WO2023053315A1

WO2023053315A1 - Pd-1 expression inhibitor, pharmaceutical composition for immune checkpoint inhibition, and method for inhibiting pd-1 expression

Info

Publication number: WO2023053315A1
Application number: PCT/JP2021/036056
Authority: WO
Inventors: 宗夫沼崎
Original assignee: 国立大学法人東北大学
Priority date: 2021-09-30
Filing date: 2021-09-30
Publication date: 2023-04-06

Abstract

This PD-1 expression inhibitor contains at least one component selected from the group consisting of the following (a) through (d): (a) interleukin 36 or a modified form thereof; (b) a polynucleotide having a nucleotide sequence encoding either interleukin 36 or a modified form thereof; (c) a vector containing the polynucleotide described in the abovementioned (b); and (d) cells that secrete either interleukin 36 or a modified form thereof.

Description

PD-1 expression inhibitor, pharmaceutical composition for immune checkpoint inhibition, and method for inhibiting PD-1 expression

The present invention relates to a PD-1 expression inhibitor, a pharmaceutical composition for immune checkpoint inhibition, and a method for inhibiting PD-1 expression.

Cancer is the number one cause of death in Japan, and there are many patients worldwide. Therefore, development of a better therapeutic agent is desired. In recent years, immune checkpoint inhibitors that inhibit the action of immune checkpoint molecules such as PD-1 (programmed cell death 1) have been introduced as therapeutic agents for cancer (Patent Document 1).
PD-1 is a molecule expressed on the surface of activated lymphocytes such as cytotoxic T cells (CTL). It is known that when PD-1 binds to a cancer cell surface ligand (such as PD-L1), CTL activation is suppressed, and cytotoxic activity against cancer cells is suppressed. Immune checkpoint inhibitors targeting PD-1 inhibit the reduction of cytotoxic activity of CTL by inhibiting ligand binding to PD-1.

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

IL-36 has three isoforms, IL-36α, IL-36β and IL-36γ, as splice variants. IL-36β is mainly produced by epithelial cells, monocytes, B cells and the like. It has been reported that truncated IL-36β, in which 30 amino acids at the N-terminus have been removed, has about 1000-fold stronger activity than native 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 cell surface MHC class II, CD80, CD86, etc. (Non-Patent Document 2).

WO2004/004771

Monoclonal antibodies against immune checkpoint molecules such as PD-1 are used as immune checkpoint inhibitors. In order to expand the options for cancer treatment, it is necessary to develop drugs that can suppress the function of immune checkpoint molecules by different mechanisms.

Therefore, an object of the present invention is to provide a PD-1 expression suppressing agent, a pharmaceutical composition for immune checkpoint inhibition, and a method for suppressing PD-1 expression that can suppress the expression of PD-1 in lymphocytes. and

The present disclosure includes the following aspects.
[1] A PD-1 expression inhibitor comprising at least one component selected from the group consisting of the following (a) to (d): (a) interleukin 36 or a variant thereof;
(b) a polynucleotide having a nucleotide sequence encoding interleukin-36 or a variant thereof; (c) a vector comprising the polynucleotide of (b); and (d) a cell that secretes interleukin-36 or a variant thereof.
[2] The PD-1 expression inhibitor according to [1], which suppresses PD-1 expression in CD8-positive T cells or CD4-positive T cells.
[3] A pharmaceutical composition for immune checkpoint inhibition, comprising the PD-1 expression inhibitor of [1] or [2] and a pharmaceutically acceptable carrier.
[4] A method of suppressing PD-1 expression in lymphocytes, comprising culturing lymphocytes in the presence of interleukin 36 or a variant thereof.

According to the present invention, a PD-1 expression inhibitor, a pharmaceutical composition for immune checkpoint inhibition, and a method for inhibiting PD-1 expression are provided, which are capable of suppressing PD-1 expression in lymphocytes.

FIG. 4 shows the results of analyzing the PD1 expression inhibitory effect of truncated mouse IL-36β on CD8-positive T cells and CD4-positive T cells by flow cytometry. FIG. 4 shows the results of analyzing the PD1 expression inhibitory effect of truncated mouse IL-36β on CD8-positive T cells and CD4-positive T cells by flow cytometry. FIG. 2 shows the mean fluorescence intensity (MFI) of PE (PD-1 positive) based on the flow cytometry analysis results of FIGS. 1 and 2. FIG. FIG. 4 shows the results of analyzing the PD1 expression inhibitory effect of truncated mouse IL-36β on CD8-positive T cells and CD4-positive T cells by flow cytometry. FIG. 4 shows the results of analyzing the PD1 expression inhibitory effect of truncated mouse IL-36β on CD8-positive T cells and CD4-positive T cells by flow cytometry. FIG. 5 shows the mean fluorescence intensity (MFI) of PE (PD-1 positive) based on the flow cytometry analysis results of FIGS. 4 and 5. FIG. Results for CD8-positive T cells are shown. Splenocytes of mice transplanted with B16-F10Neo cells, B16-F10-PTH-truncated IL-36β cells, or B16-F10-GH-truncated IL-36β cells were harvested, and PD-1 expression was analyzed by flow cytometry. Analyzed results are shown. 12 after inoculation of MCA205 Neo cells, MCA205-PTH-truncated IL-36β cells, MCA205-GH-truncated IL-36β cells, MC38Neo cells, MC38-PTH-truncated IL-36β cells, or MC38-GH-truncated IL-36β cells The results of PD-1 molecule immunostaining of day-old tumor tissues are shown.

The term "comprise" means that it may include components other than the target components. The term "consist of" means containing no elements other than the subject element. The term "consistently of" means that it does not include constituent elements other than the subject constituent elements in a mode that exhibits a special function (such as a mode that completely loses the effects of the invention). means. As used herein, the term "comprise" includes aspects that "consist of" and aspects that "consist essentially of."

Proteins, peptides, polynucleotides (DNA, RNA), vectors, and cells can be isolated. "Isolated" means separated from the natural state or other components. "Isolated" can be substantially free of other components. "Substantially free of other components" means that the content of other components contained in the isolated component is negligible. The content of other components contained in the isolated component is, for example, 10% by mass or less, 5% by mass or less, 4% by mass or less, 3% by mass or less, 2% by mass or less, 1% by mass or less, 0.5% by mass or less. It may be 5% by mass or less, or 0.1% by mass or less. The proteins, peptides, polynucleotides (DNA, RNA), vectors and cells described herein may be isolated proteins, isolated peptides, isolated polynucleotides (isolated DNA, isolated RNA), isolated vectors, and isolated cells.

"Polynucleotide" refers to a nucleotide polymer in which nucleotides are linked by phosphodiester bonds. A "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, including natural nucleotides and non-natural nucleotides (analogs of natural nucleotides, nucleotides in which at least one of the base, sugar and phosphate moieties is modified). (for example, a phosphorothioate skeleton), etc.), or a polymer of non-natural nucleotides.
Nucleotide sequences of polynucleotides are written in the generally accepted single-letter code unless otherwise indicated. Unless otherwise indicated, nucleotide sequences are written 5' to 3'. Nucleotide residues constituting a polynucleotide may be simply described as adenine, thymine, cytosine, guanine, uracil, or the like, or their one-letter codes.

"Gene" refers to a polynucleotide containing at least one open reading frame that encodes a particular protein. A gene may contain both exons and introns.

