Compositions for expanding and / or activating CD4-CD8-T cells

Abstract

Novel CD4 - CD8 - The present invention provides a composition for expanding and / or activating CD4 T cells, which comprises a spore-forming bacterium or a culture thereof as an active ingredient. - CD8 - The present invention relates to a composition for expanding and / or activating T cells.

Description

[Technical Field]

[0001] The present invention relates to CD4 - CD8 - The present invention relates to a composition for expanding and / or activating T cells. [Background technology]

[0002] T cells are a type of lymphocyte that express T cell receptors on their surface. Most T cells express either CD4 or CD8. However, there are also CD4 T cells that do not express both CD4 and CD8. - CD8 - (double negative) T cells are present.

[0003] CD4 - CD8 - T cells have been reported to be associated with antitumor effects. - CD8 - It has been reported that T cells play an important role in immune homeostasis under healthy conditions, and that they act as Treg cells, cytotoxic T cells, or Th cells, affecting the innate and adaptive immune systems in distinct pathologies closely related to inflammatory diseases, autoimmune diseases, tumorigenesis, and tumor development. - CD8 - It has been reported that T cells can be potential targets for cancer immunotherapy. [Prior art documents] [Non-patent literature]

[0004] [Patent Document 1] Wu Z, Zheng Y, Sheng J, Han Y, Yang Y, Pan H and Yao J(2022) CD3+CD4-CD8- (Double-Negative) T Cells in Inflammation, Immune Disorders and Cancer. Front. Immunol. 13:816005. doi: 10.3389 / fimmu.2022.816005 [Patent Document 2] Okamura K, Nagayama S, Tate T, Kiyotani K, and Yusuke Nakamura (2020) The potential target of double negative T cells in cancer immunotherapy, Journal of Clinical Oncology 38:15_suppl, e15180 Summary of the Invention

[0005] The present invention provides a novel CD4 - CD8 - The present invention aims to provide a composition for expanding and / or activating T cells.

[0006] The present inventors have surprisingly found that spore-forming bacteria are CD4 - CD8 - The present inventors have found that the present invention can proliferate and / or activate T cells.

[0007] That is, according to one embodiment of the present invention, there is provided a method for treating CD4 containing spore-forming bacteria or a culture thereof as an active ingredient. - CD8 - Compositions for expanding and / or activating T cells are provided. [Brief explanation of the drawings]

[0008] [Figure 1]The following shows an outline of the experiment and the results of Test Example 1. A: Schematic diagram of the experiment, B: Scatter plot showing the results of flow cytometry when Miya BM (registered trademark) suspension was used, C: Graph showing the results of flow cytometry for Test Example 1. [Figure 2] 1 shows the experimental results of Test Example 2. A: Scatter plot showing the results of flow cytometry when a suspension of spores without heat treatment was used, B: Graph showing the results of flow cytometry. [Figure 3] 1 shows the experimental results of Test Example 3. A: Scatter plot showing the results of flow cytometry, B: Graph showing the results of flow cytometry. [Figure 4] 1 shows an outline of the experiment and the results of Test Example 4. A: Schematic diagram of the experiment, B: Scatter plot showing the results of flow cytometry, C: Graph showing the results of flow cytometry. [Figure 5] 1 shows an outline of the experiment and the results of Test Example 5. A: Schematic diagram of the experiment, B: Scatter plot showing the results of flow cytometry. DETAILED DESCRIPTION OF THE INVENTION

[0009] An embodiment of the present invention will be described below, but the present invention is not limited to the following embodiment.

[0010] In this specification, the range "X to Y" means "X or more and Y or less." Unless otherwise specified, operations and measurements of physical properties are performed at room temperature (20 to 25°C) and a relative humidity of 40 to 50% RH. In this specification, "A and / or B" means at least one of A and B, and includes both A and B, or either A or B.

[0011] <CD4 - CD8 - Compositions for Proliferating and / or Activating T Cells One aspect of the present invention is a CD4 - CD8 -According to one aspect of the present invention, a novel CD4 T cell proliferation and / or activation composition is provided. - CD8 - Compositions for expanding and / or activating T cells are provided.

[0012] As used herein, CD4 - CD8 - The composition for expanding and / or activating T cells is also simply referred to as the "composition of the present invention."

[0013] Spore-forming bacteria are bacteria that form spores that are extremely resistant to physical and chemical treatments. Spore formation can be confirmed by microscopic observation, staining by spore staining methods, heat resistance based on survival after heating, and resistance to organic solvents or disinfectants based on survival after exposure.

[0014] In the composition of the present invention, the type of spore-forming bacteria is not particularly limited, and examples of spore-forming bacteria include bacteria of the genera Clostridium, Bacillus, Amphibacillus, and Sporosarcina.

[0015] In one embodiment, the spore-forming bacterium is at least one bacterium selected from the group consisting of bacteria of the genera Clostridium and Bacillus.

