Anti-tumor effect enhancer

Abstract

The present invention provides a novel method for activating immune response in tumor immunotherapy using tumor immune checkpoint inhibitors. The present invention relates to an anti-tumor effect enhancer when using tumor immune checkpoint inhibitors, which contains probiotics and prebiotics as active ingredients.

Description

Technical Field

[0001] The present invention relates to an anti-tumor effect enhancer and a tumor immunity activator for activating tumor immune response and enhancing the anti-tumor effect caused thereby. Background Art

[0002] Tumor immunotherapy is a therapy that acts on the immune surveillance mechanism that cancer patients already have, and by strengthening immunity against cancer, inhibits or treats the development of cancer. In recent years, it has become clear that cancer cells themselves have a system that evades immune surveillance mechanisms during the progression of cancer. As molecules used for such an evasion system, immune checkpoint molecule groups such as CTLA-4, PD-1, or PD-L1 as its ligand are known. In addition, immune checkpoint inhibitors that block the functions of these immune checkpoint molecule groups have been reported to be extremely useful for strengthening immunity against cancer (Patent Document 1).

[0003] In addition, since regulatory T cells (Tregs) that achieve suppressive control of immune responses suppress the induction of immune responses when suffering from cancer or infectious bacteria, various attempts have been made to control the operation of regulatory T cells in tumor immunotherapy.

[0004] However, there are cancer patients for whom sufficient therapeutic effects are not seen even by activation of the immune system using immune checkpoint inhibitors or the like, and the development of further treatment methods is needed.

[0005] On the other hand, it is known that lactic acid bacteria represented by Lactobacillus bacteria and Bifidobacterium bacteria have various effects such as improvement of intestinal flora, improvement of bowel habits, improvement of intestinal function, resistance to infection, and immune activation. These bacteria are considered to contribute to human health by improving the intestinal environment and are called so-called probiotics.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent document 1: Japanese Patent No. 5885764. Summary of the invention

[0009] The present invention relates to providing a novel method for activating immune response in tumor immunotherapy using immune checkpoint inhibitors.

[0010] The present inventors have repeatedly studied in view of such problems and found that when probiotics such as Lactobacillus bacteria and Bifidobacterium bacteria are combined with prebiotics such as galacto-oligosaccharides and used in combination with immune checkpoint inhibitors, the anti-tumor effect begins to be significantly enhanced and tumor immunity is activated.

[0011] That is, the present invention relates to the following technical solutions 1) to 19).

[0012] 1) An anti-tumor effect enhancer when an immune checkpoint inhibitor is used, comprising probiotics and prebiotics as active ingredients.

[0013] 2) The anti-tumor effect enhancer according to 1), wherein the probiotic is selected from at least one of bacteria belonging to the genus Lactobacillus and bacteria belonging to the genus Bifidobacterium.

[0014] 3) The anti-tumor effect enhancer according to 1) or 2), wherein the probiotic is at least one selected from the group consisting of Lactobacillus casei and Bifidobacterium breve.

[0015] 4) The antitumor effect enhancer according to any one of 1) to 3), wherein the probiotic is at least one selected from the group consisting of Lactobacillus casei YIT9029 strain (FERM BP-1366) and Bifidobacterium breve YIT12272 strain (FERM BP-11320).

[0016] 5) The anti-tumor effect enhancer according to any one of 1) to 4), wherein the prebiotic is galacto-oligosaccharide.

[0017] 6) The anti-tumor effect enhancer according to any one of 1) to 5), wherein the prebiotic is a galacto-oligosaccharide containing β-1,4-galactosyl lactose as a main component.

[0018] 7) The anti-tumor effect enhancer according to any one of 1) to 6), wherein the immune checkpoint inhibitor is one or more selected from anti-PD-1 antibodies, anti-PD-L1 antibodies, and PD-1 antagonists.

[0019] 8) A tumor immune activator, comprising probiotics, prebiotics and immune checkpoint inhibitors as active ingredients.

[0020] 9) The tumor immunity activating agent according to 8), wherein tumor immunity is activated by releasing the immunosuppressive state mediated by controlling T cells and / or activating NK cells.

[0021] 10) A pharmaceutical product is a combination of probiotics, prebiotics and immune checkpoint inhibitors.

