Composition and kit for inducing memory-like natural killer cells

Abstract

To provide a composition for inducing memory-like natural killer (NK) cells in vivo.SOLUTION: A composition for inducing memory-like NK cells according to the present invention comprises a stimulator of interferon genes (STING) agonist and a Toll-like receptor 9 (TLR9) agonist.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition and a kit for inducing memory-like natural killer (NK) cells. [Background technology]

[0002] NK cells are cells that control natural immunity and have high cytotoxicity and cytokine production. In particular, unlike T cells, they do not require prior sensitization with antigen stimulation, and are therefore expected to be a target for cancer immunotherapy. Strategies to enhance the effector function of NK cells using cytokine stimulation and antibodies against NK cell inhibitory receptors are being developed. Development of cell therapy using NK cells is also being advanced.

[0003] NK cells were thought to be short-lived and to exhibit antigen-independent cytotoxicity, but it has become clear that there are NK cells that have an antigen-specific memory against haptens or viruses, that is, memory NK cells (Non-Patent Document 1). It has also been reported that memory-like NK cells can be induced by treating isolated NK cells with IL-12, IL-15, and IL-18 (Non-Patent Document 2), and research and development into cell therapy for cancer is progressing (Non-Patent Document 3).

[0004] On the other hand, it has been reported that administration of an agonist of the stimulator of interferon genes (STING) pathway, a natural immune sensor essential for the initiation of cancer immune responses, loaded onto lipid nanoparticles (LNPs) efficiently activates NK cells in vivo via the production of type I IFN, inducing strong antitumor activity (Non-Patent Documents 4 and 5). It has also been reported that STING agonists are used in combination with FasL expression inhibitors or angiogenesis inhibitors for the treatment of angiogenesis-dependent diseases (Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2022 / 097634 [Non-patent literature]

[0006] [Non-Patent Document 1] Mujal AM, et al., Annu Rev Immuol 39: 417-447, 2021 [Non-Patent Document 2] Cooper MA, et al., PNAS 106: 1915-1919, 2009 [Non-Patent Document 3] Terren I, et al., Front Immunol 13: 884648, 2022 [Non-Patent Document 4] Nakamura T, et al., J Control Release 216: 149-157, 2015 [Non-Patent Document 5] Nakamura T, et al., J Immunother Cancer 9: e002852, 2021 Summary of the Invention [Problem to be solved by the invention]

[0007] Conventionally, no method has been reported for inducing memory-like NK cells in vivo. NK cells activated with STING agonists do not have the properties of memory-like NK cells, and long-term persistence of effects cannot be expected. If memory-like NK cells can be efficiently induced in vivo by administering a drug, it would contribute greatly to the development of new cancer immunotherapy, as well as to the cost and complexity of formulations. Therefore, the present invention aims to provide a composition for inducing memory-like NK cells in vivo. [Means for solving the problem]

[0008] As a result of intensive research aimed at solving the above problems, the present inventors discovered that memory-like NK cells can be induced in vivo by using a Toll-like receptor 9 (TLR9) agonist in combination with a STING agonist, and completed the present invention. That is, the present invention provides the following compositions and kits for inducing memory-like NK cells, and compositions and kits for treating or preventing cancer. [1] A composition for inducing memory-like NK cells, comprising a STING agonist and a TLR9 agonist. [2] A composition for inducing memory-like NK cells, comprising either a STING agonist or a TLR9 agonist, for use in combination with another composition comprising the other. [3] A composition for treating or preventing cancer, comprising a STING agonist and a TLR9 agonist. [4] A composition for treating or preventing cancer, comprising either a STING agonist or a TLR9 agonist, for use in combination with another composition comprising the other. [5] The composition described in any one of [1] to [4] above, wherein the STING agonist comprises one or more selected from the group consisting of cyclic dinucleotides, nucleic acids that bind to cyclic GMP-AMP synthase (cGAS), and STING-activating non-nucleic acid small molecule compounds. [6] The composition according to any one of [1] to [5] above, wherein the TLR9 agonist comprises an oligonucleotide and / or a plasmid having an unmethylated CpG motif. [7] The composition described in any one of [1] to [6] above, wherein the STING agonist is encapsulated in a lipid nanoparticle. [8] The composition according to any one of [1] to [7] above, for inducing IL-12 production. [9] The composition described in [1] or [2] above for treating or preventing cancer and / or infectious diseases.