The terms "peptide", "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acids linked by amide bonds. "Peptides", "polypeptides" and "proteins" may be polymers of naturally occurring amino acids or of naturally occurring and non-natural amino acids (chemical analogues, modified derivatives, etc. of naturally occurring amino acids). It may also be a polymer of unnatural amino acids.
Amino acid sequences are written in the generally accepted one-letter or three-letter code unless otherwise specified. Unless otherwise specified, amino acid sequences are written from the N-terminal side to the C-terminal side.

The term "operably linked" when used in reference to polynucleotides means that a first nucleotide sequence is placed sufficiently close to a second nucleotide sequence such that the first nucleotide sequence is either the second nucleotide sequence or the second nucleotide sequence. means that it can affect areas under the control of For example, that a polynucleotide is operably linked to a promoter means that the polynucleotide is linked so that it is expressed under the control of the promoter.
The term "expressible state" refers to a state in which a gene of interest can be transcribed in a cell into which the gene of interest has been introduced.
The term "expression vector" refers to a vector containing a gene of interest and equipped with a system that renders the gene of interest expressible in cells into which the vector has been introduced.
“A promoter can function” means that a polynucleotide operably linked to the promoter can be expressed in the cells of the subject organism.
"In-frame" means that the reading frame of the codon is not shifted.

"Sequence identity" between amino acid sequences or nucleotide sequences is defined by aligning the two amino acid sequences or nucleotide sequences so that the corresponding amino acids or bases match the most, leaving gaps for insertions and deletions, It is determined as the ratio of matched amino acids or bases to the entire amino acid sequence or nucleotide sequence excluding gaps in the resulting alignment. Sequence identity between amino acid sequences or between nucleotide sequences can be determined using various homology search software known in the art. For example, the sequence identity value of an amino acid sequence can be obtained by calculation based on alignments obtained by the known homology search software BLASTP, and the sequence identity value of a nucleotide sequence can be obtained by a known homology search. It can be obtained by calculation based on alignments obtained by the software BLASTN. Sequence identity is also referred to as homology.

"Stringent conditions" means conditions under which two polynucleotides with high sequence identity can specifically hybridize. Two polynucleotides with high sequence identity, the sequence identity between the two polynucleotides, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% Above, 98% or more, or 99% or more. Stringent conditions include, for example, conditions described in Molecular Cloning-A Laboratory Manual Third Edition (Sambrook et al., Cold Spring Harbor Laboratory Press). Specific examples of stringent conditions include, for example, 6 x SSC (composition of 20 x SSC: 3 M sodium chloride, 0.3 M citric acid solution, pH 7.0), 5 x Denhardt's solution (composition of 100 x Denhardt's solution: 42 in a hybridization buffer consisting of 2% by weight bovine serum albumin, 2% by weight Ficoll, 2% by weight polyvinylpyrrolidone), 0.5% by weight SDS, 0.1 mg/mL salmon sperm DNA, and 50% formamide. Conditions include incubation at ~70°C for several hours to overnight. Washing buffers used for washing after incubation include, for example, 0.1% by mass SDS-containing 1×SSC solution and 0.1% by mass SDS-containing 0.1×SSC solution.

[PD-1 expression inhibitor]
A first aspect of the present disclosure is a PD-1 expression inhibitor. The PD-1 expression inhibitor contains at least one component selected from the group consisting of the following (a) to (d):
(a) IL-36 or variant thereof;
(b) a polynucleotide having a nucleotide sequence encoding IL-36 or a variant thereof;
(c) a vector comprising the polynucleotide of (b); and (d) a cell secreting IL-36 or a variant thereof.

As shown in the examples below, PD-1 expression is suppressed in lymphocytes cultured in the presence of IL-36 compared to lymphocytes cultured in the absence of IL-36. was confirmed. In lymphocytes from mice transplanted with tumor cells that produce IL-36, the expression of PD-1 is suppressed compared to lymphocytes from mice transplanted with tumor cells that do not produce IL-36. confirmed. These results demonstrate that lymphocyte PD-1 expression is suppressed by IL-36 in vitro and in vivo. Therefore, each of the above components (a) to (d) can be used as a PD-1 expression inhibitor.

The PD-1 expression inhibitor of this embodiment can be added to a medium for culturing lymphocytes and used to prepare lymphocytes in which PD-1 expression is suppressed in vitro or ex vivo. Lymphocytes targeted for suppression of PD-1 expression are not particularly limited as long as they express PD-1. Lymphocytes targeted for suppression of PD-1 expression include, for example, T cells and natural killer cells. Examples of T cells include CD8-positive T cells (CTL and the like), CD4-positive T cells (helper T cells and the like), and the like. Organisms from which lymphocytes are derived are not particularly limited, but include, for example, humans and mammals other than humans. Mammals other than humans include, for example, primates (monkeys, chimpanzees, gorillas, etc.), rodents (mice, hamsters, rats, etc.), rabbits, dogs, cats, cows, goats, sheep, horses, pigs, and the like. include but are not limited to:
Lymphocytes may be genetically modified. The type of genetic modification is not particularly limited. Specific examples of genetically modified lymphocytes include lymphocytes modified to express chimeric antigen receptors (CAR-T cells, etc.), lymphocytes modified to express exogenous TCR (TCR- T cells, etc.) and the like.

The PD-1 expression inhibitor of the present embodiment may be administered to a living body and used in vivo to suppress PD-1 expression of lymphocytes present in the living body. The organism to which the PD-1 inhibitor is administered is not particularly limited as long as it has lymphocytes expressing PD-1. Organisms to be administered include the same organisms as those described above.

<(a) IL-36>
IL-36 is a cytokine belonging to the IL-1 cytokine family. IL-36 has three isoforms, IL-36α, IL-36β and IL-36γ, as splice variants. IL-36 may be any of IL-36α, IL-36β, and IL-36γ, with IL-36β being preferred.

Although the organism from which IL-36 is derived is not particularly limited, it is preferably the same species as the organism from which the lymphocytes whose PD-1 expression is to be suppressed are derived. For example, when human lymphocytes are targeted for suppression of PD-1 expression, it is preferable to use human IL-36. Mouse IL-36 is preferably used when the target for suppression of PD-1 expression is mouse lymphocytes.

IL-36 may be naturally occurring or modified. Native IL-36 is IL-36 produced in nature. Native IL-36 is, for example, IL-36 produced in mammals as described above. Specific examples of native IL-36 include human IL-36 and mouse IL-36.
Examples of the amino acid sequence of human IL-36β (NCBI Gene ID: 27177) include the amino acid sequence described in SEQ ID NO: 2 (nucleotide sequence: SEQ ID NO: 1), the amino acid sequence described in SEQ ID NO: 4 (nucleotide sequence: SEQ ID NO: 3) and the like.
The amino acid sequence of mouse IL-36β (NCBI Gene ID: 69677) includes, for example, the amino acid sequence set forth in SEQ ID NO: 6 (nucleotide sequence: SEQ ID NO: 5). Native IL-36 is not limited to those having the above amino acid sequences, and may be homologues (orthologs or paralogs) thereof. The amino acid sequence of native IL-36 can be obtained from sequence databases such as GenBank.

Modified IL-36 (hereinafter also referred to as "variants of IL-36") is a polypeptide obtained by modifying native IL-36, and is a polypeptide that maintains the function of native IL-36. . A variant of IL-36 has an amino acid sequence in which one or more amino acids are altered (substitution, deletion, addition, insertion, or a combination thereof) as compared with the amino acid sequence of native IL-36. The number of amino acids to be modified is not particularly limited. , 1 to 3, or 1 to 2, and the like. Modified IL-36 may have an amino acid sequence with 80% or more sequence identity with the amino acid sequence of native IL-36. Sequence identity can be, for example, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater.

