated IL-36β-IRES-Neo)
FIG. 1 shows an outline of a method for constructing pDFG-hPTH-truncated IL-36β-IRES-Neo.

We asked Takara Bio to synthesize human parathyroid hormone signal sequence (SEQ ID NO: 7) -added truncated IL-36β DNA (hPTH-truncated IL-36β DNA; SEQ ID NO: 17), and the DNA was inserted into the NcoI-BamHI site. T-vector pMD19 (TaKaRa Bio) was obtained. HPTH-truncated IL-36β DNA was cut out from the vector with restriction enzymes NcoI and BamHI (New England BioLabs Japan, Tokyo, Japan), and was donated by the retrovirus vector pMFG-Tst DNA (Tokyo University of Pharmacy and Technology). It was inserted into the BamHI site (pMFG-hPTH-truncated IL-36β). It was confirmed from the size of the DNA fragment cleaved with the restriction enzyme EcoRI that hPTH-truncated IL-36β was inserted into the pMFG-Tst DNA.

Restriction enzyme BamHI (TakaRa) from pDFG-mIL-10-IRES-Neo DNA (provided by Professor Hideaki Tahara, Institute of Medical Sciences, University of Tokyo) for internal ribosome entry site (IRES) and DNA of neomycin resistance gene (IRES-Neo) And inserted into the BamHI site of pMFG-hPTH-truncated IL-36β (pDFG-hPTH-truncated IL-36β-IRES-Neo; FIG. 2). It was confirmed from the size of the DNA fragment cut with the restriction enzyme EcoRI that IRES-Neo was inserted in the BamHI site of pMFG-hPTH truncated-IL-36β in the positive direction.

(Construction of pDFG-hGH-truncated IL-36β-IRES-Neo)
FIG. 3 shows an outline of a method for constructing pDFG-hGH-truncated IL-36β-IRES-Neo.
We asked Eurofins Genomics to synthesize human growth hormone signal sequence (SEQ ID NO: 9) -added truncated IL-36β DNA (hGH-truncated IL-36β DNA; SEQ ID NO: 19), and pTAKN in which the DNA was inserted into the NcoI-BamHI site. -2 Vectors (Eurofins Genomics, Tokyo, Japan) were obtained. The hGH-truncated IL-36β DNA was excised from the vector with restriction enzymes NcoI and BamHI and inserted into the NcoI-BamHI site of the pMFG-Tst DNA (pMFG-hGH-truncated IL-36β). It was confirmed from the size of the DNA fragment cleaved with the restriction enzyme EcoRI that hGH-truncated IL-36β was inserted into the pMFG-Tst DNA.

pDFG-hPTH-truncated IL-36β-IRES-Neo was treated with restriction enzymes SalI and BamHI (TaKaRa) to obtain a DNA fragment of 3444bp. pMFG-hGH-truncated IL-36β was treated with restriction enzymes BamHI, SphI and SalI (TaKaRa) to obtain two DNA fragments, 1143 bp and 5399 bp. Three DNA fragments of 3444 bp, 1143 bp and 5399 bp were bound by ligase to construct pDFG-hGH-truncated IL-36β IRES-Neo (Fig. 4). It was confirmed from the size of the DNA fragment cleaved with the restriction enzyme EcoRI that IRES-Neo was inserted into pMFG-hGH-truncated IL-36β.

(Construction of pMFG-IRES-Neo)
IRES-Neo was excised from pDFG-mIL-10-IRES-Neo DNA with the restriction enzyme BamHI (TaKaRa) and inserted into the BamHI site of the retroviral vector pMFG-Tst DNA to construct pMFG-IRES-Neo (Fig. 5). ..

4. Production of transgenic retroviruses pMEI-5-Neo-full-transfection IL-36β, pDFG-hPTH tunnelated IL-36β-IRES-Neo, pDFG-hGH-transfected IL-36β-IRES-Neo and pMFG-IRES-Neo Was transfected into packaging cells BOSC23 or Ecopack 2-293 using TransIT ^TM- 293 Transfection Reagent (Clontech).

Each cell was seeded in 2 × 10 ⁶ cells / 10 cm dish (TPP) and 24 hours later with a transfection reagent, pMEI-5-Neo-full-length IL-36β and pDFG-hPTH trunkated IL-36β-IRES-Neo, respectively. , PDFG-hGH-truncated IL-36β-IRES-Neo and pMFG-IRES-Neo were added (Table 22).