[0016] Bacteria in the genus Clostridium include Clostridium butyricum, Clostridium absonum, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium cadaveris, Clostridium clostridiiforme, Clostridium cochlearium, Clostridium difficile, Clostridium innocuum, Clostridium novyi, and Clostridium perfringens. perfringens, Clostridium putrificum, Clostridium ramosum, Clostridium rectum, Clostridium scatologenes, Clostridium (Paeniclostridium) sordellii, Clostridium sporogenes, Clostridium tertium, Clostridium tyrobutyricum, Clostridium aerotolerans, Clostridium aminophilum, Clostridium aminovalericum aminovalericum, Clostridium celeriacense (Clostridiumcelerecrescens, Clostridium cellulosi, Clostridium coccoides, Clostridium hiranonis, Clostridium leptum, Clostridium nexile, Clostridium oroticum, Clostridium polysaccharolyticum, Clostridium populeti, Clostridium scindens, Clostridium sphenoides, Clostridium sporosphaeroides sporosphaeroides, Clostridium symbiosum, Clostridium xylanolyticum, and Clostridium hylemonae.

[0017] Examples of bacteria in the genus Bacillus include Bacillus cereus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus licheniformis, Bacillus polyfermenticus, Bacillus pumilus, and Bacillus subtilis.

[0018] These bacteria can be obtained from, for example, the National Institute of Technology and Evaluation (NITE), the RIKEN Joint Microbial Research Center (JCM), the National BioResource Project (NBRP), the American Type Culture Collection (ATCC), and the German Collection of Microbial Cell Cultures (DSMZ).

[0019] The spore-forming bacterium is preferably a bacterium of the genus Clostridium, more preferably Clostridium butyricum, Clostridium sordellii, or Clostridium putrificum, and even more preferably Clostridium butyricum MIYAIRI 588 (FERM BP-2789), Clostridium sordellii JCM3814 strain, or Clostridium putrificum ATCC25784 strain. Clostridium butyricum MIYAIRI 588 (FERM BP-2789) was deposited on May 1, 1981, at the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently the Patent Organism Depositary, National Institute of Technology and Evaluation, Room 120, Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) under the accession number FERM BP-2789, and was transferred to an international depository organization under the Budapest Treaty on March 6, 1990, where it is deposited under the accession number FERM BP-2789.

[0020] The spore-forming bacteria may be used alone or in combination of two or more kinds.

[0021] The spore-forming bacteria may be in the form of spores or vegetative forms. The spore-forming bacteria are preferably in the form of spores, from the viewpoint of being able to more effectively exhibit the effects of the present invention. The spore-forming bacteria contained in the composition of the present invention may be in the form of spores only, vegetative forms only, or both spores and vegetative forms.

[0022] The method for forming spores is not particularly limited, and a conventionally known method can be adopted depending on the spore-forming bacterium used.

[0023] In the present invention, a culture of spore-forming bacteria refers to a culture medium in which spore-forming bacteria are cultured, a supernatant obtained by centrifuging the culture medium, a residue containing spore-forming bacteria obtained by centrifuging the culture medium, a purified product from the residue, and a dried product of the residue.

[0024] The purified product from the residue can be obtained according to a conventionally known method depending on the target component.

[0025] The dried residue can be obtained by centrifuging the culture solution, and then subjecting the residue containing the bacteria to air drying or vacuum drying.

[0026] The culture of spore-forming bacteria according to the present invention can be obtained by a conventionally known culture method depending on the spore-forming bacteria used. For example, the medium used to culture the spore-forming bacteria may be either a solid or liquid medium, and may be either a synthetic medium or a natural medium, so long as it contains a carbon source, an appropriate amount of a nitrogen source, inorganic salts, and other nutrients that can be assimilated by the spore-forming bacteria used. Typically, the medium contains a carbon source, a nitrogen source, and inorganic substances.

[0027] Carbon sources that can be used in the cultivation of spore-forming bacteria according to the present invention are not particularly limited as long as they can be assimilated by the strain used. Specific examples, taking into consideration the assimilation ability of spore-forming bacteria, include organic acids and salts thereof, such as gluconic acid, capric acid, adipic acid, malic acid, citric acid, phenyl acetate, acetic acid, lactic acid, succinic acid, glucuronic acid, and pyruvic acid; hydrocarbons, such as hexadecane; natural products, such as wheat and rice; alcohols, such as glycerol, methanol, and ethanol; and sugars. The carbon source is appropriately selected in consideration of the assimilation ability of the spore-forming bacteria to be cultured. One or more of the above carbon sources can be selected and used.