The variant of IL-36 preferably has at least one activity of native IL-36 that is equal to or greater than that of native IL-36. The activity of native IL-36 (hereinafter also referred to as “IL-36 activity”) includes, for example, the activity of suppressing PD-1 expression in lymphocytes. IL-36 activity includes binding activity to a receptor consisting of a heterodimer of IL-36R and IL-1RAcp, activity to cause cells having the receptor (e.g., lung fibroblasts) to produce IL-6, and It may be antitumor activity or the like. A variant of IL-36 having an activity equal to or greater than that of native IL-36β has a relative activity of, for example, 80 or more, 85 or more, 90 or more, or 95 or more when the activity exhibited by natural IL-36β is taken as 100. It shows activity.

Mouse IL-36β has been reported to exhibit approximately 1,000-fold stronger activity compared to native IL-36β in a variant in which 30 amino acids at the N-terminus have been removed (Towne JE, et al. 2011 Dec 9; 286(49):42594-602.). In human IL-36β, it has been reported that a variant in which 4 amino acids at the N-terminus have been removed exhibits stronger activity than wild-type IL-36β (Towne JE, et al., J Biol. Chem. 2011 Dec 9; 286(49):42594-602.). Therefore, variants of IL-36β include those from which amino acids in the N-terminal region of native IL-36β have been removed (hereinafter also referred to as "truncated IL-36β"). The upper limit of the number of N-terminal amino acids to be removed is, for example, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, or 4 Below are listed. The lower limit for the number of N-terminal amino acids to be removed is 1 or more. The above upper limit and lower limit can be arbitrarily combined. For murine IL-36β, the number of N-terminal amino acids removed includes, for example, 1-40, 5-35, or 10-30. For human IL-36β, the number of N-terminal amino acids removed includes, for example, 1-40, 2-30, 3-20, or 4-10. The amino acid sequence of truncated human IL-36β includes, for example, the amino acid sequence set forth in SEQ ID NO: 8 (nucleotide sequence: SEQ ID NO: 7), the amino acid sequence set forth in SEQ ID NO: 10 (nucleotide sequence: SEQ ID NO: 9), and the like. . The amino acid sequence of truncated mouse IL-36β is, for example, the amino acid sequence set forth in SEQ ID NO: 12 (nucleotide sequence: SEQ ID NO: 11).

<(b) Polynucleotide>
The polynucleotide of (b) has a nucleotide sequence encoding IL-36 or a variant thereof (hereinafter referred to as "IL-36 coding sequence"). An IL-36 coding sequence is the nucleotide sequence of the IL-36 gene. The polynucleotide (b) can also be said to be a polynucleotide containing the IL-36 gene.

<<IL-36 coding sequence>>
The IL-36 coding sequence may encode native IL-36 or variants of IL-36. The polynucleotides of (b) include, for example, the following polynucleotides.
(1) A polynucleotide encoding a polypeptide having the amino acid sequence of native IL-36 (eg, SEQ ID NO: 2, 4 or 6).
(2) an amino acid sequence in which one or more amino acids are modified (deleted, substituted, added, inserted, or a combination thereof) in the amino acid sequence of native IL-36 (e.g., SEQ ID NO: 2, 4 or 6); and which has IL-36 activity.
(3) A polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence of native IL-36 (e.g., SEQ ID NO: 2, 4 or 6) and having IL-36 activity A polynucleotide that encodes a peptide.
(4) A polynucleotide having the nucleotide sequence of a native IL-36 gene (eg, SEQ ID NO: 1, 3 or 5).
(5) A nucleotide sequence in which one or more nucleotides are modified (deleted, substituted, added, inserted, or a combination thereof) in the nucleotide sequence of a native IL-36 gene (e.g., SEQ ID NO: 1, 3 or 5) and which encodes a polypeptide having IL-36 activity.
(6) A polynucleotide having a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence of a native IL-36 gene (e.g., SEQ ID NO: 1, 3 or 5), and having IL-36β activity A polynucleotide that encodes a polypeptide.
(7) A polynucleotide that hybridizes under stringent conditions with a polynucleotide having the nucleotide sequence of a native IL-36 gene (e.g., SEQ ID NO: 1, 3 or 5) and has IL-36 activity A polynucleotide that encodes a peptide.

The number of modified amino acids in (2) above and the value of sequence identity in (3) above are as exemplified in <(a) IL-36β> above.
The number of modified nucleotides 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 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2, and the like.
The sequence identity in (6) may be, for example, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

The IL-36 coding sequence may encode truncated IL-36β. Examples include the following polynucleotides.
(8) A polynucleotide encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:8, 10 or 12.
(9) A polypeptide having an amino acid sequence in which one or more amino acids are altered (deleted, substituted, added, inserted, or a combination thereof) in the amino acid sequence set forth in SEQ ID NO: 8, 10 or 12, and a polypeptide that has IL-36β activity.
(10) A polynucleotide encoding a polypeptide consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 8, 10 or 12, and having IL-36β activity .
(11) A polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 7, 9 or 11.
(12) A polynucleotide having a nucleotide sequence in which one or more bases are modified (deleted, substituted, added, inserted, or a combination thereof) in the nucleotide sequence set forth in SEQ ID NO: 7, 9 or 11, and a polynucleotide encoding a polypeptide having IL-36β activity.
(13) A polynucleotide having a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 7, 9 or 11 and encoding a polypeptide having IL-36β activity.
(14) A polynucleotide that hybridizes under stringent conditions with a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 7, 9 or 11 and that encodes a polypeptide having IL-36β activity.

The number of modified amino acids in (9) is, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5, 1 to 4, 1 to 3, or 1 to 2, and the like. The sequence identity in (10) may be, for example, 85% or more, 90% or more, or 95% or more. The number of modified bases in (12) 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 to 10 or 1 to 5, and the like. The sequence identity in (13) includes, for example, 5% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

≪Other nucleotide sequences≫
The polynucleotide of (b) may contain other nucleotide sequences in addition to the IL-36 coding sequence. Other nucleotide sequences include, for example, a nucleotide sequence encoding a signal peptide (hereinafter referred to as "signal peptide coding sequence"), a sequence controlling IL-36 gene expression (hereinafter referred to as "expression control sequence"), and the like. is mentioned.

(signal peptide coding sequence)
The polynucleotide of (b) preferably further has a signal peptide coding sequence for a secretory protein in addition to the IL-36 coding sequence. The signal peptide coding sequence is preferably directly or indirectly linked in-frame to the 5' end of the IL-36 coding sequence. A signal peptide coding sequence is ligated in-frame to the 5′ end of the IL-36 coding sequence, so that when IL-36 is produced in cells into which the polynucleotide of (b) has been introduced, IL- 36β can be secreted extracellularly.

The signal peptide coding sequence includes a sequence that encodes a signal peptide that can function as a secretory signal in cells into which the polynucleotide of (b) can be introduced (hereinafter also referred to as "introduction target cells"). From the viewpoint that IL-36 can be stably secreted extracellularly, the signal peptide coding sequence is preferably derived from the same species of organism as the target cell. For example, when the cells to be introduced are human cells, it is preferable to use the signal peptide coding sequence of a human secretory protein. When the cells to be introduced are mouse cells, it is preferable to use the signal peptide coding sequence of a mouse secretory protein.