[10] The memory-like NK cells are + CD27 -The composition according to claim 1 or 2, comprising a cell.

[11] A kit for inducing memory-like NK cells, comprising a STING agonist and a TLR9 agonist.

[12] A kit for treating or preventing cancer, comprising a STING agonist and a TLR9 agonist. Effect of the Invention

[0009] According to the present invention, by using a TLR9 agonist in combination with a STING agonist, memory-like NK cells can be induced in vivo, thereby treating or preventing cancer. STING agonists and TLR9 agonists can be prepared relatively easily and inexpensively, making it possible to provide a novel and highly efficient cancer immunotherapy. [Brief description of the drawings]

[0010] [Figure 1] Serum cytokine concentrations after intravenous administration of each test sample to mice are shown. [Figure 2A] Mice were administered B16-F10 melanoma, and photographs of the lungs after administration of each test sample are shown. [Figure 2B] A graph of luciferase levels from B16-F10 melanoma cells engrafted in the lungs is shown. [Figure 3A] Mice were administered B16-F10 melanoma, and photographs of the lungs after administration of each test sample are shown. [Figure 3B] A graph of luciferase levels from B16-F10 melanoma cells engrafted in the lungs is shown. [Figure 4] The figures show the percentage of CD11b positive cells (left), CD27 positive cells (center), or KLRG1 positive cells (right) in the CD3 negative NK1.1 positive spleen cells (NK cell group) after administration of each test sample. ("+ve" stands for "positive.") [Diagram 5] The results of expression analysis of CD11b and CD27 in CD3-negative NK1.1-positive spleen cells (NK cell group) after administration of each test sample are shown. [Figure 6] The figures show the percentage of CD11b-negative CD27-negative double negative (DN) cells (upper left), CD11b-negative CD27-positive single positive (SP) cells (upper right), CD11b-positive CD27-positive double positive (DP) cells (lower left), or CD11b-positive CD27-negative SP cells (lower right) in the CD3-negative NK1.1-positive spleen cells (NK cell group) after administration of each test sample. The arrows indicate the tendency of increase or decrease compared to the control group (PBS). [Figure 7] The graph shows the change in the ratio of CD11b-positive CD27-negative SP cells among CD3-negative NK1.1-positive spleen cells (NK cell group) after administration of each test sample. "28 (tumor)" is the measurement data 4 days after administration of B16-F10 melanoma on the 24th day. [Figure 8A] Photographs of the lungs of mice after administration of each test sample and administration of B16-F10 melanoma are shown. [Figure 8B] A graph of luciferase levels from B16-F10 melanoma cells engrafted in the lungs is shown. [Figure 9A] Photographs of the lungs of mice after administration of each test sample and administration of B16-F10 melanoma are shown. [Figure 9B] A graph of luciferase levels from B16-F10 melanoma cells engrafted in the lungs is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention will now be described in further detail. The composition for inducing memory-like NK cells of the present invention is characterized by containing a STING agonist and a TLR9 agonist. The "memory-like NK cells" described herein refer to NK cells that express the NK cell marker NK1.1 and have a CD11b-positive, CD27-negative phenotype. Memory-like NK cells exist in the body for a longer period of time than normal NK cells, and can attack cancer cells, virus-infected cells, and the like as targets.

[0012] The STING agonist can be any one that can activate STING, without any particular limitation. For example, the STING agonist may be one or more types selected from the group consisting of cyclic dinucleotides, nucleic acids that bind to cyclic GMP-AMP synthase (cGAS), and STING-activating non-nucleic acid small molecule compounds.