[0028] Nitrogen sources that can be used in the cultivation of spore-forming bacteria according to the present invention include organic nitrogen sources such as meat extract, fish extract, peptone, polypeptone, tryptone, yeast extract, malt extract, soybean hydrolysate, soybean powder, casein, milk casein, casamino acids, various amino acids such as glycine, glutamic acid, and aspartic acid, corn steep liquor, and hydrolysates of other animals, plants, and microorganisms; and inorganic nitrogen sources such as ammonia, ammonium salts such as ammonium nitrate, ammonium sulfate, and ammonium chloride, nitrates such as sodium nitrate, nitrites such as sodium nitrite, and urea. The above nitrogen source is appropriately selected taking into consideration the assimilation ability of the spore-forming bacteria to be cultivated. One or more of the above nitrogen sources can be selected and used.

[0029] Inorganic substances that can be used in the cultivation of spore-forming bacteria according to the present invention include halides such as phosphates, hydrochlorides, sulfates, acetates, carbonates, and chlorides of magnesium, manganese, calcium, sodium, potassium, copper, iron, and zinc. The inorganic substances are appropriately selected in consideration of the assimilation ability of the spore-forming bacteria to be cultivated. One or more of the inorganic substances can be selected and used. Furthermore, surfactants and the like may be added to the medium as needed.

[0030] The spore-forming bacteria of the present invention can be cultured by conventional methods. For example, depending on the type of spore-forming bacteria, the spore-forming bacteria are cultured under aerobic or anaerobic conditions. In the former case, the spore-forming bacteria are cultured by shaking or aeration / agitation. The spore-forming bacteria may also be cultured continuously or batchwise. Culture conditions are appropriately selected depending on the composition of the medium and the culture method, and are not particularly limited as long as they allow the spore-forming bacteria of the present invention to grow. They can be appropriately selected depending on the type of spore-forming bacteria to be cultured. The culture temperature is usually 20 to 42°C, preferably 35 to 40°C. The pH of the medium suitable for culture is not particularly limited, but is preferably 5 to 11, more preferably 6 to 10. The culture time is also not particularly limited and varies depending on the type of spore-forming bacteria to be cultured, the amount of medium, the culture conditions, etc. Typically, vegetative culture is continued until the exponential growth phase, while spores are cultured for, for example, 16 hours to 7 days.

[0031] As an example of a culture of spore-forming bacteria according to the present invention, a method for preparing a culture of Clostridium butyricum MIYAIRI 588 (FERM BP-2789) (hereinafter also referred to as strain 588) will be described.

[0032] The method for preparing the culture solution for the trophozoites can be the method described in Japanese Patent Application Laid-Open No. 08-252088.

[0033] Specifically, 588 strains were cultured in a medium (PYD medium) consisting of 1.0% peptone, 1.5% yeast extract, and 3% glucose for 10 min. 6 / mL and gently agitate at 20 rpm at 27°C. Using a pH controller, culture the medium until the early exponential growth phase while controlling the pH to 5.5 ± 0.2 with 5% sodium hydroxide solution. This yields a "vegetative culture solution of strain 588."

[0034] The spore suspension can be prepared using the method described in R. SATO and M. TANAKA, Multiplication of Orally Administered Clostridium Butyricum in Rats, MICROBIAL ECOLOGY IN HEALTH AND DISEASE, Vol. 9: 115-122 (1996).

[0035] Specifically, a "spore suspension of strain 588" can be obtained by culturing the strain in nutrient agar medium supplemented with 0.5% glucose and 0.3% calcium carbonate at 37°C under anaerobic conditions for 5 days, followed by suspending the strain in a liquid such as medium. Alternatively, a "purified spore suspension of strain 588 that does not contain vegetative spores" can be obtained by suspending the strain in sterile water instead of medium, centrifugally washing the spores with sterile water, purifying the spores by density gradient centrifugation using 45% urografin (common name: amidotrizoic acid), and then centrifugally washing the spores with sterile water.

[0036] The spore suspension may also be prepared by the method described in Japanese Patent Application Laid-Open No. 11-042081.

[0037] Specifically, strain 588 is inoculated into 10 ml of CS liquid medium consisting of 2.0% corn starch, 2.0% amino acid solution, and 0.75% calcium carbonate, and anaerobically cultured at 35-37°C for 16-24 hours using the carbon dioxide displacement steel wool method to prepare a preculture solution. After the specified incubation time, 0.01-1 ml of this preculture solution is inoculated into 10 ml of CS liquid medium for main culture at 60-100°C. The CS liquid medium is then cooled to 30-40°C with water, and anaerobically cultured at 35-37°C for 40-48 hours using the carbon dioxide displacement steel wool method. This produces a "spore suspension of strain 588."

[0038] The resulting culture solution is then centrifuged (2,000-6,000 g x 10-30 minutes) to separate the "residue containing the bacteria obtained by centrifuging the culture solution." This residue is then dried by air drying or the like at 0-80°C, preferably 10-20°C, for 1-24 hours, preferably 5-18 hours, or by vacuum drying at 0-80°C, preferably 10-20°C, 0.05-500 Torr (7 Pa-66.7 kPa), preferably 1-100 Torr (133 Pa-13.3 kPa), for 1-24 hours, preferably 2-15 hours, to obtain a "dried residue." Spray drying, freeze drying, or the like may also be used to obtain the dried product.