The secreted protein from which the signal peptide coding sequence is derived is not particularly limited. Examples of secretory proteins include peptide hormones such as parathyroid hormone and growth hormone; cytokines such as IFN-γ, GM-CSF and IL-6; immunoglobulin heavy or light chains; not.
Examples of the signal peptide coding sequence include a nucleotide sequence (eg, SEQ ID NO: 13) encoding the amino acid sequence set forth in SEQ ID NO: 14 (the signal peptide of human parathyroid hormone (hPTH)), and the amino acid sequence set forth in SEQ ID NO: 16. and a nucleotide sequence (eg, SEQ ID NO: 15) encoding (signal peptide of human growth hormone (hGH)).

The signal peptide coding sequence may be directly linked to the 5' end of the IL-36 coding sequence or indirectly via a linker. The linker is not particularly limited as long as it does not impair IL-36 activity when the signal peptide coding sequence-linker sequence-IL-36 coding sequence is translated. The length of the linker may be, for example, 3 nucleotides, 6 nucleotides, 9 nucleotides, or 12 nucleotides.
Preferably, the signal peptide coding sequence is directly linked to the 5' end of the IL-36 coding sequence.

(expression control sequence)
The polynucleotide of (b) preferably has an expression control sequence in addition to the IL-36 coding sequence. Examples of expression control sequences include 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 to be introduced. Promoters include, for example, CMV (cytomegalovirus) promoter, SRα promoter, SV40 early promoter, retroviral LTR, RSV (Rous sarcoma virus) promoter, HSV-TK (herpes simplex virus thymidine kinase) promoter, EF1α promoter, metallothionein promoters, heat shock promoters, etc., but are not limited thereto.
The IL-36 coding sequence is preferably operably linked to a suitable promoter. If the polynucleotide of (b) has a signal peptide coding sequence, it is preferred that the signal peptide coding sequence and the IL-36 coding sequence are operably linked to a suitable promoter.

The polynucleotide of (b) may be DNA or RNA. When the polynucleotide of (b) is DNA, it may be in the form of a vector described below. When the polynucleotide of (b) is RNA, it may be in the form of mRNA. In this case, the polynucleotide (b) may have a 5'Cap structure, poly-A-tail, or the like.

<(c) Vector>
The vector of (c) contains the polynucleotide of (b). The type of vector is not particularly limited, and an expression vector or the like generally used for gene expression can be used. Vectors may be linear or circular. Examples of vectors include non-viral vectors such as plasmids, viral vectors, transposon vectors, episomal vectors, artificial chromosome vectors and the like.

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

Viral vectors include, for example, Sendai virus vectors, retrovirus (including lentivirus) vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, pox virus vectors, polio virus vectors, silvis virus vectors , rhabdovirus vector, paramyxovirus vector, orthomyxovirus vector and the like.

An episomal vector is a vector that can replicate autonomously outside the chromosome. Examples of episomal vectors include vectors containing, as vector elements, sequences necessary for autonomous replication derived from EBV, SV40, and the like. Vector elements required for autonomous replication specifically include genes encoding replication origins and proteins that bind to replication origins to control replication. For example, EBV includes the replication origin oriP and the EBNA-1 gene, and SV40 includes the replication origin ori and the SV40LT gene.

Examples of artificial chromosome vectors include YAC (Yeast artificial chromosome) vectors, BAC (Bacterial artificial chromosome) vectors, PAC (P1-derived artificial chromosome) vectors, and the like.

Vectors can be produced using commercially available products. For example, a vector containing the polynucleotide (b) can be produced by inserting the polynucleotide (b) into the multicloning site of a commercially available vector. Insertion of the polynucleotide (b) into a commercially available vector can be performed by, for example, a combination of restriction enzyme treatment, ligase treatment, and the like.

When the vector (c) is a targeting vector that is integrated into the genome of the target cell by genome editing technology or the like, it preferably contains homology arms for homologous recombination. A homology arm has a sequence homologous to any genomic region of the cell to be introduced. The genomic region homologous to the homology arms is the region into which the polynucleotide of (b) is inserted. The homology arms are located flanking the 5' and 3' sides of the polynucleotide of (b).
The sequence of homology arms can be appropriately designed according to the type of cell to be introduced and the target region. For example, when the cells to be introduced are tumor cells, an oncogene possessed by the tumor cells may be selected as a target region and homology arms may be designed.

<(d) cells>
The cells of (d) are cells that secrete IL-36 or variants thereof (hereinafter referred to as "IL-36-secreting cells"). The IL-36-secreting cells may be native IL-36-secreting cells or transgenic IL-36-secreting cells. When IL-36-secreting cells are administered to a living organism, the IL-36-secreting cells are preferably cells of the organism to which they are administered.

Native IL-36 secreting cells are cells that endogenously produce and secrete IL-36. Native IL-36-secreting cells include, for example, epithelial cells, monocytes, B cells, and the like.
Transgenic IL-36-secreting cells are cells into which the polynucleotide of (b) or the vector of (c) has been introduced. Host cells are not particularly limited, but include, for example, cells collected from an administration subject, cells having the same MHC as that of the administration subject, and the like. Examples of cell types include, but are not limited to, tumor cells, immune cells (dendritic cells, T cells, natural killer cells, etc.), fibroblasts, and the like. A host cell can be, for example, a T cell. Examples of T cells include CD8-positive T-cells (CTL etc.), CD4-positive T-cells (helper T-cells etc.) and the like.

Transgenic IL-36-secreting cells can be prepared using known gene transfer methods depending on the type of vector. Gene introduction methods include, for example, the virus infection method, lipofection method, microinjection method, calcium phosphate method, DEAE-dextran method, electroporation method, method using transposon, particle gun method and the like.
Transgenic IL-36-secreting cells may be prepared using known gene editing techniques and the like. Gene editing techniques include, for example, techniques using endonucleases such as zinc finger nucleases, TALENs (transcription activation-like effector nucleases), and CRISPR-Cas systems.

When producing transgenic IL-36-secreting cells, the vector (c) may contain a marker gene. A marker gene can be used to select IL-36β-transfected cells. Commonly used marker genes (eg, drug resistance genes, fluorescent dye genes) can be used without particular limitations. Examples of marker genes include, but are not limited to, neomycin resistance gene, ampicillin resistance gene, hygromycin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene, and the like.
The marker gene may be linked to the IL-36 coding sequence (IL-36 gene) via a nucleotide sequence encoding a self-cleaving terminal peptide such as 2A peptide, or an IRES (internal ribozyme entry site) sequence or the like. . By interposing these sequences, the IL-36 gene and the marker gene can be expressed independently from one promoter.

Secretion of IL-36 from IL-36-secreting cells can be confirmed by a known method. For example, IL-36 secretion from IL-36-secreting cells is confirmed by collecting the culture supernatant of IL-36-secreting cells and measuring IL-36 by Western blotting or an immunological method such as ELISA. can do. Alternatively, IL-36-secreting cells by allowing the culture supernatant of IL-36-secreting cells to act on IL-6-producing cells such as fibroblasts and measuring the amount of IL-6 secreted from the fibroblasts. IL-36 secretion from .