[0022] 11) A pharmaceutical composition comprising probiotics and prebiotics for use in combination with an immune checkpoint inhibitor.

[0023] 12) Use of probiotics and prebiotics in the manufacture of agents to enhance the anti-tumor effects of immune checkpoint inhibitors.

[0024] 13) Application of probiotics, prebiotics and immune checkpoint inhibitors in the manufacture of tumor immune activators.

[0025] 14) Use of a combination of probiotics, prebiotics and immune checkpoint inhibitors in the manufacture of pharmaceuticals.

[0026] 15) A probiotic and a prebiotic for enhancing the anti-tumor effect when using an immune checkpoint inhibitor.

[0027] 16) A probiotic, a prebiotic and an immune checkpoint inhibitor for tumor immune activation.

[0028] 17) A combination of probiotics, prebiotics and immune checkpoint inhibitors for use in medicines.

[0029] 18) A method for enhancing the anti-tumor effect when using an immune checkpoint inhibitor, wherein probiotics and prebiotics are administered to a subject in need thereof.

[0030] 19) A method for tumor immune activation, comprising administering probiotics, prebiotics, and immune checkpoint inhibitors to a subject in need thereof.

[0031] Effects of the Invention

[0032] According to the present invention, tumor immunity can be activated, and the development of cancer, the recurrence of cancer or the cancer can be inhibited or treated. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The anti-tumor effect is brought about by the combined use of synbiotics / immune checkpoint inhibitors.

[0034] Figure 2 It is the effect on immune cells caused by the combined use of synbiotics / immune checkpoint inhibitors.

[0035] Figure 3 It is the effect on regulatory T cells (Treg) brought about by the combined use of synbiotics / immune checkpoint inhibitors.

[0036] Figure 4 It is the effect on the immunosuppressive state caused by the combined use of synbiotics / immune checkpoint inhibitors.

[0037] Figure 5 The combined use of synbiotics and immune checkpoint inhibitors has an effect on tumor-derived medullary immunosuppressive cells (MDSCs).

[0038] Figure 6 It is the impact on immune status caused by the combined use of synbiotics / immune checkpoint inhibitors.

[0039] Figure 7It is the NK cell activity enhancement effect brought about by the combined use of various intestinal bacterial preparations / immune checkpoint inhibitors. C: control group, P: anti-PD-1 antibody administration group, LP: probiotics + anti-PD-1 antibody administration group, GP: prebiotics + anti-PD-1 antibody administration group, LG: synbiotics administration group, LGP: synbiotics + anti-PD-1 antibody administration group. DETAILED DESCRIPTION

[0040] In the present invention, "probiotics" refers to living microorganisms that have beneficial effects on the health of the host when ingested in appropriate amounts. "Prebiotics" refers to substances that serve as "food" for bifidobacteria and lactic acid bacteria, increase these bacteria, and have beneficial effects.

[0041] Specific examples of the probiotic bacteria of the present invention include one or more microorganisms selected from the group consisting of bacteria of the genus Lactobacillus, bacteria of the genus Bifidobacterium, bacteria of the genus Streptococcus, and bacteria of the genus Lactococcus.

[0042] Examples of the bacteria of the genus Lactobacillus include Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus gallinarum, Lactobacillus amylovorus, Lactobacillus brevis, Lactobacillus rhamnosus, Lactobacillus kefir, Lactobacillus curvatus, Lactobacillus zeae, Lactobacillus helveticus, Lactobacillus salivalius, Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus crispatus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. subsp.delbrueckii), Lactobacillus johnsonii, etc.

[0043] Among them, from the viewpoint of enhancing the antitumor effect, Lactobacillus casei and Lactobacillus acidophilus are preferred, Lactobacillus casei YIT 9018 (FERM BP-665), Lactobacillus casei YIT 9029 (FERM BP-1366), Lactobacillus casei YIT 10003 (FERM BP-7707), and Lactobacillus acidophilus YIT 0198 are more preferred, and Lactobacillus casei YIT 9029 (FERM BP-1366) is further preferred.

[0044] Examples of the bacteria belonging to the genus Bifidobacterium include Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium suis, Bifidobacterium infantis, Bifidobacterium adolescentis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium lactis, and Bifidobacterium globosum.