[0039] The spore-forming bacterium or a culture thereof according to the present invention is a CD4 - CD8 - T cells can be expanded and / or activated.

[0040] As used herein, CD4 - CD8 - T cell proliferation can be confirmed by CTV (Cell Trace Violet) assay. - CD8 - T cell activation can be confirmed by AIM (Activation-Induced Marker) assay.

[0041] As used herein, CD4 - CD8 - T cells refer to CD4-negative CD8-negative T cells. In a preferred embodiment, CD4 - CD8 - The T cells include γδ T cells, which include Vγ9Vδ2 T cells.

[0042] The composition of the present invention contains the spore-forming bacterium of the present invention or a culture thereof in an amount sufficient to exert the desired effect (i.e., an effective amount). The composition of the present invention may be the spore-forming bacterium of the present invention or a culture thereof itself, or may further contain other components. Examples of other components include those described below in the context of pharmaceutical compositions and food and beverage compositions.

[0043] In one embodiment, the compositions of the present invention are used in combination with an anti-tumor agent.

[0044] As used herein, the term "combination therapy" refers to simultaneous administration of the composition of the present invention and an antitumor agent, or sequential administration of the composition of the present invention and an antitumor agent at predetermined intervals over the course of treatment (combination therapy). The administration routes and means of the composition of the present invention and the antitumor agent may be the same or different.

[0045] As mentioned above, CD4 - CD8 - T cells have been reported to be associated with antitumor effects. The composition of the present invention is expected to further enhance the effects of an antitumor agent when used in combination with the antitumor agent.

[0046] Antitumor agents are not particularly limited, and examples thereof include tyrosine kinase inhibitors, immune checkpoint inhibitors, alkylating agents, antimetabolites, antitumor antibiotics, plant-derived antitumor agents, antitumor platinum coordination compounds, antitumor camptothecin derivatives, serine-threonine kinase inhibitors, kinase inhibitors, antitumor monoclonal antibodies, interferon preparations, biological response modifiers, hormone preparations, angiogenesis inhibitors, epigenetics-related molecule inhibitors, protein post-translational modification inhibitors, proteasome inhibitors, etc. The antitumor agent is preferably a tyrosine kinase inhibitor or an immune checkpoint inhibitor.

[0047] Tyrosine kinase inhibitors are drugs that exert antitumor effects by inhibiting cellular tyrosine kinases, enzymes involved in many cellular functions, including cell signaling, growth, and division.

[0048] The tyrosine kinase inhibitor is not particularly limited, and any known tyrosine kinase inhibitor can be used. The tyrosine kinase inhibitor is preferably at least one selected from the group consisting of an EGFR tyrosine kinase inhibitor, a c-kit receptor tyrosine kinase inhibitor, a BCR-ABL tyrosine kinase inhibitor, and a multikinase inhibitor, and more preferably a multikinase inhibitor.

[0049] Specific examples of tyrosine kinase inhibitors include imatinib, dasatinib, nilotinib, afatinib, erlotinib, osimertinib, gefitinib, axitinib, cabozantinib, sunitinib, sorafenib, pazopanib, regorafenib, and lenvatinib.

[0050] Immune checkpoint inhibitors inhibit the function of immune checkpoint receptors or ligands, and examples include antagonists of inhibitory receptors and agonists of costimulatory immune checkpoint receptors.

[0051] The term "antagonist" includes various substances that block receptor activation through binding between a receptor and a ligand, such as substances that bind to a receptor and block the receptor-ligand binding, and substances that bind to a ligand and block the receptor-ligand binding.

[0052] Examples of antagonists against inhibitory immune checkpoints include antagonistic antibodies that bind to inhibitory immune checkpoint molecules (inhibitory receptors or ligands for said receptors), soluble polypeptides designed based on inhibitory immune checkpoint ligands that do not activate receptors, and vectors capable of expressing said polypeptides.

[0053] The immune checkpoint inhibitor is not particularly limited, and any conventionally known immune checkpoint inhibitor can be used. The immune checkpoint inhibitor is preferably at least one selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor, and more preferably a PD-1 inhibitor.

[0054] Specific examples of immune checkpoint inhibitors include, but are not limited to, at least one selected from the group consisting of nivolumab, ipilimumab, pembrolizumab, semipilimab, durvalumab, daclizumab, avelumab, and atezolizumab, and more preferably at least one selected from the group consisting of nivolumab, pembrolizumab, and semipilimab.

[0055] The dosage and administration method of the composition and antitumor agent according to the present invention can be appropriately determined taking into consideration the type of tumor, symptoms, age, sex, weight, condition, etc. of the subject.

[0056] In one embodiment, the composition of the present invention is a pharmaceutical composition. - CD8 - T cells have been reported to be associated with anti-tumor effects and can be used in pharmaceutical applications.