The PD-1 expression inhibitor of the present embodiment may contain any one of the components (a) to (d) alone, or may contain two or more in combination.
The PD-1 expression inhibitor can contain at least one component selected from the group consisting of (a) to (d) as an active ingredient for the PD-1 expression inhibitory effect. The "PD-1 expression inhibitory effect" means that when the PD-1 expression inhibitor is exposed to the lymphocytes, compared with the case where the PD-1 expression inhibitor is not exposed to the lymphocytes, PD-1 of the lymphocytes. It refers to the effect of suppressing expression. For example, culturing lymphocytes in the presence of a PD-1 expression inhibitor suppresses PD-1 expression in lymphocytes compared to culturing in the absence of a PD-1 expression inhibitor. For example, when the PD-1 expression level is 100 when lymphocytes are cultured in the absence of a PD-1 expression inhibitor, when the lymphocytes are cultured in the presence of a PD-1 expression inhibitor, PD- The relative expression level of 1 may be 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, or 40 or less. The PD-1 expression level of lymphocytes can be confirmed by a known method. Methods for confirming the PD-1 expression level include, for example, flow cytometry analysis, RT-qPCR and the like.

PD-1 expression in lymphocytes can be effectively suppressed by the PD-1 expression inhibitor of the present embodiment.
The PD-1 expression inhibitor of this embodiment can be used to prepare lymphocytes used for adoptive immunotherapy or CAR-T therapy for cancer. For example, lymphocytes with suppressed PD-1 expression can be prepared by culturing lymphocytes in the presence of the PD-1 expression inhibitor of the present embodiment. By administering such lymphocytes to cancer patients, they can bypass the immune checkpoint system and express cytotoxic activity against cancer cells. When the PD-1 expression inhibitor of the present embodiment is used in vitro, it is preferable to use component (a) or (d).

The PD-1 expression inhibitor of the present embodiment may be administered to a living body in order to suppress PD-1 expression of lymphocytes in the living body. When the PD-1 expression inhibitor is the component (a), IL-36 or a variant thereof contacts lymphocytes in vivo and PD-1 expression in the lymphocytes is inhibited. When the PD-1 expression inhibitor is the component (b) or (c), cells incorporating the polynucleotide of (b) or the vector of (c) produce IL-36 or a variant thereof. The produced IL-36 or its variant comes into contact with lymphocytes in vivo, and PD-1 expression in the lymphocytes is suppressed. When the PD-1 expression inhibitor is a component of (d), IL-36 produced by the cells of (d) or a variant thereof contacts lymphocytes in vivo, and PD-1 expression of the lymphocytes is suppressed.

[Pharmaceutical composition]
A second aspect of the present disclosure is a pharmaceutical composition for immune checkpoint inhibition. The pharmaceutical composition for immune checkpoint inhibition contains the PD-1 expression inhibitor of the first aspect and a pharmaceutically acceptable carrier.

The pharmaceutical composition of this embodiment is administered to a subject to suppress the immune checkpoint system. When the pharmaceutical composition of this embodiment is administered, the administered or produced IL-36 or its variant acts on lymphocytes in vivo. This suppresses PD-1 expression in lymphocytes and suppresses the PD-1-mediated immune checkpoint system.

The subject of administration of the pharmaceutical composition of the present embodiment is a subject requiring inhibition of the immune checkpoint system via PD-1. Administration subjects include, for example, cancer patients. When administering the pharmaceutical composition of the present embodiment to a cancer patient, the pharmaceutical composition of the present embodiment may be administered alone or in combination with an anticancer agent.

The pharmaceutical composition of this embodiment contains a pharmaceutically acceptable carrier in addition to the PD-1 expression inhibitor of the first aspect. "Pharmaceutically acceptable carrier" means a carrier that does not inhibit the physiological activity of the active ingredient and does not exhibit substantial toxicity to the subject to which it is administered. The phrase "substantially non-toxic" means that the component does not show toxicity to the subject at the dose normally used. In the pharmaceutical composition of this embodiment, the pharmaceutically acceptable carrier does not inhibit the PD-1 expression inhibitory activity of the components (a) to (d), and is substantially It is a non-toxic carrier. Pharmaceutically acceptable carriers include any known pharmaceutically acceptable ingredients that are typically considered non-active ingredients. Pharmaceutically acceptable carriers include, but are not limited to, solvents, diluents, vehicles, excipients, glidants, binders, granulating agents, dispersing agents, suspending agents, wetting agents, Lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, fillers, preservatives (e.g., antioxidants), chelating agents, flavoring agents, sweeteners, thickeners, buffering agents, coloring agents, etc. mentioned. One type of pharmaceutically acceptable carrier may be used alone, or two or more types may be used in combination. When the pharmaceutical composition contains (b) or (c) above, pharmaceutically acceptable carriers may be solvents, diluents, vehicles, buffers and the like. When the pharmaceutical composition contains (d) above, the pharmaceutically acceptable carrier may be a cell culture medium, a buffer (physiological saline, PBS, citrate buffer, etc.), or the like.

In the pharmaceutical composition of the present embodiment, in addition to those listed as pharmaceutically acceptable carriers, additives commonly used in the pharmaceutical field can be used without particular limitation. When the pharmaceutical composition contains (b) or (c), the pharmaceutical composition may contain a transfection promoter, a genome editing reagent (CRISPR/Cas9 system, etc.) and the like.

The dosage form of the pharmaceutical composition is not particularly limited, and may be a dosage form commonly used as a pharmaceutical preparation. The pharmaceutical composition may be an oral formulation or a parenteral formulation. Examples of oral preparations include tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, liquids, drops, emulsions and the like. Examples of parenteral formulations include injections, suppositories, ointments, sprays, nasal drops, and inhalants. Pharmaceutical compositions in these dosage forms can be formulated according to standard methods (eg, methods described in the Japanese Pharmacopoeia).
The pharmaceutical composition of this embodiment is preferably a parenteral formulation, more preferably an injection.

The pharmaceutical composition of this embodiment may contain active ingredients other than the PD-1 expression inhibitor of the first aspect. The active ingredient is not particularly limited. Animal/microorganism extract, antipruritic, anti-inflammatory analgesic, antifungal agent, antihistamine, sedative hypnotic, tranquilizer, antihypertensive, hypotensive diuretic, antibiotic, anesthetic, antibacterial, antiepileptic, coronary artery Examples include, but are not limited to, dilating agents, herbal medicines, antipruritic agents, keratin softening exfoliants, and the like. The active ingredient may also be another anti-tumor agent. Other antitumor agents may be appropriately selected according to the type of cancer to be applied. Other active ingredients may be used alone or in combination of two or more.

The pharmaceutical composition of this embodiment can contain a therapeutically effective amount of the PD-1 expression inhibitor of the first aspect. By "therapeutically effective amount" is meant an amount of drug effective to achieve the purpose of the pharmaceutical composition. For example, a therapeutically effective amount of any of components (a)-(d) can be an amount capable of suppressing PD-1 expression in lymphocytes. When the pharmaceutical composition contains any one of components (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 dosage unit form. up to 5 mg, or 1 μg to 1 mg, and the like. When the pharmaceutical composition contains component (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 ⁵ cells per dosage unit form. 10 ⁸ etc. are mentioned.