After 48 hours, the culture supernatant was collected and passed through a 0.2 μm filter (ADVANTEC, Tokyo, Japan) to remove debris to prepare a recombinant retrovirus solution. ^{ΦCRIP was inoculated in two} flasks of 1.5 × 10 ⁶ cells / 75 cm, and after 24 hours, a mixed solution of 8 μg / mL of polybrene (SIGMA-ALDRICH) was added to 5 mL of the recombinant retrovirus solution. After 5 hours, fresh medium was added. After 24 hours, subculture was performed, and a mixture of the recombinant retrovirus solution and the polybrene solution was added again. After culturing for 48 hours or more, 700 μg / mL G418 solution (Roche Diagnostics, Tokyo, Japan) was added to establish a virus-producing cell line. After culturing the virus-producing cells for 1 month, the cells were ^{seeded in 2.0 × 10 6} cells / 10 mL medium / 75 cm ² flasks, and the culture supernatant was collected 48 hours later. The collected supernatant was passed through a 0.2 μm filter to prepare a recombinant retrovirus solution.

5. Retrovirus infection of target cells MCA205, B16-F10 and MC38 ^{were seeded in flasks at 1.3 × 10 6} cells / 75 cm ² and 24 hours later, polybrene (final concentration 8 μg /) was added to 5 mL of the recombinant retrovirus solution. A mixed solution mixed with mL) was added. After 5 hours, 10 mL of fresh medium was added. Substitution was performed after 24 hours, and after another 24 hours, a mixture of a recombinant retrovirus solution and a polybrene solution was added. After culturing for 48 hours or more, 700 μg / mL G418 solution was added to select cells. After culturing the cells for 1 month, RT-PCR was performed (Table 23) to confirm the expression of IL-36β mRNA.
Primer pairs were purchased from eurofins Genomics. The primer pairs used and the PCR conditions are shown below.

Mouse IL-36β (SEQ ID NO: 28, 29)
forward: 5'-GGTATGGGTCCTGACTGGAA-3'
reverse: 5'-CCAGCCAGGATAGAGACTG-3'
PCR product size: 316bp
β-actin (SEQ ID NO: 30, 31)
forward: 5'-TTCTACAAATTGAGCTGCGTGTG-3'
reverse: 5'-TTCTACAAATGAGCTGCGTGTG-3'
PCR product size: 627bp

RT-PCR conditions Mouse IL-36β
95 ° C, 5 minutes; 95 ° C, 30 seconds; 61 ° C, 30 seconds; 72 ° C, 30 seconds; 72 ° C, 1 minute 27 cycles
β-actin
94 ° C, 5 minutes; 94 ° C, 45 seconds; 60 ° C, 45 seconds; 72 ° C, 45 seconds; 72 ° C, 1 second 27cycle

The PCR product was prepared using a 1% agarose gel (TaKaRa) containing 0.5 μg / mL ethidium bromide (Nippon Gene, Tokyo, Japan) and a Mupid-2 plus (Advance, Tokyo, Japan) electrophoresis tank using 1 × TAE. Electrophoresed. As a result, the expression of introduced full-length IL-36β, hPTH-truncated IL-36β or hGH-truncated IL-36β was confirmed in all of the cells of MCA205, B16-F10, and MC38 (FIG. 6). ..
Hereinafter, cells into which pMEI-5-Neo-full-length IL-36β has been introduced into MCA205 are referred to as "MCA205 full-length IL-36β", and cells into which pDFG-hPTH truncated IL-36β-IRES-Neo has been introduced are referred to as "MCA205". The cells into which hPTH-truncated IL-36β and pDFG-hGH-truncated IL-36β-IRES-Neo have been introduced are referred to as "MCA205 hGH-truncated IL-36β", and the cells into which pMFG-IRES-Neo has been introduced are referred to as "MCA205 Neo". ..
Cells in which pMEI-5-Neo-full-length IL-36β was introduced into B16-F10 were introduced with "B16-F10 full-length IL-36β", and cells into which pDFG-hPTH trunkated IL-36β-IRES-Neo was introduced. Cells into which "B16-F10 hPTH-truncated IL-36β" and pDFG-hGH-truncated IL-36β-IRES-Neo have been introduced are referred to as "B16-F10 hGH-truncated IL-36β" and cells into which pMFG-IRES-Neo has been introduced. Is called "B16-F10 Neo".
The cells into which pMEI-5-Neo-full-length IL-36β was introduced into MC38 were referred to as "MC38 full-length IL-36β", and the cells into which pDFG-hPTH trunkated IL-36β-IRES-Neo was introduced were referred to as "MC38 hPTH-". The cells introduced with "truncated IL-36β" and pDFG-hGH-truncated IL-36β-IRES-Neo are referred to as "MC38 hGH-truncated IL-36β", and the cells introduced with pMFG-IRES-Neo are referred to as "MC38 Neo".