[0057] The pharmaceutical composition of the present invention contains a sufficient amount of the spore-forming bacterium of the present invention or a culture thereof to exert the desired effect (i.e., an effective amount).

[0058] The pharmaceutical composition of the present invention may be the spore-forming bacterium or a culture thereof of the present invention itself, or may be prepared as an oral or parenteral formulation in accordance with conventional methods by combining additives acceptable for formulation. Examples of additives acceptable for formulation include excipients, stabilizers, preservatives, humectants, emulsifiers, lubricants, sweeteners, colorants, flavorings, buffers, antioxidants, pH adjusters, binders, thickeners, dispersants, suspending agents, disintegrants, bacteriostatic agents, surfactants, etc. The dosage form is not particularly limited and may be appropriately selected, and examples include tablets, powders, fine granules, granules, capsules, pills, sustained-release preparations, solutions, suspensions, emulsions, lotions, injections, infusions, topical preparations, suppositories, and patches.

[0059] In one embodiment, the composition of the present invention is a food or beverage composition.

[0060] The food and drink may be prepared by adding conventional additives such as stabilizers to the composition of the present invention, or by further blending various proteins, sugars, fats, trace elements, vitamins, etc., into the composition, or in the form of a liquid, semi-liquid, or solid, or in the form of a paste, or by adding the composition of the present invention to a general food or drink.

[0061] In this specification, "food and drink" refers to anything other than medicine that can be orally ingested by mammals, and is not particularly limited as long as it is in a form that can be taken orally by mammals, etc., and the form may be any of liquids (solutions, suspensions, emulsions, etc.), semi-liquids, powders, or solid moldings. Therefore, food and drink may be in the form of, for example, a drink, or may be in the form of a tablet of a nutritional supplement such as a supplement.

[0062] Specific examples of food and beverage products include instant noodles, retort pouch foods, canned foods, microwaveable foods, instant soups, miso soups, freeze-dried foods, and other instant foods; soft drinks, fruit juice drinks, vegetable drinks, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, nutritional drinks, alcoholic drinks, and other beverages; flour products such as bread, pasta, noodles, cake mix, fried chicken powder, and breadcrumbs; and sweets such as candy, caramel, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, and dessert sweets. Condiments such as sauces, processed tomato seasonings, flavor seasonings, cooking mixes, sauces, dressings, soups, curry and stew bases; processed oils and fats such as butter, margarine, mayonnaise; dairy products such as milk drinks, yogurts, lactic acid bacteria drinks, ice creams, and creams; processed seafood products such as fish ham and sausages and fish paste products; processed livestock products such as meat ham and sausages; processed agricultural products such as canned agricultural products, jams and marmalades, pickles, boiled beans, and cereals; frozen foods; and nutritional foods.

[0063] As used herein, "food and drink" includes categories such as health foods, functional foods, foods for specified health uses, nutritional supplements, foods labeled as reducing disease risk, and foods for medical patients. Furthermore, when used for mammals other than humans, the term "food and drink" can also be used to mean feed.

[0064] The food and drink compositions of the present invention may further contain ingredients with other functions. For example, by incorporating the active ingredient of the present invention into foods consumed in daily life, health foods, functional foods, and supplements (e.g., foods containing one or more minerals such as calcium and magnesium, or vitamins such as vitamin K), it is possible to provide foods and drinks that have the effects of the present invention as well as the functions based on the other ingredients.

[0065] <CD4 - CD8 - Methods for Expanding and / or Activating T Cells Another aspect of the invention is a method for producing a CD4 spore-forming bacterium comprising contacting peripheral blood mononuclear cells with spore-forming bacteria or a culture thereof. - CD8 - The spore-forming bacterium or a culture thereof according to the present invention is a method for expanding and / or activating T cells. - CD8 - T cells can be significantly expanded and / or activated.

[0066] The method for preparing and isolating peripheral blood mononuclear cells (PBMCs) is not particularly limited and can be performed according to a conventionally known method. Peripheral blood mononuclear cells can be isolated by removing plasma components, red blood cells, platelets, granulocytes, etc. from a blood sample collected from a subject, for example, by using density gradient centrifugation.

[0067] The subject from which peripheral blood mononuclear cells are derived is not particularly limited as long as it is a mammal. Here, mammals include both primates such as humans, monkeys, gorillas, chimpanzees, and orangutans, as well as non-human mammals such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, pigs, cows, horses, sheep, camels, and goats. Among these, the subject is preferably a primate, and more preferably a human.

[0068] In this embodiment, the "spore-forming bacteria or cultures thereof" are the same as those explained above, and therefore further explanation will be omitted.