<Administration method>
Pharmaceutical compositions can be administered to subjects in need thereof by known methods. The administration method may be oral administration or parenteral administration, but parenteral administration is preferred. A parenteral route may be any route of administration other than the oral route, such as intravenous administration, intramuscular administration, subcutaneous administration, intranasal administration, intradermal administration, intraocular administration, intracerebral administration, intrarectal administration. , vaginal administration, and intraperitoneal administration. The administration method may be local administration or systemic administration. Preferred routes of administration include intravenous administration, subcutaneous administration, intramuscular administration, intratumoral administration, and the like.

The dosage and administration interval of the pharmaceutical composition can be appropriately selected depending on the age, sex, weight, etc. of the administration subject, the type, progression, symptoms, etc. of the disease, administration method, and the like. The dosage can be a therapeutically effective amount of the PD-1 expression inhibitor of the first aspect. When the pharmaceutical composition contains any one of components (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. up to 5 mg, or 1 μg to 1 mg, and the like. When the pharmaceutical composition contains component (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 ⁵ cells per administration. 10 ⁸ etc. are mentioned.

The pharmaceutical composition may be a single dose or multiple doses. In the case of multiple administrations, the administration interval can be, for example, every day, every 2-3 days, every week, every 10-30 days, every month, every 3-6 months, or every year. .

[Method for Suppressing PD-1 Expression]
A third aspect of the present disclosure is a method of inhibiting PD-1 expression in lymphocytes. The method includes culturing lymphocytes in the presence of IL-36 or a variant thereof.

The method of this embodiment is a method performed in vitro or ex vivo. Lymphocytes with reduced PD-1 expression can be produced by the method of the present embodiment.

<IL-36 or variant thereof>
As IL-36 or a variant thereof, the same ones as listed in [PD-1 expression inhibitor] can be used.

<Lymphocytes>
Lymphocytes are not particularly limited as long as they express PD-1. Examples of lymphocytes include those listed in [PD-1 expression inhibitor]. Preferred lymphocytes include CD8-positive T cells (CTL, etc.), CD4-positive T cells (helper T cells, etc.), modified cells thereof (CAR-T, TCR-T, etc.), and the like.
Lymphocytes may be included in a cell population containing multiple types of cells. Cell populations containing lymphocytes include, for example, peripheral blood mononuclear cells (PBMC), splenocytes, thymocytes, and the like.

<Culture>
Culturing is performed in the presence of IL-36 or a variant thereof. More specifically, lymphocytes are cultured in a medium containing IL-36 or a variant thereof.

(Culture medium)
The medium includes, for example, a medium commonly used for culturing animal cells (hereinafter also referred to as "animal cell medium") supplemented with IL-36 or a variant thereof.

Animal cell media include basal media for animal cell culture. Examples of basal media include Doulbecco's modified Eagle's Medium (DMEM) medium, DMEM/F12 medium, Advanced DMEM/F12 medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Examples include Ham's F12 medium, RPMI1640 medium, Fischer's medium, and mixed medium thereof.

The animal cell medium may be a basal medium to which any component has been added. Additional components include, for example, serum (fetal bovine serum (FBS), etc.), serum substitutes, and the like. Serum replacements include, for example, albumin, transferrin, sodium selenite, ITS-X (Invitrogen), knockout serum replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, Insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3′-thiolglycerol, etc. Additional components include lipids, amino acids, L-glutamine, Glutamax, non-essential amino acids, vitamins, growth factors, Antibiotics (penicillin, streptomycin, etc.), antioxidants, pyruvic acid, buffers, inorganic salts, etc. These additive components may be used singly or in combination of two or more. good.

The concentration of IL-36 or its variant in the medium is not particularly limited as long as it is a concentration capable of suppressing PD-1 expression in lymphocytes. Concentrations of IL-36 or variants thereof include, for example, 0.001 to 1000 ng/mL. The lower limit of the concentration of IL-36 or its variant in the medium is, for example, 0.001 ng/mL or more, 0.003 ng/mL or more, 0.05 ng/mL or more, 0.07 ng/mL or more, or 0 .01 ng/mL or greater, 0.03 ng/mL or greater, 0.05 ng/mL or greater, 0.07 ng/mL or greater, 0.1 ng/mL or greater, 0.3 ng/mL or greater, or 0.5 ng/mL or greater. be done. The upper limit of the concentration of IL-36 or its variant in the medium is, for example, 1000 ng/mL or less, 500 ng/mL or less, 300 ng/mL or less, 100 ng/mL or less, 70 ng/mL or less, 50 ng/mL or less, 40 ng/mL or less, or 30 ng/mL or less. These upper and lower limits can be combined arbitrarily.
The concentration range of IL-36 or its variants in the medium is, for example, 0.001 to 500 ng/mL, 0.005 to 300 ng/mL, 0.01 to 100 ng/mL, 0.01 to 50 ng/mL, 0.01 to 40 ng/mL, 0.01 to 30 ng/mL, 0.005 to 0.03 ng/mL, 0.3 to 40 ng/mL, or 0.5 to 30 ng/mL.

(Culture conditions)
Culturing can be performed under culture conditions generally used for culturing lymphocytes. Culture conditions include, for example, a culture temperature of 32-40° C. (preferably 35-38° C., typically 37° C.) and a CO ₂ concentration of 2-5% (preferably 5%).

The culture period should be sufficient to suppress PD-1 expression. Examples of the culture period include 1 to 150 hours, 5 to 100 hours, 10 to 80 hours, 15 to 70 hours, 20 to 60 hours, 25 to 50 hours, or 30 to 50 hours.

Lymphocyte activation treatment may be performed during the culture period or before the culture. Lymphocyte activation treatment can be performed by a known method. Methods of activation treatment of lymphocytes include methods of stimulating the TCR/CD3 complex and co-stimulatory molecules (such as CD28). Stimulation of the TCR/CD3 complex can be done using a CD3 binding agent. A co-stimulatory molecule can be stimulated using a co-stimulatory molecule-binding substance. CD3 binding agents include anti-CD3 antibodies or antigen-binding fragments thereof. Co-stimulatory molecule binding agents include anti-CD28 antibodies or antigen-binding fragments thereof. An antigen-binding fragment is an antibody fragment that retains the antigen-binding properties of the original antibody. Antigen-binding fragments include, for example, Fab, Fab', F(ab') ₂ , Fv, scFv and the like. Anti-CD3 antibody and anti-CD28 antibody. Monoclonal antibodies are preferred.
Lymphocyte activation treatment can be performed by culturing lymphocytes in the presence of a CD3-binding substance and a CD28-binding substance.

　The activation treatment of lymphocytes can be performed simultaneously with culturing in the presence of IL-36 or its variants. In this case, for example, a method of culturing lymphocytes in wells coated with a CD3-binding substance using a medium containing IL-36 or a variant thereof and a CD28-binding substance can be mentioned. Examples of the medium, culture conditions, and culture period are the same as those described above.

Lymphocyte activation treatment may be performed prior to culturing in the presence of IL-36 or its variants. In this case, for example, a method of culturing lymphocytes in a well coated with a CD3 binding substance using a medium containing a CD28 binding substance can be mentioned. Examples of the medium and culture conditions are the same as those described above. The culture period is, for example, 15 to 100 hours, or 20 to 80 hours. After lymphocyte activation treatment, lymphocytes are collected. The method of this embodiment can then be carried out by culturing the collected lymphocytes in the presence of IL-36 or a variant thereof.

The method of the present embodiment can suppress PD-1 expression in lymphocytes. When the PD-1 expression level in lymphocytes cultured in the absence of IL-36 or a variant thereof is set to 100, the relative expression level of PD-1 in lymphocytes obtained by the method of the present embodiment is, for example, It can be 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, or 40 or less.