[0045] Among them, from the viewpoint of enhancing the antitumor effect, Bifidobacterium breve, Bifidobacterium bifidum, and Bifidobacterium pseudocatenulatus are preferred, and Bifidobacterium breve YIT12272 strain (FERM BP-11320), Bifidobacterium breve YIT10001 strain (FERM BP-8205), and Bifidobacterium bifidum YIT10347 (FERM BP-10613) are more preferred.

[0046] Examples of bacteria belonging to the genus Streptococcus include Streptococcus thermophilus and Streptococcus lactis.

[0047] Among them, from the viewpoint of enhancing the antitumor effect, Streptococcus thermophilus is preferred, and Streptococcus thermophilus YIT 2021 strain (FERM BP-7537) is more preferred.

[0048] Examples of the bacteria belonging to the genus Lactococcus include Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus plantarum, and Lactococcus raffinolactis.

[0049] Among them, from the viewpoint of enhancing the antitumor effect, Lactococcus lactis is preferred, and Lactococcus lactis YIT 2027 (FERM BP-6224) is more preferred.

[0050] Prebiotics of the present invention are preferably substances that impart beneficial effects to the host by preferentially acting on the probiotics of the present invention, and specifically include one or more selected from oligosaccharides such as galacto-oligosaccharides, fructo-oligosaccharides, lactofructo-oligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, niger oligosaccharides, gentio-oligosaccharides, pectin oligosaccharides, cyclodextrins, polydextrose, inulin, germinated barley, inulin, indigestible dextrins, and dietary fibers such as resistant starch.

[0051] In the present invention, galacto-oligosaccharides are preferred among these prebiotics from the viewpoint of enhancing the anti-tumor effect.

[0052] Here, galacto-oligosaccharide is a general term for oligosaccharides having at least one galactose residue in the molecule, and examples thereof include sugars formed by 2 to 9, preferably 3 to 4 monosaccharide bonds. In addition, examples of galacto-oligosaccharides include sugars formed by β1-2 bonds of galactose, sugars formed by β1-3 bonds, sugars formed by β1-4 bonds, and sugars formed by β1-6 bonds of galactose, but β1-4 bonds and β1-6 bonds of galacto-oligosaccharides are particularly preferred.

[0053] More preferred galacto-oligosaccharides include β-1,4-galactosyl lactose (Galβ1-4Galβ1-4Glc), β-1,6-galactosyl lactose (Galβ1-6Galβ1-4Glc), β-1,3-galactosyl lactose (Galβ1-3Galβ1-4Glc), etc. From the viewpoint of enhancing the anti-tumor effect, galacto-oligosaccharides containing β-1,4-galactosyl lactose as the main component are more preferred. For example, Oligomate 55N (Yakult Pharmaceutical Co., Ltd.; the content of β-1,4-galactosyl lactose in the solid component is 18.5%) can be used.

[0054] In the present invention, probiotics and prebiotics may be used in combination as synbiotics. Synbiotics are preferably used as a preparation (intestinal bacteria preparation) prepared by mixing the two.

[0055] The content of probiotics in the preparation is not particularly limited, and is, for example, 0.1×10 11 ~100×10 11 CFU (colony forming unit), preferably 1×10 11 ~10×10 11 CFU.

[0056] In addition, the ratio of the content of probiotics to prebiotics in the preparation is, for example, 0.01×10 11 ~20×10 11 CFU, preferably 0.2×10 11~2×10 11 CFU.

[0057] The content of the prebiotic in the preparation is not particularly limited, but is 0.5 to 40% by mass, preferably 2 to 10% by mass, and more preferably 3 to 6% by mass in the preparation.

[0058] Synbiotics may contain other ingredients within the scope of not impairing the effects of the present invention. As other ingredients, minerals such as calcium, magnesium, zinc, iron, dolomite or their salts; acids such as citric acid, malic acid, ascorbic acid, lactic acid, acetic acid, amino acids, collagen, chondroitin sulfate, hydroxyproline, flavonoids, flavonols, isoflavones, anthocyanidins, catechins, proanthocyanidins and other additives, vitamin A, vitamin B, vitamin C, vitamin E, vitamin D, vitamin K, beta-carotene, retinoic acid, folic acid and other vitamins, etc. These can be used alone or in combination of two or more.

[0059] The administration form of synbiotics is not particularly limited, and examples thereof include forms that can be administered orally or enterally, and an oral administration form is preferred.