[0013] The cyclic dinucleotide may be a natural one or a chemically synthesized one, and any one commonly used in the art may be used without any particular limitation. For example, the cyclic dinucleotide may be a natural cyclic dinucleotide such as 2'3'-cyclic GAMP (CAS No.: 1441190-66-4), 3'3'-cyclic GAMP (CAS No.: 849214-04-6), cyclic-di-AMP (CAS No.: 54447-84-6), and cyclic-di-GMP (CAS No.: 61093-23-0); as well as cyclic AIMP (CL592) (CAS No.: 1507367-51-2), cyclic AIMP Difluor (CL614) (CAS No.: 1951464-78-0), cyclic AIM (PS) 2 Difluor (Rp / Sp) (CL656) (CAS No.: 1951464-79-1), 2'2'-cyclic GAMP, 2'3'-cyclic GAM (PS) 2 (Rp / Sp) (CAS No.:1637675-05-8), 3'3'-cyclicGAMP Fluorinated, cyclic-di-AMP Fluorinated(CAS No.:1427269-47-3), 2'3'-cyclic-di-AMP(CAS No.:1638241-77-6), 2'3'-cyclic-di-AM(PS)2(Rp,Rp)(ADU-S100)(CAS No.:1638750-95-4), cyclic-di-GMP Fluorinated(CAS No.:1334145-18-4), 2'3'-cyclic-di-GMP(CAS No.:1638241-12-9), cyclic-di-IMP(CAS No.:79940-41-3), and DMXAA(CAS The nucleic acid may contain one or more types selected from the group consisting of synthetic cyclic dinucleotides such as cyclic dinucleotides (N.S. No. 117570-53-3).

[0014] The cGAS binds to double-stranded DNA present in the cytoplasm in a sequence-independent manner to activate it, synthesizing 2',3'-cyclic-GMP-AMP and activating the STING pathway. Therefore, the nucleic acid that binds to the cGAS used as the STING agonist is not particularly limited as long as it is formulated to provide double-stranded DNA in the cytoplasm, but may be, for example, a double-stranded DNA having a length of about 20 base pairs or more, preferably a double-stranded DNA having a length of about 20 to about 20,000 base pairs.

[0015] The STING-activating non-nucleic acid small molecule compound is a small molecule compound that does not have a nucleic acid structure but can activate STING, and as the STING-activating non-nucleic acid small molecule compound, any compound commonly used in the art can be used without any particular limitation. For example, the STING agonistic non-nucleic acid small molecule compound may include one or more selected from the group consisting of SR-717 (CAS No.: 2375421-09-1), SR-717 free acid (CAS No.: 2375420-34-9), MSA-2 (CAS No.: 129425-81-6), diABZI STING agonist-1 (CAS No.: 2138299-33-7), diABZI STING agonist-1 trihydrochloride (CAS No.: 2138299-34-8), STING agonist-1 (CAS No.: 702662-50-8), STING agonist-3 (CAS No.: 2138299-29-1), and STING agonist-4 (CAS No.: 2138300-40-8).

[0016] The TLR9 agonist can be used without any particular limitation as long as it can activate TLR9. For example, the TLR9 agonist may include an oligonucleotide and / or a plasmid having an unmethylated CpG motif. A CpG motif is a DNA sequence in which cytosine and guanine are bound by a phosphodiester bond, and is not methylated in bacteria or viruses. This unmethylated CpG motif is known to activate TLR9. The oligonucleotide and / or plasmid having an unmethylated CpG motif may include a natural and / or synthetic oligonucleotide and / or plasmid. In one embodiment, the oligonucleotide having an unmethylated CpG motif may include a CpG-oligodeoxynucleotide (CpG-ODN). The length and sequence of the oligonucleotide and / or plasmid having an unmethylated CpG motif may be any condition commonly used in the art. For example, the oligonucleotide having an unmethylated CpG motif may be about 10 to about 40 bases long or about 15 to about 30 bases long.

[0017] The ratio between the amount of the STING agonist used and the amount of the TLR9 agonist used is not particularly limited, and may be, for example, about 1:20 to about 1:200 or about 1:40 to about 1:100 by mass ratio.

[0018] Without being bound by any particular theory, for example, it is believed that when the STING agonist and the TLR9 agonist are used in combination, the concentrations of IL-12, IL-18, and IL-1β in the blood of a subject are increased, and in particular, the concentration of IL-12 is synergistically increased, thereby promoting the induction of memory-like NK cells in vivo. That is, in one embodiment, the composition for inducing memory-like NK cells of the present invention may be for inducing IL-12 production. In addition, since memory-like NK cells can attack cancer cells and infected cells, the composition for inducing memory-like NK cells of the present invention may be for treating or preventing cancer and / or infectious diseases.