According to the method of the present embodiment, PD-1 expression in lymphocytes can be suppressed to obtain lymphocytes with a reduced PD-1 expression level. Lymphocytes with a reduced PD-1 expression level easily evade the immune checkpoint system when administered to a living body. Therefore, it can exhibit cytotoxic activity against target cells (for example, cancer cells) without being inhibited by the immune checkpoint system.

The lymphocytes obtained by the method of the present embodiment have a reduced PD-1 expression level, so they can be suitably used for cancer therapy and the like. The method of the present embodiment can also be said to be a method for producing lymphocytes with reduced PD-1 expression level.

In one embodiment, the present invention provides at least one component selected from the group consisting of (a) to (d), which is used for PD-1 expression suppression.

In one embodiment, the present invention provides a method of suppressing PD-1 expression in lymphocytes, comprising administering to a subject at least one component selected from the group consisting of (a) to (d). offer. The subject is, for example, a cancer patient.
In one embodiment, the present invention provides at least one component selected from the group consisting of (a) to (d), which is used in the production of PD-1 expression inhibitors.
In one embodiment, the present invention provides at least one component selected from the group consisting of (a) to (d), which is used for the production of a pharmaceutical composition for immune checkpoint inhibition.
In one embodiment, the present invention provides in vitro use of at least one component selected from the group consisting of (a) to (d) for suppressing PD-1 expression in lymphocytes. do.

The present invention will be explained based on examples. However, embodiments of the present invention are not limited to the description of these examples.

[Example 1]
(Preparation of splenocytes)
Spleens were removed from 10-week-old female C57BL/6 mice and immediately immersed in 20 mL of RPMI1640 solution (RPMI1640 culture medium) without drying. The RPMI1640 solution was RPMI1640 medium supplemented with 10% feat-inactivated fetal bovine serum (FBS), 0.1 mM non-essential amino acids, 100 IU/ml penicillin, and 100 μg/mL streptomycin. A spleen cell suspension was prepared by gently mashing the cells with a slide glass so as not to damage the cells. The splenocyte suspension was aspirated with a 5 mL pipette and passed through a cell strainer with a pore size of 40 μm placed on the mouth of a 50 mL centrifuge tube to remove connective tissue debris.

A 50 mL centrifuge tube was centrifuged at 270 xg (about 1,200 rpm) for 5 minutes to collect the cells at the bottom of the centrifuge tube, and the supernatant was removed by aspiration with an aspirator. After the cells were washed once with phosphate-buffered saline (PBS), 10 mL of Tris-buffered ammonium chloride for erythrocyte removal that had been kept at room temperature in advance was added. The cell pellet was loosened by gentle pipetting with a pipette, and the cells were completely suspended and allowed to react at room temperature for 10 minutes. Cells were pelleted by centrifugation at 270 xg for 5 minutes and the supernatant was aspirated off. 10 mL of RPMI1640 culture medium was added, and the cell pellet was gently loosened with a pipette to suspend the cells. Next, erythrocyte debris was removed by passing it through a cell strainer with a pore size of 40 μm placed on a 50 mL centrifugal tube mouth. 10 mL of RPMI1640 culture medium was added, the cells were gently loosened with a pipette to suspend the cells, the cells were precipitated by centrifugation at 270×g for 5 minutes, and the supernatant was removed by aspiration. This washing operation was repeated twice to completely remove ammonium chloride from the cells. After washing twice, the cells were suspended in 20 mL of RPMI1640 culture medium to prepare a cell suspension.

(stimulation of splenocytes)
A cover glass was placed on the hemocytometer, and a cell suspension mixed with trypan blue was gently put into the gap between the counting board and the cover glass using a micropipette. This was observed under a microscope and the number of viable cells was counted. Based on the calculated cell density, the concentration of the cell suspension was adjusted to 1.0×10 ⁶ cells/mL. Cells were placed in a 15 mL centrifuge tube at 8.0×10 ⁶ cells/centrifuge tube, centrifuged at 270×g for 5 minutes to precipitate the cells, and the supernatant was removed by aspiration.
RPMI1640 solutions containing 0-50 ng/mL of truncated mouse IL-36β (SEQ ID NO: 12) were added with a 5 mL pipette to suspend the splenocytes. Concentrations of truncated mouse IL-36β were 50 nm/mL, 30 ng/mL, 10 ng/mL, 5 ng/mL, 1 ng/mL, 0.5 ng/mL, 0.1 ng/mL, 0.05 ng/mL, 0.01 ng /mL, and 0 ng/mL. To each well of a 12-well culture plate precoated with 0.1 μg of rat anti-mouse CD3 monoclonal antibody (mAb), 2 mL of cell suspension was added for each concentration group of mouse IL-36β. rice field. Finally, rat anti-mouse CD28 mAb was added to a concentration of 2 μg/mL. Then, it was cultured for 48 hours under conditions of 5% CO ₂ and 37°C.

(PD-1 expression analysis)
After culturing for 48 hours, proliferated splenocytes were collected from 2 wells of each group, and the number of cells was counted using a hemocytometer. Then added to Eppendorf tubes at 1.0×10 ⁶ cells/tube. Cells were collected at the bottom of an Eppendorf tube by centrifugation at 270×g (approximately 1,200 rpm) for 5 minutes, and the supernatant was removed by aspiration with an aspirator. After washing twice with 0.1% sodium azide-containing PBS, the cells were suspended in 100 μL of PBS. To this, rat anti-mouse CD16/32 mAb was added to block the Fc receptor. Then, add APC-conjugated rat anti-mouse CD8a mAb, FITC-conjugated anti-mouse CD4 mAb, and PE-conjugated anti-mouse CD279 (PD-1) mAb or PE-conjugated rat bat IgG2b κ Isotype Acon, The reaction was allowed to proceed for 30 minutes at room temperature. After washing twice with 1 mL of PBS, the cells were suspended in 500 μL of PBS, and PD-1 expression in CD8-positive T cells and CD4-positive T cells was measured using a flow cytometer.

(result)
The results are shown in Figures 1-6. FIG. 1 shows splenocytes cultured in the presence of 0 ng/mL, 50 ng/mL, or 30 ng/mL IL-36β. In FIG. 1, "no stimulation" means that stimulation with CD3 mAb and CD28 mAb was not performed, and IL-36β was not added. FIG. 2 shows splenocytes cultured in the presence of 10 ng/mL, 5 ng/mL, or 1 ng/mL IL-36β. FIG. 3 shows the mean fluorescence intensity (MFI) of PE (PD-1 positive) in CD8-positive T cells and CD4-positive T cells in FIGS. FIG. 4 shows splenocytes cultured in the presence of 0 ng/mL, 10 ng/mL, 1 ng/mL, or 0.5 ng/mL IL-36β. FIG. 5 shows splenocytes cultured in the presence of 0.1 ng/mL, 0.05 ng/mL, or 0.01 ng/mL IL-36β. FIG. 6 shows the mean fluorescence intensity (MFI) of PE (PD-1 positive) in the CD8-positive T cells of FIGS.

From the results shown in Figures 1 to 6, it was confirmed that truncated IL-36β suppresses the expression of PD-1 in activated T cells (especially activated CD8-positive T cells). PD-1 expression was strongly suppressed even at relatively low concentrations (eg, 5 ng/ml, 1 ng/ml, 0.5 ng/ml) of truncated IL-36β.