[0060] Examples of oral administration include tablets (including sugar-coated tablets, enteric-coated tablets, buccal tablets), powders, capsules (including enteric-coated capsules, soft capsules), granules (including coated types), pills, lozenges, liposome-encapsulated agents, liquids, and sustained-release preparations thereof. In addition, the oral administration may be in the form of food (e.g., beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit juice drinks, and lactic acid bacteria drinks; dairy products such as processed milk, milk drinks, fermented milk, and butter; functional foods such as health supplements, etc.).

[0061] In the formulation, carriers commonly used in general pharmaceutical preparations, excipients (such as lactose, glucose, saccharide, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, etc.), binders (such as starch, gelatin, glucose, galactose, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypropyl cellulose, ethyl cellulose, methyl cellulose and carboxymethyl cellulose, etc.), disintegrants (such as starch, agar, gelatin, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, crystalline cellulose, calcium carbonate, sodium bicarbonate, and sodium alginate, etc.), lubricants (such as magnesium stearate, hydrogenated vegetable oils and polyethylene glycol, etc.), stabilizers, flavoring deodorants, diluents, surfactants, solvents and other additives can be used.

[0062] In the present invention, the “immune checkpoint inhibitor” refers to a molecule that inhibits the function of immune checkpoint molecules such as CTLA-4, PD-1, or PD-L1, which is its ligand.

[0063] Examples of immune checkpoint inhibitors include anti-PD-1 antibodies (e.g., Nivolumab, Pembrolizumab), anti-PD-L1 antibodies (e.g., Atezolizumab, Avelumab, Durvalumab), PD-1 antagonists (e.g., AUNP-12), anti-CTLA-4 antibodies (e.g., Ipilimumab and Tremelimumab), and anti-LAG-3 antibodies (e.g., BMS-986016 and LAG525).

[0064] Among them, from the viewpoint of enhancing the anti-tumor effect, anti-PD-1 antibodies, anti-PD-L1 antibodies, and PD-1 antagonists are preferred, and anti-PD-1 antibodies are more preferred.

[0065] As shown in the examples described below, the combination of probiotics and prebiotics involved in the present invention, when used in combination with immune checkpoint inhibitors, enhances the anti-tumor effect brought about by immune checkpoint inhibitors. Therefore, the combination of probiotics and prebiotics can become an anti-tumor effect enhancer when using immune checkpoint inhibitors.

[0066] In addition, the combination of probiotics and prebiotics and immune checkpoint inhibitors can enhance the survival of Th1 cells (CD3 + / CD4 + / IFN-γ + 、CD3 + / CD8 + / IFN-γ + ), NK cell activation, and inhibit the expression and function of controlling T cells (Treg), bone marrow-derived immunosuppressive cells (MDSC), and immunosuppressive cytokines (TGF-β), thereby inhibiting the tumor immunosuppressive system. Therefore, the combination of probiotics and prebiotics and immune checkpoint inhibitors can become an anti-tumor immune activator.

[0067] Such anti-tumor effect enhancers and tumor immunity activators when using immune checkpoint inhibitors can be used to inhibit the progression, recurrence, and treatment of cancer.

[0068] In addition, according to the anti-tumor effect enhancer and tumor immunity activator when using an immune checkpoint inhibitor of the present invention, it is also possible to reduce the dosage of the immune checkpoint inhibitor and alleviate side effects.

[0069] In the anti-tumor effect enhancer or anti-tumor immune activator using immune checkpoint inhibitors of the present invention, the cancer as the object is not particularly limited, and may include any solid cancer and blood cancer. For example, head and neck cancer, esophageal cancer, gastric cancer, colon and rectal cancer, liver cancer, gallbladder and bile duct cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, testicular cancer, bone and soft tissue sarcoma, malignant lymphoma, leukemia, cervical cancer, skin cancer, brain tumors, etc., among which, cancers for which tumor immunotherapy based on immune checkpoint inhibitors is more effective are preferably cited.

[0070] The probiotics and prebiotics of the present invention and the immune checkpoint inhibitors can be administered simultaneously, or the probiotics and prebiotics can be administered first and then the immune checkpoint inhibitors, or the immune checkpoint inhibitors can be administered first and then the probiotics and prebiotics.