[0019] In another aspect, the present invention also relates to a composition for inducing memory-like NK cells, which comprises either the STING agonist or the TLR9 agonist, and is used in combination with another composition comprising the other. That is, in order to induce memory-like NK cells, it is necessary to use the STING agonist and the TLR9 agonist in combination, but these active ingredients do not necessarily need to be contained in the same composition, and memory-like NK cells can be induced by simultaneously or consecutively administering a composition containing only the former and a composition containing only the latter. As the STING agonist or the TLR9 agonist contained in the composition for inducing memory-like NK cells of the present invention, those described above in relation to the composition of one aspect of the present invention can be appropriately adopted.

[0020] In another aspect, the present invention also relates to a composition for treating or preventing cancer and / or infectious diseases, comprising a STING agonist and a TLR9 agonist, or comprising either a STING agonist or a TLR9 agonist, for use in combination with another composition comprising the other. When the STING agonist and the TLR9 agonist are used in combination, memory-like NK cells are induced, and the memory-like NK cells attack cancer cells or infected cells, thereby making it possible to treat or prevent cancer or infectious diseases. These active ingredients do not necessarily have to be contained in the same composition, and a composition comprising only the former and a composition comprising only the latter can be administered simultaneously or consecutively to treat or prevent cancer or infectious diseases. As the STING agonist or the TLR9 agonist contained in the composition for treating or preventing cancer and / or infectious diseases of the present invention, those mentioned above in relation to the composition of one aspect of the present invention can be appropriately adopted.

[0021] The type of cancer to which each composition of the present invention can be applied is not particularly limited, but for example, the cancer may include solid cancer and / or blood cancer. The solid cancer may include skin cancer, oral cancer, bladder cancer, tongue cancer, pharyngeal cancer, esophageal cancer, lung cancer, liver cancer, stomach cancer, thyroid cancer, pancreatic cancer, small intestine cancer, large intestine cancer (such as colon cancer or rectal cancer), breast cancer, uterine cancer (such as cervical cancer or uterine cancer), ovarian cancer, prostate cancer, and brain cancer. The blood cancer may include leukemia (such as acute lymphocytic leukemia or acute myeloid leukemia), lymphoma (such as Hodgkin's lymphoma or non-Hodgkin's lymphoma), and myeloma (such as multiple myeloma).

[0022] The type of infectious disease to which each composition of the present invention can be applied is not particularly limited, but for example, the infectious disease may include a viral infection. The viral infection may be an infectious disease caused by a virus such as influenza virus, coronavirus, rhinovirus, adenovirus, enterovirus, human hepatitis virus (such as hepatitis B virus, hepatitis C virus, hepatitis A virus, or hepatitis E virus), human retrovirus, human immunodeficiency virus (such as HIV1 or HIV2), human T-cell leukemia virus or human T-lymphotropic virus (such as HTLV1 or HTLV2), herpes simplex virus (such as type 1 or type 2), Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, human herpes virus, poliovirus, measles virus, rubella virus, Japanese encephalitis virus, mumps virus, Ebola virus, and West Nile virus.

[0023] Each composition of the present invention can be appropriately formulated by a method commonly used in the art. The specific formulation form can be appropriately selected according to the administration route, and may be, for example, oral solids such as powders, granules, capsules, tablets, chewables, and sustained release agents, oral liquids such as solutions and syrups, injections, enemas, sprays, patches, and ointments. In one embodiment, the STING agonist and / or the TLR9 agonist are contained in lipid nanoparticles. The lipid nanoparticles can protect the active ingredient loaded therein from degradation and can also efficiently deliver the active ingredient into cells. The lipids constituting the lipid membrane of the lipid nanoparticles can be those commonly used in the art without particular limitation, and for example, the lipid nanoparticles may contain cationic lipids, phospholipids, sterols, and / or saturated or unsaturated fatty acids.