[0069] The method for contacting peripheral blood mononuclear cells with spore-forming bacteria or a culture thereof is not particularly limited, and examples thereof include a method of culturing peripheral blood mononuclear cells and spore-forming bacteria or a culture thereof in a medium containing the cells. When culturing peripheral blood mononuclear cells and spore-forming bacteria or a culture thereof in a medium containing the cells, the culture conditions, such as the culture temperature and the culture time, can be adjusted to the known culture conditions for peripheral blood mononuclear cells.

[0070] In this embodiment, T cells isolated from a blood sample or peripheral blood mononuclear cells can be used instead of peripheral blood mononuclear cells. T cells can be isolated by conventionally known techniques.

[0071] One embodiment of this aspect is a CD4 T cell immunotherapy comprising contacting T cells with spore-forming bacteria or a culture thereof. - CD8 - A method for expanding and / or activating T cells.

[0072] Another aspect of the present invention is a method for preventing and treating CD4+ spores comprising administering to a subject an effective amount of spore-forming bacteria or a culture thereof. - CD8 - A method for expanding and / or activating T cells.

[0073] In this embodiment, the "spore-forming bacteria or cultures thereof" are the same as those described above, and therefore their explanation will be omitted. The "subject" is the same as the "subject from which peripheral blood mononuclear cells are derived" described above, and therefore their explanation will be omitted.

[0074] In this embodiment, "effective amount" refers to a CD4 - CD8 - It refers to the amount of active ingredient (i.e., spore-forming bacteria or a culture thereof) required to proliferate and / or activate T cells.

[0075] In this form, the spore-forming bacterium or a culture thereof can be administered orally, intravenously, intramuscularly, intrathecally, intraperitoneally, transdermally (for example, as a topical ointment), or by inhalation. Depending on each of these administration modes, the spore-forming bacterium or a culture thereof may be prepared as an oral or parenteral formulation in accordance with standard methods using additives acceptable for formulation. The additives acceptable for formulation are the same as those described above, and therefore further description will be omitted.

[0076] The following examples illustrate embodiments of the present invention. [1] CD4, containing spore-forming bacteria or their cultures as the active ingredient -CD8 - A composition for expanding and / or activating T cells. [2] The composition described in [1], wherein the spore-forming bacterium is at least one bacterium selected from the group consisting of bacteria of the genus Clostridium and Bacillus. [3] The spore-forming bacteria include Clostridium butyricum, Clostridium absonum, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium cadaveris, Clostridium clostridiiforme, Clostridium cochlearium, Clostridium difficile, Clostridium innocuum, Clostridium novyi, and Clostridium perfringens. perfringens, Clostridium putrificum, Clostridium ramosum, Clostridium rectum, Clostridium scatologenes, Clostridium sordellii, Clostridium sporogenes, Clostridium tertium, Clostridium tyrobutyricum, Clostridium aerotolerans, Clostridium aminophilum, Clostridium aminovalericum, Clostridium celerecrescences celerecrescens, Clostridium cerulosicellulosi, Clostridium coccoides, Clostridium hiranonis, Clostridium leptum, Clostridium nexile, Clostridium oroticum, Clostridium polysaccharolyticum, Clostridium populeti, Clostridium scindens, Clostridium sphenoides, Clostridium sporosphaeroides, Clostridium symbiosum symbiosum, Clostridium xylanolyticum, Clostridium hylemonae, Bacillus cereus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus licheniformis, Bacillus polyfermenticus, Bacillus pumilus, and Bacillus subtilis. [4] The composition described in [3], wherein the spore-forming bacterium is at least one bacterium selected from the group consisting of Clostridium butyricum, Clostridium sordellii, and Clostridium putrificum. [5] The composition according to any one of [1] to [4], wherein the spore-forming bacteria are in the form of spores. [6] CD4 - CD8 - The composition according to any one of [1] to [5], wherein the T cells include γδ T cells. [7] The composition described in [6], wherein the γδ T cells include Vγ9Vδ2 T cells. [8] The composition according to any one of [1] to [7], which is used in combination with an antitumor agent. [9] The composition according to any one of [1] to [8], which is a pharmaceutical composition.

[10] The composition according to any one of [1] to [7], which is a food or drink composition.

[11] A CD4 - CD8 - A method for expanding and / or activating T cells.

[12] A method for treating CD4 - CD8 - A method for expanding and / or activating T cells.

[13] CD4 - CD8 - A spore-forming bacterium or culture thereof used to grow and / or activate T cells. [Example]

[0077] The present invention will be described in detail below using examples, but the present invention is not limited to the following examples.

[0078] All of the following experiments were approved by the Ethics Committee of Kumamoto University. Peripheral blood mononuclear cells (PBMCs) separated from heparin-treated blood collected from healthy volunteers were used as human cell samples. All blood samples were collected from humans after informed consent was obtained and the blood donor's willingness confirmed. Patient samples were analyzed using coding that did not include personal information.