[Examination of IL-36β protein production]
<Materials and methods>
1. 1. Cells used Mouse normal cell line Lung fibroblasts (NMLF) were established by Numazaki (Tohoku University).
The culture method was the same as the method described in 2 of [Introduction of mouse IL-36β gene into tumor cells by retroviral vector].

2. ELISA
MCA205 parent strain (MCA205WT), MCA205 full-length IL-36β, MCA205 hPTH-truncated IL-36β and MCA205 hGH-truncated IL-36β; B16-F10 WT, B16-F10 full-Length truncated IL-36β and B16 F10 hGH-truncated IL-36β ; and MC38WT, MC38 full-length IL- 36β, MC38 the hPTH-truncated IL-36β and MC38 hGH-truncated IL-36β, each 1.0 × ¹⁰ 6 cells ^{Seed in 2} flasks of / 75 cm, and the culture supernatant was collected after 48 hours. NMLF was seeded and cultured on a 48-well plate (Sumitomo Bakelite, Tokyo, Japan) so as to have a ratio of 3.0 × 10 ⁴ cells / 500 μL / well. After 3 hours, IL-36β protein (R & D Systems, Minneapolis, MN) (500 μL) prepared to each concentration, or MCA205WT, MCA205full-length IL-36β, MCA205 hPTH-cultured IL-36β, MCA205hGH-trunc B16-F10WT, B16-F10full-lingth IL-36β, B16-F10hPTH-cultated IL-36β, B16-F10hGH-truncated IL-36β, MC38WT, MC38full-lingth IL-36β, MC38hPT NMLF was stimulated with any of the culture supernatants of hGH-proteinated IL-36β (250 μL culture supernatant + 250 μL medium), and the culture supernatant of NMLF was collected 24 hours later. Mouse IL-6 ELISA Ready-SET-Go! ^{Using TM} (eBioscience, San Diego, CA), the concentration of IL-6 in the recovered NMLF culture supernatant was measured.

3. 3. Statistical analysis processing Statistical analysis processing was performed using an unpaired two-sided student's t-test, assuming that the data were parametric. If the P value is 0.05 or less, there is a statistically significant difference. The statistical analysis process was performed in the same manner in the subsequent experiments.

<Result>
The production of IL-6 was induced in NMLF in which the culture supernatant of cells introduced with hPTH-truncated IL-36β or hGH-truncated IL-36β was allowed to act. (Fig. 7). On the other hand, the production of IL-6 was hardly confirmed in NMLF in which the culture supernatant of the cells into which WT and full length IL-36β was introduced was allowed to act.

[Effect of gene transfer on in vitro cell proliferation]
<Materials and methods>
1. 1. MTT assay
MCA205WT, MCA205hPTH-truncated IL-36β, MCA205 hGH-truncated IL-36β, B16-F10WT, B16-F10 hPTH-truncated-IL-36β, B16-F10 hGH-truncated Or, MC38 hGH-trunkated IL-36β was seeded on a 96-well plate (Sumitomo Bakelite) at 1.0 × 10 ³ cells / 100 μL / well and cultured for 4 days. Then, 5.0 mg / mL MTT solution (Nakalitesk, Kyoto, Japan) was added, and the supernatant was removed after 2 hours. Dimethyl sulfoxide (DMSO) (Wako) was added to dissolve the formazan formed on the cell membrane, and then the absorbance at 590 nm was measured using an EMax Precision Microplatate Reader (Molecular device, Tokyo, Japan).