[0024] The cationic lipid may include, for example, pH-sensitive cationic lipids such as YSK12-C4 and YSK05 (see Patent Document 1). The phospholipid may include, for example, glycerophospholipids such as phosphatidylethanolamine (PE), phosphalidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylglycerol (PG), cardiolipin, sphingomyelin, ceramide phosphorylethanolamine, ceramide phosphorylglycerol, ceramide phosphorylglycerol phosphate, 1,2-dimyristoyl-1,2-deoxyphosphatidylcholine, plasmalogen, and / or phosphatidic acid (PA). The phospholipid may be modified with, for example, polyalkylene glycols such as polyethylene glycol, polypropylene glycol, polytetramethylene glycol, and polyhexamethylene glycol. The sterols may include, for example, animal-derived sterols such as cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol, and dihydrocholesterol; plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, campesterol, and brassicasterol; and / or microbial-derived sterols such as zymosterol and ergosterol.

[0025] Each composition of the present invention may further contain any inactive ingredient that may be commonly used in the art, as long as it does not impair the purpose of the present invention, and may further contain an additional active ingredient. The inactive ingredient may include, but is not limited to, for example, an excipient, a binder, a lubricant, a disintegrant, a fluidizing agent, a flavoring agent, a solvent, a solubilizing agent, a buffer, a suspending agent, an emulsifying agent, an isotonic agent, a stabilizer, a preservative, an antioxidant, and / or a coloring agent. The additional active ingredient may be, but is not limited to, for example, an ingredient useful for the induction of memory-like NK cells, the treatment or prevention of cancer, or the treatment or prevention of infectious diseases, or may have other effects.

[0026] In another aspect, the present invention also relates to a method for inducing memory-like NK cells, and a method for treating or preventing cancer and / or infectious diseases, each of which includes a step of administering the STING agonist and the TLR9 agonist to a subject. When the STING agonist and the TLR9 agonist are used in combination, memory-like NK cells are induced, and the memory-like NK cells attack cancer cells or infected cells, thereby making it possible to treat or prevent cancer or infectious diseases. The STING agonist and the TLR9 agonist do not necessarily need to be administered simultaneously, and may be administered sequentially. As the STING agonist or the TLR9 agonist used in the method of the present invention, those described above in relation to the composition of one aspect of the present invention can be appropriately adopted.

[0027] The subject is not particularly limited, and may be, for example, a human or a non-human animal, such as a mammal, cow, pig, horse, sheep, goat, monkey, dog, cat, rabbit, mouse, rat, hamster, or guinea pig, or a bird, such as a chicken, quail, or duck.

[0028] Each method of the present invention may further include any step that may be commonly used in the art, as long as it does not impair the object of the present invention, and may further include a step of administering an additional active ingredient.

[0029] The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited to these examples. EXAMPLES

[0030] <Experimental Method> 1. Preparation of STING agonist-loaded LNPs (STING-LNPs) One of the constituent lipids of LNP, (6Z,9Z,28Z,31Z)-19-(4-(dimethylamino)butyl)heptatriaconta-6,9,28,31-tetraen-19-ol (YSK12-C4), was synthesized by a standard method. Cholesterol was purchased from Avanti Polar Lipids Inc. 1,2-Dimyristoyl-sn-glycerol methoxyethylene glycol 2000 ether (DMG-PEG 2000 ) were purchased from NOF Corporation. These were used to prepare STING-LNPs by standard methods. Typically, YSK12-C4, cholesterol, and DMG-PEG were used. 2000 STING-LNPs were prepared by mixing the above in a tert-butanol solution at a molar ratio of 85:15:1, mixing this with 1 mM citrate buffer (pH 4.5) containing the STING agonist cyclic-di-GMP (Cayman Chemical), and ultrafiltration.

[0031] 2. Measurement of cytokine production in blood STING-LNP (containing 4 μg of cyclic di-GMP; the same applies below), CpG-ODN (250 μg), or a combination of STING-LNP (4 μg) and CpG-ODN (250 μg) was intravenously administered to mice, and serum was collected 2 or 4 hours later. Cytokine concentrations in the collected serum were quantified using ELISA. As the CpG-ODN, a synthetic ODN (Hokkaido System Science Co., Ltd.) having the sequence shown in SEQ ID NO: 1 was used. SEQ ID NO: 1: 5'-ggGGTCAACGTTGAgggggg-3' (However, lowercase bases indicate phosphorothioate bonds.)