[0071] In addition, the administration form of probiotics and prebiotics and immune checkpoint inhibitors can be the same or different, and can be administered in a form suitable for each. In addition, probiotics and prebiotics and immune checkpoint inhibitors can also be combined as a kit to prepare a pharmaceutical preparation.

[0072] In the anti-tumor effect enhancer or tumor immune activator of the present invention using an immune checkpoint inhibitor, the dosage of probiotics and prebiotics varies according to age, weight, symptoms, therapeutic effect, administration method, treatment time, etc., and is generally 3×10 7 ~3×10 11 CFU, preferably 3×10 8 ~3×10 10 CFU, the prebiotic is 1 to 10 g, preferably 3 to 9 g, and is administered once or several times a day.

[0073] The dosage of the immune checkpoint inhibitor can be appropriately selected based on the dosage used clinically. In addition, any two or more immune checkpoint inhibitors can be administered in combination.

[0074] Example

[0075] The present invention will be further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.

[0076] <Test product>

[0077] 1) Probiotics

[0078] The strains used were Lactobacillus casei YIT9029 (FERM BP-1366) and Bifidobacterium breve YIT12272 (FERM BP-11320).

[0079] 2) Prebiotics

[0080] Galacto-oligosaccharides with β-1,4-galactosyl lactose as the main component

[0081] 3) Biostime

[0082] Mix 1mL GOS (100mg / 1mL), 0.5mL (20×10 8 CFU / 1mL) Lactobacillus casei YIT9029 strain, 0.5mL (20×10 8 CFU / 1mL) Bifidobacterium breve YIT12272 strain.

[0083] 4) Anti-PD-1 antibodies

[0084] "Anti-PD1 (J43) Hamster IgG ms" (BioxCell) (control antibody "IgG Isotype Hamster IgG ms" (BioxCell))

[0085] Example 1 Anti-tumor effect of synbiotics / immune checkpoint inhibitors in vivo

[0086] Mouse colon cancer cell line MC38 cells were transplanted into 7-week-old male C57BL / 6NCrSlc mice to make cell line transplantation model mice. They were divided into control (Vehicle) group, synbiotics administration group, anti-PD-1 antibody administration group, synbiotics + anti-PD-1 antibody administration group {n=10 (Vehicle group), 5 (synbiotics administration group, anti-PD-1 antibody administration group), 6 (synbiotics + anti-PD-1 antibody administration group)}.

[0087] Synbiotics were administered as follows: 0.2 mL of synbiotics / mouse was orally administered once a day for 28 days after transplantation. Anti-PD-1 antibodies were administered as follows: 20 mg / kg of anti-PD-1 antibodies were administered intraperitoneally for the first time starting from the 7th day after tumor transplantation, and then every 6 days, with 10 mg / kg administered intraperitoneally for the 2nd to 4th time.

[0088] Tumor volume was measured with calipers (Tukey-Kramer test).

[0089] Tumor volume is measured by the long and short diameters of the tumor using {(long diameter)×(short diameter) 2} / 2 calculation formula.

[0090] The results are shown in Figure 1 .Depend on Figure 1It shows that the tumor volume of the group using anti-PD-1 antibody and synbiotics together was significantly smaller. On the other hand, no significant reduction in tumor volume was observed in the group using both drugs alone. In addition, the tumor size when used alone was larger than the midpoint of the Vehicle group and the synbiotic + anti-PD-1 antibody administration group, so it was also different from the simple additive effect. Therefore, it can be judged that the anti-PD-1 antibody and synbiotics specifically exerted an inhibitory effect on tumor growth when used together.

[0091] Example 2 Effects of synbiotics / immune checkpoints on immune cells in vivo

[0092] Mouse colon cancer cell line MC38 cells were transplanted into 7-week-old male C57BL / 6NCrSlc mice to create cell line transplantation model mice. The mice were divided into control (Vehicle) group, synbiotics-administered group, anti-PD-1 antibody-administered group, and synbiotics + anti-PD-1 antibody-administered group.

[0093] Synbiotics were administered as follows: 0.2 mL of synbiotics / mouse was orally administered once a day for 28 days after transplantation. Anti-PD-1 antibodies were administered as follows: 20 mg / kg of anti-PD-1 antibodies were administered intraperitoneally for the first time starting from the 7th day after tumor transplantation, and then every 6 days, with 10 mg / kg administered intraperitoneally for the 2nd to 4th time.