<Result>
It was revealed that there was almost no difference in in vitro proliferation of MCA205WT, MCA205 hPTH-truncated IL-36β and MCA205 hGH-truncated IL-36β (Fig. 8). Similarly, it was revealed that there was almost no difference in in vitro proliferation of B16-F10 WT, B16-F10 hPTH-truncated IL-36 and B16-F10 hGH-truncated IL-36β (Fig. 8). Similarly, it was revealed that there was almost no difference in in vitro proliferation of MC38WT, MC38hPTH-truncated IL-36β and MC38hGH-truncated IL-36β (Fig. 8).

[Effect of gene transfer on in vivo tumor growth]
<Materials and methods>
1. 1. Mouse used C57BL / 6J Female mouse was purchased from Claire Japan. Animals were allowed free intake of water and solid feed. The age of the mice at the time of the experiment was 9 to 10 weeks. Similar mice were used in subsequent experiments.

2. Tumor burden mice produced MCA205WT, MCA205Neo, the MCA205hPTH-truncated IL-36β and MCA205hGH-truncated IL-36β, and suspended in PBS to a respective 1.0 × ¹⁰ 5 cells / 100μL. MC38WT, MC38Neo, MC38hPTH-truncated IL-36β and MC38hGH-truncated IL-36β were suspended in PBS at ^{1.5 × 10 5 cells / 100 μL, respectively.} B16-F10 WT, B16-F10 Neo, B16-F10 hPTH-truncated IL-36β and B16 F10-hGH-truncated IL-36β were suspended in PBS to a ^{concentration of 3.0 × 10 5 cells / 100 μL, respectively.} .. 100 μL of the PBS suspension of each cell was inoculated subcutaneously into the abdomen of the mouse (Day 0). The major and minor diameters of the tumor formed subcutaneously were measured with a caliper at intervals of 2 days from Day 4, and the tumor volume (mm ³ ) = major axis (mm) x minor axis ² (mm ² ) / 2. The tumor volume was calculated using the formula. The measurement was performed for one month.

<Result>
Tumor growth was significantly suppressed in mice administered with MCA205 hPTH-truncated IL-36β or MCA205 hGH-truncated IL-36β as compared with mice administered with MCA205WT or MCA205Neo (FIG. 9 (A)). In the MCA205hGH trainated IL-36β-administered mice, the tumor regressed in one mouse each on day 18 and day 30, and finally eradication of the tumor was observed. Tumor growth was significantly suppressed in mice administered with MC38 hPTH-truncated IL-36β or MC38 hGH-truncated IL-36β as compared with mice administered with MC38WT or MC38Neo (FIG. 9B).
Tumor growth was significantly suppressed in mice administered with B16-F10 hPTH-truncated IL-36β or hGH-truncated IL-36β as compared with mice administered with B16-F10WT or B16-F10Neo (FIG. 10). ). FIG. 10 (A) shows the results in mice inoculated with ^{3 × 10 5 cells.} FIG. 10B shows the results in mice inoculated with ^{6 × 10 5 cells.}

[Examination of the presence or absence of additive / synergistic antitumor action between IL-36β and IL-12]
<Materials and methods>
1. 1. Administration MCA205Neo making and IL-12 protein in tumor burden mice and MCA205hGH-truncated IL-36β, were suspended in PBS to a respective 1.0 × ¹⁰ 5 cells / 100μL, the PBS suspension 100 [mu] L of mouse Inoculated subcutaneously (Day 0). 0.2 μg of IL-12 protein (R & D Systems) was intraperitoneally administered to mice from Day 7 for 1 week. In addition, the major axis and minor axis of the tumor formed subcutaneously were measured with a caliper at intervals of once every two days from day 4, and the tumor volume (mm ³ ) = major axis (mm) × minor axis ² (mm ² ) / The tumor volume was calculated by using the formula of 2. The measurement was performed for one month.

<Result>
Mice to which IL-12 was administered in addition to MCA205 hGH-truncated IL-36β had significantly smaller tumor volumes than mice to which MCA205 hGH-truncated IL-36β was administered alone (FIG. 11). On day22, tumor regression occurred in all mice treated with IL-12 in addition to MCA205 hGH-truncated IL-36β, and finally tumor eradication was observed.