[0079] <Test Example 1> Using PBMCs derived from healthy individuals, we investigated whether stimulation with MiyaBM® tablets (manufactured by Miyarisan Pharmaceutical Co., Ltd.) induces the activation of specific T cell subsets. MiyaBM® tablets contain Clostridium butyricum MIYAIRI 588 (FERM BP-2789) (588 strain) as the active ingredient. Specifically, an AIM (Activation-Induced Marker) assay was performed, and analysis using the activation marker (CD137) as an indicator was performed by flow cytometry. 1 × 10 6PBMCs per well were suspended in 100 μL of RPMI 1640 medium (Thermo Fisher Scientific, Cat. No. 200-02) containing 10% FBS. One MiyaBM® tablet was dissolved in 3 mL of purified water and suspended. 3 μL, 10 μL, and 30 μL of the MiyaBM® suspension were added to each well. A well without MiyaBM® tablet stimulation served as a negative control. After pulsing, the cells were cultured for 24 hours at 37°C in 2.5% CO2. After culture, the PBMCs were washed and their surfaces were stained with antibodies. The antibodies used were CD3 FITC (UCHT1, 1:100 dilution, Biolegend), CD8 APCcy7 (RPA-T8, 1:100 dilution, Biolegend), CD14 PerCP / Cy5.5 (HCD14, 1:100 dilution, Biolegend), CD19 PerCP / Cy5.5 (HIB19, 1:10 dilution, Biolegend), CD25 PEcy7 (M-A251, 1:50 dilution, Biolegend), and CD137 APC (4B4-1, 1:50 dilution, Biolegend). 7-aminoactinomycin D (7-AAD, Biolegend, Cat No. 420404) was also added to stain for dead cells. After addition, the cells were incubated on ice for 20 minutes. After incubation, PBMCs were fixed with 1% paraformaldehyde (Nacalai Tesque, Cat. No. 09154-85). After fixation, the expression levels of surface molecules were measured by flow cytometry using a FACS Canto II (BD Biosciences). Data acquired by flow cytometry were analyzed using FACS Diva v9.0 (BD Biosciences) and FlowJo software v10 (Tree Star). The results are shown in Figures 1B and 1C. Figure 1B shows the results when MiyaBM® suspension (10 μL) was used. Figure 1C shows the results when MiyaBM® suspension (10 μL) was used (CBM588) and the control (No stimuli).

[0080] As shown in Figures 1B and 1C, stimulation with Miya BM® tablets increased CD4 - CD8 - Significant activation of T cells was observed, and CD4 - CD8 - Elevated activation markers of T cell subsets were observed.

[0081] <Test Example 2> Culture solutions or suspensions of strain 588, a component of MiyaBM, were prepared. The following culture solutions or suspensions of strain 588 were prepared: a suspension of spores without heat treatment, a suspension of spores with heat treatment, a culture solution of trophozoites without heat treatment, and a culture solution of trophozoites with heat treatment. Heat treatment was performed at 90°C for 90 minutes for spores and at 70°C for 30 minutes for trophozoites. The samples were added to PBMCs derived from the same healthy individuals as in Test Example 1, and an AIM assay was performed to verify whether subset activation similar to that in Test Example 1 could be achieved. Spores of strain 588 were prepared as follows: They were cultured on nutrient agar medium supplemented with 0.5% glucose and 0.3% calcium carbonate under anaerobic conditions at 37°C for 5 days, then centrifuged and washed with sterile water. They were then purified by density gradient centrifugation using 45% urografin (common name: amidotrizoic acid). A suspension of strain 588 spores was prepared by further centrifuging and washing with sterile water. In the following test examples, spores of 588 strains were similarly formed.

[0082] Specifically, an activation-induced marker (AIM) assay was performed, and analysis using activation markers (CD69, OX40, and CD137) as indicators was performed by flow cytometry. 6 PBMCs / well were prepared in 100 μL of RPMI 1640 medium (Thermo Fisher Scientific, Cat. No. 200-02) containing 10% FBS. 1 μL and 3 μL of the culture medium of the 588 strain were added to each well. The spore or trophozoite concentration in the culture medium was 1 × 10 7The concentration was 1000 cells / mL. An unstimulated well was placed as a negative control. After pulsing, the wells were cultured for 24 hours at 37°C and 2.5% CO2. After culture, the PBMCs were washed, and the surface was stained with the same antibody as in Test Example 1 and OX40 (Ber-ACT35, 50-fold diluted, Biolegend). 7-aminoactinomycin D (7-AAD, Biolegend, Cat No. 420404) was also added to stain dead cells. After addition, the wells were incubated on ice for 20 minutes. After incubation, the PBMCs were fixed with 1% paraformaldehyde (Nacalai Tesque, Cat No. 09154-85). After fixation, the expression levels of surface molecules were measured by flow cytometry using a FACS Canto II (BD Biosciences). Flow cytometry data were analyzed using FACS Diva v9.0 (BD Biosciences) and FlowJo software v10 (Tree Star). The results are shown in Figures 2A and 2B. Figure 2A shows the results when a suspension of spores (3 μL) without heat treatment was used. In Figure 2B, "No stimuli" indicates the control, "G" indicates the suspension of spores without heat treatment, "G inact" indicates the suspension of spores with heat treatment, "E" indicates the culture medium of trophozoites without heat treatment, and "E inact" indicates the culture medium of trophozoites with heat treatment.