[0094] During the autopsy, various tissues (spleen, tumor) were collected and the cells were separated into single cells. The separated cells were labeled with specific antibodies for various immune cell marker proteins and identified using flow cytometry. Based on the identification data, the Th1 cells (CD3 + / CD4 + / IFN-γ + 、CD3 + / CD8 + / IFN-γ + ), the cell ratio of each Treg cell group. {n=8 (Vehicle group), 5 (Synbiotics group, anti-PD-1 antibody group), 6 (Synbiotics + anti-PD-1 antibody group), Student's t test}

[0095] The results are shown in Figure 2 .Depend on Figure 2It was confirmed that the number of Tregs that inhibit the activity of surrounding immune cells showed a significant decrease only in the synbiotic + anti-PD-1 antibody administration group. Since Tregs are also used as an anti-immune mechanism for tumor cells, the results can be inferred that the anti-immune defense of the tumor was suppressed by the combined use of two doses. On the other hand, the number of activated T cells in the spleen (activation markers CD8, CD4 positive) did not specifically increase in the synbiotic + anti-PD-1 antibody administration group, suggesting that the effect brought about by the combined use is due to the activation of immune cells.

[0096] Example 3 Effects of synbiotics / immune checkpoint inhibitors on small intestinal regulatory T cells (Treg) in vivo

[0097] Mouse colon cancer cell line MC38 cells were transplanted into 7-week-old male C57BL / 6NCrSlc mice to create cell line transplantation model mice. The mice were divided into control (Vehicle) group, synbiotics group, anti-PD-1 antibody group, synbiotics + anti-PD-1 antibody group {n = 10 (Vehicle group), 5 (synbiotics group, anti-PD-1 antibody group), 6 (synbiotics + anti-PD-1 antibody group)}.

[0098] Synbiotics were administered as follows: 0.2 mL of synbiotics / mouse was orally administered once a day for 28 days after transplantation. Anti-PD-1 antibodies were administered as follows: 20 mg / kg of anti-PD-1 antibodies were administered intraperitoneally for the first time starting from the 7th day after tumor transplantation, and then every 6 days, with 10 mg / kg administered intraperitoneally for the 2nd to 4th time.

[0099] During autopsy, the small intestine was recovered and Treg was evaluated by real-time PCR using FOXP3 as an indicator. (Mann Whitney Utest)

[0100] The results are shown in Figure 3 .Depend on Figure 3 The results showed that the expression level of FOXP3 gene, the master transcription factor of Treg, was significantly reduced in the synbiotics + anti-PD-1 antibody administration group. This result supports the reduction in the number of Tregs shown above.

[0101] Example 4 Effect of combined use of synbiotics / immune checkpoint inhibitors on systemic immunosuppression in vivo

[0102] Mouse colon cancer cell line MC38 cells were transplanted into 7-week-old male C57BL / 6NCrSlc mice to make cell line transplantation model mice. They were divided into control (Vehicle) group, synbiotics administration group, anti-PD-1 antibody administration group, synbiotics + anti-PD-1 antibody administration group {n=10 (Vehicle group), 5 (synbiotics administration group, anti-PD-1 antibody administration group), 6 (synbiotics + anti-PD-1 antibody administration group)}.

[0103] Synbiotics were administered as follows: 0.2 mL of synbiotics / mouse was orally administered once a day for 28 days after transplantation. Anti-PD-1 antibodies were administered as follows: 20 mg / kg of anti-PD-1 antibodies were administered intraperitoneally for the first time starting from the 7th day after tumor transplantation, and then every 6 days, with 10 mg / kg administered intraperitoneally for the 2nd to 4th time.

[0104] During autopsy, various tissues (small intestine, Peyer's patches) were recovered, and the immunosuppression status was evaluated by real-time PCR using TGFβ as an index. (Mann Whitney U test)

[0105] The results are shown in Figure 4 .according to Figure 4 , TGFβ is a cytokine that exerts an immunosuppressive effect and is induced by substances derived from intestinal bacteria. Figure 4 In the small intestine as a whole and in the Peyer's patches, the central tissue of small intestinal immunity, the gene expression level of TGFβ was significantly lower in the synbiotic + anti-PD-1 antibody administration group compared with the vehicle group, suggesting the activation of small intestinal immunity.