[Effect of IL-36β on liver metastasis]
<Materials and methods>
1. 1. Cell administration and removal of liver Liver metastatic mice were prepared transspleenically. Before the operation, the surgical instruments were disinfected by infiltrating with 5% Hibitene solution (Sumitomo Dainippon Pharma, Osaka, Japan). Mice were not stimulated with cytokines or bacterial toxins prior to cell injection.
In the operation, mice were induced under deep anesthesia by using intraperitoneal administration of somnopentil (Kyoritsu Seiyaku, Tokyo, Japan) and inhalation administration of diethyl ether (Wako) in combination. After confirming deep anesthesia, the left abdomen of the mouse was disinfected with povidone iodine (Meiji, Tokyo, Japan), the abdomen was opened 2 cm, and the spleen was abducted. MC38Neo and MCA38hGH-truncated IL-36β were ^{suspended in PBS at 1.5 × 10 5} cells / 100 μL, respectively, and 100 μL of the PBS suspension was administered to the spleen. After administration, hemostasis was performed with a cotton swab sprayed with 70% ethanol (Wako). After confirming hemostasis, a nylon suture (Akiyama Seisakusho, Saitama, Japan) was used to quickly suture the abdominal muscle and skin in two layers. After suturing, the abdomen was disinfected with Hibitene. After the operation, the mice were rested on a simple warm mat to maintain their body temperature, and after confirming their arousal, they were returned to their cages.
The day of cell administration was set to day 0, and the mice were sacrificed to death on day 14 and the liver was removed. Then, the number of tumor nodules confirmed on the surface of the excised liver was measured. Furthermore, liver weight was measured.

<Result>
A significant decrease in the number of nodules was confirmed in the MC38hGH-truncated IL-36β-administered mice as compared with the MC38Neo-administered mice. However, there was no difference in liver weight between the two groups (FIGS. 12 and 13). From the above results, it was confirmed that IL-36β markedly suppresses liver metastasis of colorectal cancer cells.

According to the present invention, a novel antitumor agent and a novel pharmaceutical composition for treating a tumor are provided.

Claims (9)

(A) Interleukin (IL) -36β or a variant thereof;
(B) A polynucleotide having a base sequence encoding IL-36β or a variant thereof;
(C) A vector containing the polynucleotide of (b) above; and (d) a cell secreting IL-36β or a variant thereof.
An antitumor agent containing at least one component selected from the group consisting of. The polynucleotide of (b) is
It also has a base sequence encoding a signal peptide of a secretory protein,
The base sequence encoding the signal peptide is directly or indirectly linked in-frame to the 5'end of the base sequence encoding IL-36β or a variant thereof.
The antitumor agent according to claim 1. The antitumor agent according to claim 1 or 2, which is administered in combination with IL-12. (A) Interleukin (IL) -36β or a variant thereof;
(B) A polynucleotide having a base sequence encoding IL-36β or a variant thereof;
(C) A vector containing the polynucleotide of (b) above; and (d) a cell secreting IL-36β or a variant thereof.
A pharmaceutical composition for treating a tumor, which comprises at least one component selected from the group consisting of, and a pharmaceutically acceptable carrier. The polynucleotide of (b) is
It also has a base sequence encoding a signal peptide of a secretory protein,
The base sequence encoding the signal peptide is directly or indirectly linked in-frame to the 5'end of the base sequence encoding IL-36β or a variant thereof.
The pharmaceutical composition according to claim 4. The pharmaceutical composition according to claim 4 or 5, which is administered in combination with IL-12. The pharmaceutical composition according to claim 4 or 5, further comprising IL-12. (A) Interleukin (IL) -36β or a variant thereof;
(B) A polynucleotide having a base sequence encoding IL-36β or a variant thereof;
(C) A vector containing the polynucleotide of (b) above; and (d) a cell secreting IL-36β or a variant thereof.
An antitumor effect enhancer used for enhancing the antitumor effect of IL-12, which contains at least one component selected from the group consisting of. (A) Interleukin (IL) -36β or a variant thereof;
(B) A polynucleotide having a base sequence encoding IL-36β or a variant thereof;
(C) A vector containing the polynucleotide of (b) above; and (d) a cell secreting IL-36β or a variant thereof.
It contains at least one component selected from the group consisting of, and a pharmaceutically acceptable carrier. A pharmaceutical composition used to enhance the antitumor effect of IL-12.

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