[0032] 3. Examination of therapeutic antitumor activity A lung metastasis model was prepared by intravenously administering B16-F10-luc cells (cells in which luciferase gene was introduced into B16-F10 melanoma) to mice. Test samples were administered intravenously on the 6th and 10th days after cancer cell administration, and lungs were collected on the 14th day. When performing a depletion assay, anti-NK1.1 antibody (200 μg) or anti-CD8a antibody (250 μg) was administered intraperitoneally on the 5th and 9th days after cancer cell administration (i.e., the day before administration of the test sample). Photographs of the collected lungs were taken, and luciferase activity of the lungs was measured by a conventional method. As test samples, PBS, STING-LNP (4 μg), CpG-ODN (250 μg), or a combination of STING-LNP (4 μg) and CpG-ODN (250 μg) were used.

[0033] 4. Phenotype analysis of NK cells A lung metastasis model was prepared in the same manner as in item 3 above, the test sample was administered intravenously, and the spleens were collected 14 days after the administration of the cancer cells. When examining persistence, the test sample was administered intravenously to the mice on the day the test started (day 0) and on day 4 after the start of the test, and the spleens were collected on days 7, 14, 21, or 28 after the start of the test. For some groups, B16-F10-luc cells were administered intravenously on day 24 after the start of the test, and the spleens were collected on day 28 after the start of the test. A cell suspension was prepared from the collected spleens, and immunostaining was performed using a fluorescently labeled antibody by standard methods. The stained cells were analyzed using a flow cytometer, and CD3 - NK1.1 + The NK cell population was gated to examine the expression profiles of CD11b and CD27, or the expression profile of KLRG1. Test samples used were PBS, STING-LNP (4 μg), CpG-ODN (250 μg), or a combination of STING-LNP (4 μg) and CpG-ODN (250 μg).

[0034] 5. Examination of preventive antitumor activity Test samples were administered intravenously to mice on the first day of the test (day 0) and on day 4 after the start of the test, and B16-F10-luc cells were administered intravenously on days 14 and 24 after the start of the test. Lungs were collected 14 days after the administration of cancer cells (days 28 and 38 after the start of the test, respectively). Photographs of the collected lungs were taken, and luciferase activity in the lungs was measured by a standard method. Test samples used were PBS, STING-LNP (4 μg), CpG-ODN (250 μg), or a combination of STING-LNP (4 μg) and CpG-ODN (250 μg).

[0035] <Experimental Results> 1. Cytokine Production In Vivo Each test sample was intravenously administered to mice, and serum cytokine concentrations were measured 2 or 4 hours later. In the group administered STING-LNP alone, the production of IL-12 and IL-18 was low, but when it was administered in combination with CpG-ODN, the production of IL-12 and IL-18 was enhanced compared to the group administered alone, and the production of IL-1b was also enhanced (Figure 1). In particular, the production of IL-12 exceeded the total production of IL-12 when STING-LNP or CpG-ODN was administered alone, confirming the synergistic effect of the combined administration.

[0036] 2. Therapeutic antitumor activity The therapeutic antitumor activity against B16-F10 melanoma was examined. Each test sample was intravenously administered to the mouse model. In the group administered STING-LNP and CpG-ODN in combination, the amount of cancer cells engrafted in the lungs was visually reduced (Figure 2A), and the luciferase activity derived from the cancer cells was significantly reduced only in the group administered STING-LNP and CpG-ODN in combination (Figure 2B). This demonstrated that the combination of STING-LNP and CpG-ODN exerts therapeutic antitumor activity.

[0037] In addition, administration of anti-NK1.1 antibody or anti-CD8a antibody increased the expression of NK cells or CD8 +When T cells were depleted, the antitumor activity was lost in the former case, but was maintained in the latter case (Figures 3A and 3B). This suggests that the antitumor activity of the combination of STING-LNP and CpG-ODN is related to the CD8 + It was revealed that NK cells, rather than T cells, are involved.