[0106] Example 5 Effects of Synbiotics / Immune Checkpoint Inhibitor Combination on Tumor-Derived Medullary Immunosuppressive Cells (MDSC) in Vivo

[0107] Mouse colon cancer cell line MC38 cells were transplanted into 7-week-old male C57BL / 6NCrSlc mice to make cell line transplantation model mice. They were divided into control (Vehicle) group, synbiotics administration group, anti-PD-1 antibody administration group, synbiotics + anti-PD-1 antibody administration group {n=10 (Vehicle group), 5 (synbiotics administration group, anti-PD-1 antibody administration group), 6 (synbiotics + anti-PD-1 antibody administration group)}.

[0108] Synbiotics were administered as follows: 0.2 mL of synbiotics / mouse was orally administered once a day for 28 days after transplantation. Anti-PD-1 antibodies were administered as follows: 20 mg / kg of anti-PD-1 antibodies were administered intraperitoneally for the first time starting from the 7th day after tumor transplantation, and then every 6 days, with 10 mg / kg administered intraperitoneally for the 2nd to 4th time.

[0109] During autopsy, the tumor was recovered and MDSCs were evaluated by real-time PCR using Bv8 as an indicator. (Mann Whitney Utest)

[0110] The results are shown in Figure 5 Bv8 is a marker for MDSCs, and a reduction in the number of MDSCs present in tumors suggests an anti-tumor effect.

[0111] Example 6 Effects of combined use of synbiotics / immune checkpoint inhibitors on systemic immune status in vivo

[0112] Mouse colon cancer cell line MC38 cells were transplanted into 7-week-old male C57BL / 6NCrSlc mice to make cell line transplantation model mice. They were divided into control (Vehicle) group, synbiotics administration group, anti-PD-1 antibody administration group, synbiotics + anti-PD-1 antibody administration group {n=10 (Vehicle group), 5 (synbiotics administration group, anti-PD-1 antibody administration group), 6 (synbiotics + anti-PD-1 antibody administration group)}.

[0113] Synbiotics were administered as follows: 0.2 mL of synbiotics / mouse was orally administered once a day for 28 days after transplantation. Anti-PD-1 antibodies were administered as follows: 20 mg / kg of anti-PD-1 antibodies were administered intraperitoneally for the first time starting from the 7th day after tumor transplantation, and then every 6 days, with 10 mg / kg administered intraperitoneally for the 2nd to 4th time.

[0114] During autopsy, each tissue (small intestine, tumor) was recovered and the expression level of each cytokine (IFN-β, IL-17) gene was evaluated by real-time PCR. (Mann Whitney U test)

[0115] The results are shown in Figure 6 .according to Figure 6 In the small intestine of the synbiotics + anti-PD-1 antibody group, the gene expression of the anti-inflammatory cytokine IFN-β was specifically high, and combined with Figure 4 The TGFβ shown in the figure is low, indicating a high possibility of immune hyperfunction in the small intestine. On the other hand, IL-17 is a cytokine highly expressed by immune cells, and its value was significantly high in the synbiotic + anti-PD-1 antibody administration group, suggesting an increase in immune cells in the tumor. These results all support the activation of tumor immunity.

[0116] Example 7: In vivo enhancement of NK cell activity by various intestinal bacterial preparations / immune checkpoint inhibitors combined use

[0117] Mouse colon cancer cell line MC38 cells were transplanted into 7-week-old male C57BL / 6NCrSlc mice to make transplantation model mice. The mice were divided into control (Vehicle) group (C), anti-PD-1 antibody administration group (P), probiotics + anti-PD-1 antibody administration group (LP), prebiotics + anti-PD-1 antibody administration group (GP), synbiotics administration group (LG), synbiotics + anti-PD-1 antibody administration group (LGP) {n=9 (C, LG, LGP), 8 (P, GP, LP)}.