[Example 2]
(Preparation of IL-36β-expressing melanoma cells)
According to the method described in JP-A-2021-70683, three retroviral vectors (DFG-IRES-Neo, DFG-PTH-truncated IL-36β-IRES-Neo, DFG-GH-truncated IL-36β-IRES- Neo) was produced. DFG-IRES-Neo is a retroviral vector that expresses only the neomycin phosphotransferase gene (Neo). DFG-PTH-truncated IL-36β-IRES-Neo is a retroviral vector expressing truncated IL-36β (SEQ ID NOS: 17, 18) to which a human parathyroid hormone signal sequence (SEQ ID NOS: 13, 14) is added and Neo. is. DFG-GH-truncated IL-36β-IRES-Neo is a retroviral vector that expresses truncated IL-36β (SEQ ID NOS: 19 and 20) to which a human growth hormone signal sequence (SEQ ID NOS: 15 and 16) is added and Neo. be.

DFG-IRES-Neo, DFG-PTH-truncated IL-36β-IRES-Neo, and DFG-GH-truncated IL-36β-IRES-Neo were introduced into B16-F10 melanoma cells, respectively, to obtain B16-F10 Neo cells, B16-F10-PTH-truncated IL-36β cells and B16-F10-GH-truncated IL-36β cells were established, respectively.

(Cell transplantation into mice and collection of splenocytes)
B16-F10Neo cells, B16-F10-PTH-truncated IL-36β cells, or B16-F10-GH-truncated IL-36β cells were inoculated into the abdomen of 10-week-old female C57BL/6 mice (n=3). . The inoculum amount was 3×10 ⁵ cells/mouse. Twelve days after cell inoculation, the spleen of each mouse was harvested.

(PD-1 expression analysis)
The collected spleen was treated in the same manner as described in Example 1 (Preparation of splenocytes) to prepare a cell suspension of splenocytes.

A cover glass was placed on the hemocytometer, and a cell suspension mixed with trypan blue was gently put into the gap between the counting board and the cover glass using a micropipette. This was observed under a microscope and the number of viable cells was counted. Based on the calculated cell density, the concentration of the cell suspension was adjusted to 1.0×10 ⁶ cells/mL, and added to the Eppendorf tube so that 1.0×10 ⁶ cells/tube. Cells were collected at the bottom of an Eppendorf tube by centrifugation at 270×g (approximately 1,200 rpm) for 5 minutes, and the supernatant was aspirated off with an aspirator. After washing twice with 0.1% sodium azide-containing PBS, the cells were suspended in 100 μL of PBS. To this, rat anti-mouse CD16/32 mAb was added to block the Fc receptor. Then, PE-conjugated anti-mouse CD279 (PD-1) mAb or PE-conjugated rat IgG2b κ Isotype control mAb was added and allowed to react at room temperature for 30 minutes. After washing twice with 1 mL of PBS, the cells were suspended in 500 μL of PBS, and PD-1 expression in splenocytes was measured using a flow cytometer.

(result)
The results are shown in FIG. Splenocytes collected from B16-F10-PTH-truncated IL-36β cell-inoculated mice or B16-F10-GH-truncated IL-36β cell-inoculated mice were compared with splenocytes collected from B16-F10Neo cell-inoculated mice, PD-1 expression was markedly reduced. These results confirmed that IL-36β suppresses PD-1 expression in lymphocytes even in vivo.

[Example 3]
(Generation of IL-36β-expressing tumor cells)
DFG-IRES-Neo, DFG-PTH-truncated IL-36β-IRES-Neo, and DFG-GH-truncated IL-36β-IRES-Neo were each introduced into MCA205 cells (mouse fibrosarcoma cells) to obtain MCA205Neo cells. , MCA205-PTH-truncated IL-36β cells, and MCA205-GH-truncated IL-36β cells were established, respectively. DFG-IRES-Neo, DFG-PTH-truncated IL-36β-IRES-Neo, and DFG-GH-truncated IL-36β-IRES-Neo were each introduced into MC38 cells (mouse colon adenocarcinoma cells) to obtain MC38Neo. cells, MC38-PTH-truncated IL-36β cells, and MC38-GH-truncated IL-36β cells were established, respectively.

(Cell transplantation into mice and collection of tumor tissue)
MCA205Neo cells, MCA205-PTH-truncated IL-36β cells, or MCA205-GH-truncated IL-36β cells were inoculated into the abdomen of 10-week-old female C57BL/6 mice (n=3). The inoculum amount was 1.5×10 ⁵ cells/mouse. MC38Neo cells, MC38-PTH-truncated IL-36β cells, or MC38-GH-truncated IL-36β cells were inoculated into the abdomen of 10-week-old female C57BL/6 mice (n=3). The inoculum amount was 2×10 ⁵ cells/mouse. Twelve days after inoculation, tumor tissue was harvested from each mouse.

(Immunostaining)
The collected tumor tissue was immersed in a PBS-buffered formalin solution for 2 days and then embedded in paraffin. Sections were made from deparaffinized tissue for PD-1 staining. After treating this with normal goat serum, it was allowed to react with goat anti-mouse PD-1 antibody at 4° C. overnight. After washing with PBS, a biotinylated bovine anti-goat IgG antibody was reacted for 30 minutes. After washing with PBS, a solution containing peroxidase-conjugated streptavidin was applied. After washing with PBS, the color was developed with diaminobenzidine.

(result)
FIG. 8 shows the results of immunostaining. PD-1 expression was significantly higher in tumor tissue collected from MCA205-PTH-truncated IL-36β cell-inoculated mice or MCA205-GH-truncated IL-36β cell-inoculated mice compared to tumor tissue collected from MCA205Neo cell-inoculated mice. It was clearly lowered (Fig. 8 upper). PD-1 expression was significantly higher in tumor tissue collected from MC38-PTH-truncated IL-36β cell-inoculated mice or MC38-GH-truncated IL-36β cell-inoculated mice compared to tumor tissue collected from MC38Neo cell-inoculated mice. It was clearly lowered (Fig. 8, bottom). These results demonstrate that PD-1 expression is reduced in tumor-infiltrating lymphocytes in tumor tissue from mice implanted with tumor cells expressing truncated IL-36β. These results confirmed that IL-36β suppresses PD-1 expression in lymphocytes even in vivo.

While the preferred embodiments of the invention have been described and illustrated above, it should be understood that they are intended to be illustrative of the invention and should not be taken as limiting. Additions, omissions, substitutions, and other changes can be made without departing from the spirit or scope of the invention. Accordingly, the present invention should not be viewed as limited by the foregoing description, but only by the scope of the appended claims.

Claims

A PD-1 expression inhibitor comprising at least one component selected from the group consisting of the following (a) to (d):
(a) interleukin 36 or a variant thereof;
(b) a polynucleotide having a nucleotide sequence encoding interleukin 36 or a variant thereof;
(c) a vector comprising the polynucleotide of (b); and (d) a cell secreting interleukin-36 or a variant thereof.
The PD-1 expression inhibitor according to claim 1, which suppresses PD-1 expression in CD8-positive T cells or CD4-positive T cells.
A pharmaceutical composition for immune checkpoint inhibition, comprising the PD-1 expression inhibitor according to claim 1 or 2 and a pharmaceutically acceptable carrier.
A method for suppressing PD-1 expression in lymphocytes, comprising culturing lymphocytes in the presence of interleukin 36 or a variant thereof.