[0038] 3. Phenotype analysis of NK cells The test samples were administered intravenously on the 6th and 10th days after the intravenous administration of the cancer cells, and the spleens were collected on the 14th day after the administration of the cancer cells, and flow cytometry analysis of the spleen cells was performed. In the CD3 negative NK1.1 positive NK cell group, the group administered the STING-LNP and CpG-ODN in combination showed CD11b + Increased percentage of cells CD27 + A decrease in the proportion of cells with KLRG1 + An increase in the number of mature cells was observed (Figure 4). Furthermore, when analyzed by dot plot of CD11b and CD27, the group administered STING-LNP and CpG-ODN in combination showed that the most mature CD11b + CD27 - Single positive (CD11b + The percentage of memory-like NK cells induced by IL-12, IL-15, and IL-18 was the highest in the IL-11b SP group (Figures 5 and 6). This result indicates that memory-like NK cells induced by IL-12, IL-15, and IL-18 express CD11b + CD27 - This is consistent with the phenotype shown in (Non-Patent Document 2).

[0039] 4.CD11b + Persistence of NK cells in SP CD11b induced by combined administration of STING-LNP and CpG-ODN + We investigated how long the increase in NK cells in SP was maintained. As a result, CD11b + The proportion of NK cells in the SP increased and was maintained at least until day 28 (Figure 7). +The proportion of NK cells in the SP was maintained for at least 28 days. + The proportion of NK cells in the SP did not change with administration of cancer cells (Figure 7).

[0040] 5.Preventive antitumor activity We examined whether the combined administration of STING-LNP and CpG-ODN could induce a preventive effect, i.e., memory-like immunity (similar to memory immunity). Mice that had been administered the test sample in advance were administered B16-F10 melanoma. When cancer cells were administered on the 14th day after the start of the test, no antitumor activity was observed with STING-LNP or CpG-ODN alone, whereas the group administered STING-LNP and CpG-ODN in combination showed significant antitumor activity (Figures 8A and 8B). Furthermore, when cancer cells were administered on the 24th day after the start of the test, significant antitumor activity was observed only in the group administered STING-LNP and CpG-ODN in combination (Figures 9A and 9B). These results suggest that the combined administration of STING-LNP and CpG-ODN can induce preventive antitumor activity, i.e., memory-like immunity. Therefore, CD11b induced by the combined administration of STING-LNP and CpG-ODN + The NK cells in SP are thought to be memory-like NK cells.

[0041] These results suggest that the use of a TLR9 agonist in combination with a STING agonist can induce memory-like NK cells in vivo, thereby enabling the treatment or prevention of cancer. STING agonists and TLR9 agonists can be prepared relatively easily and inexpensively, making it possible to provide a novel and highly efficient cancer immunotherapy.

Claims

1. A composition for inducing memory-like natural killer (NK) cells, comprising a stimulator of interferon genes (STING) agonist and a Toll-like receptor 9 (TLR9) agonist.

2. A composition for inducing memory-like NK cells, comprising either a STING agonist or a TLR9 agonist, for use in combination with another composition comprising the other.

3. A composition for treating or preventing cancer, comprising a STING agonist and a TLR9 agonist.

4. A composition for treating or preventing cancer, comprising either a STING agonist or a TLR9 agonist, for use in combination with another composition comprising the other.

5. The composition of any one of claims 1 to 4, wherein the STING agonist comprises one or more selected from the group consisting of cyclic dinucleotides, nucleic acids that bind to cyclic GMP-AMP synthase (cGAS), and STING agonistic non-nucleic acid small molecule compounds.

6. The composition of any one of claims 1 to 4, wherein the TLR9 agonist comprises an oligonucleotide and / or a plasmid having an unmethylated CpG motif.

7. The composition of any one of claims 1 to 4, wherein the STING agonist is incorporated into a lipid nanoparticle.

8. The composition according to any one of claims 1 to 4, for inducing IL-12 production.

9. The composition according to claim 1 or 2 for treating or preventing cancer and / or infectious diseases.

10. The memory-like NK cells are + CD27 - The composition of claim 1 or 2, comprising a cell.

11. A kit for inducing memory-like NK cells, comprising a STING agonist and a TLR9 agonist.

12. A kit for treating or preventing cancer, comprising a STING agonist and a TLR9 agonist.

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