[0118] Synbiotics were administered as follows: after transplantation, 0.2 mL of synbiotics / mouse were orally administered once a day for six days except Sundays for a total of 15 days. Anti-PD-1 antibodies were administered as follows: starting on the 6th day after tumor transplantation, 20 mg / kg of anti-PD-1 antibodies were administered intraperitoneally for the first time, and then every 6 days, with 10 mg / kg administered intraperitoneally for the 2nd to 3rd time. Single cells were prepared from the spleen taken during dissection to form effector cells. As target cells, mouse lymphoma, namely YAC-1 cells, were used. By dividing the target cells (4×10 5 cells / mL) were added with calcein-AM (cell labeling reagent, final concentration 1 mg / mL) and incubated at 37°C for 30 minutes for fluorescent staining. 4 cells / 100mL) and pre-cultured effector cells (4×10 5 cells / 100mL) were co-cultured in a U-bottom 96-well plate for 4 hours (effector cells: target cells = 40:1). Afterwards, the cells were centrifuged at 300×g for 5 minutes, and 80 μL of the supernatant was transferred to a black 96-well plate. The amount of calcein-AM transferred to the culture supernatant was measured using a fluorescence plate reader (Ex. 488 nm, Em. 535 nm). The obtained results were used to calculate the cell toxicity based on the following formula (n = 6, standard error, Dunnet's test).

[0119] Cytotoxic activity (% cell lysis) = ((sample fluorescence intensity - natural free fluorescence intensity) × 100) / (maximum fluorescence intensity - natural free fluorescence intensity)

[0120] The results are shown in Figure 7 .according to Figure 7 It can be seen that only in the LGP group where probiotics, prebiotics, and anti-PD-1 antibodies co-existed, the cytotoxic activity of NK cells was significantly activated. Figure 2 The decrease in the number of Tregs shown in the figure suppresses the anti-immune defense function of the tumor, resulting in an increase in the activity of immune cells against the tumor, which supports the activation of tumor immunity brought about by the combined use of the two preparations.

Claims

1. The use of probiotics and prebiotics in the manufacture of anti-tumor effect enhancers in the case of immune checkpoint inhibitors, The probiotics are Lactobacillus casei strain YIT9029, i.e. FERM BP-1366, and Bifidobacterium breve strain YIT12272, i.e. FERM BP-11320. Prebiotics are galacto-oligosaccharides. The immune checkpoint inhibitor is one or more selected from anti-PD-1 antibodies and anti-PD-L1 antibodies.

2. The use according to claim 1, wherein: Prebiotics are galacto-oligosaccharides with β-1,4-galactosyl lactose as the main component.

3. A tumor immune activator, comprising probiotics, prebiotics and immune checkpoint inhibitors as active ingredients, The probiotics are Lactobacillus casei strain YIT9029, i.e. FERM BP-1366, and Bifidobacterium breve strain YIT12272, i.e. FERM BP-11320. Prebiotics are galacto-oligosaccharides. The immune checkpoint inhibitor is one or more selected from anti-PD-1 antibodies and anti-PD-L1 antibodies.

4. The tumor immunity activator according to claim 3, wherein Tumor immunity is activated by relieving the immunosuppressive state mediated by regulatory T cells and / or activating NK cells.

5. A pharmaceutical product comprising a combination of probiotics, prebiotics and immune checkpoint inhibitors. The probiotics are Lactobacillus casei strain YIT9029, i.e. FERM BP-1366, and Bifidobacterium breve strain YIT12272, i.e. FERM BP-11320. Prebiotics are galacto-oligosaccharides. The immune checkpoint inhibitor is one or more selected from anti-PD-1 antibodies and anti-PD-L1 antibodies.

6. Application of probiotics, prebiotics and immune checkpoint inhibitors in the manufacture of tumor immune activators, The probiotics are Lactobacillus casei strain YIT9029, i.e. FERM BP-1366, and Bifidobacterium breve strain YIT12272, i.e. FERM BP-11320. Prebiotics are galacto-oligosaccharides. The immune checkpoint inhibitor is one or more selected from anti-PD-1 antibodies and anti-PD-L1 antibodies.

7. Application of probiotics, prebiotics and immune checkpoint inhibitors in the manufacture of pharmaceuticals. The probiotics are Lactobacillus casei strain YIT9029, i.e. FERM BP-1366, and Bifidobacterium breve strain YIT12272, i.e. FERM BP-11320. Prebiotics are galacto-oligosaccharides. The immune checkpoint inhibitor is one or more selected from anti-PD-1 antibodies and anti-PD-L1 antibodies.

Images