[0083] As shown in Figure 2A, stimulation with 3 μL of unheat-treated spore suspension resulted in CD4 - CD8 - As shown in Figure 2B, the group stimulated with the culture medium of the 588 strain showed a significant increase in CD4 T cells compared with the group without stimulation. - CD8 - T cell activation was observed, and it was shown to be consistent with the response to MiyaBM tablets. In addition, when comparing the groups stimulated with the culture medium of strain 588, the group stimulated with the spore suspension showed a higher CD4 count than the group stimulated with trophozoites. - CD8 -T cell activation was observed. - CD8 - Regarding the effect on T cell activation, the non-heat-treated spore suspension showed a higher CD4 T cell count than the heat-treated group. - CD8 - In the culture medium of the trophozoites, there was a significant difference in CD4 T cells between the non-heat-treated and heat-treated groups. - CD8 - No effect on T cell activation was observed.

[0084] <Test Example 3> We attempted to identify immune subsets activated by spore stimulation of strain 588 using PBMCs derived from healthy individuals as an indicator of cell proliferation. Specifically, we performed a CTV (Cell Trace Violet) assay to label PBMCs with CTV dye, and evaluated cell proliferation by detecting the decrease in CTV dye concentration in response to cell division using flow cytometry.

[0085] 5 mM CTV / 20 μL DMSO from the Cell Trace Violet Cell Proliferation Kit (Thermo Fisher Scientific, Invitrogen, C34557) was diluted 5-fold with PBS to prepare a 1 mM CTV / μL solution. 10 mM CTV / 10 μL was diluted to 1 × 10 7 The PBMCs were suspended in 10 mL of PBS at a final concentration of 1 μM CTV / μL for labeling. 6 CTV-labeled PBMCs per well were suspended in 200 μL of RPMI 1640 medium (Thermo Fisher Scientific, Cat No. 200-02) containing 10% FBS. 7The cells were suspended in 100% PBS (2 μL / mL). Control wells included no stimulation and wells stimulated with anti-CD3-CD28 antibody (2 μL). After pulsing, the cells were cultured for 7 days at 37°C in 2.5% CO2. After culture, the PBMCs were washed and their surfaces were stained with antibodies: CD3 FITC (UCHT1, 1:100 dilution, Biolegend), CD8 APCcy7 (RPA-T8, 1:100 dilution, Biolegend), CD14 PerCP / Cy5.5 (HCD14, 1:100 dilution, Biolegend), and CD19 PerCP / Cy5.5 (HIB19, 1:10 dilution, Biolegend). 7-aminoactinomycin D (7-AAD, Biolegend, Cat. No. 420404) was also added to stain for dead cells. After addition, the mixture was incubated on ice for 20 minutes. After incubation, PBMCs were fixed with 1% paraformaldehyde (Nacalai Tesque, Cat. No. 09154-85). After fixation, the expression levels of surface molecules were measured by flow cytometry using Cytek Nothrem Lights (Cytec Japan). Data acquired by flow cytometry were analyzed using FlowJo software v10 (Tree Star). The results are shown in Figures 3A and 3B. In Figure 3A, "None" indicates no stimulation, "Anti-CD3 / CD28" indicates anti-CD3-CD28 antibody, and "Spores" indicates a spore suspension without heat treatment. In Figure 3B, "None" indicates no stimulation, "CD3" indicates anti-CD3-CD28 antibody, and "Spores" indicates a spore suspension without heat treatment.

[0086] As shown in Figures 3A and 3B, the group stimulated with the suspension of unheat-treated spores showed a significant increase in CD4 expression compared with the unstimulated group and the group stimulated with anti-CD3-CD28 antibody. - CD8 - A significant proliferation of T cells was observed.

[0087] <Test Example 4> CD4 cells significantly proliferated under spore stimulation using PBMCs derived from healthy individuals- CD8 - We attempted to identify the subsets that make up T cells.

[0088] 5 mM CTV / 20 μL DMSO from the Cell Trace Violet Cell Proliferation Kit (Thermo Fisher Scientific, Invitrogen, C34557) was diluted 5-fold with PBS to prepare a 1 mM CTV / μL solution. 10 mM CTV / 10 μL was diluted to 1 × 10 7 The PBMCs were suspended in 10 mL of PBS at a final concentration of 1 μM CTV / μL for labeling. 6 CTV-labeled PBMCs per well were suspended in 200 μL of RPMI 1640 medium (Thermo Fisher Scientific, Cat No. 200-02) containing 10% FBS. 7 cells / mL) or a suspension of heat-treated spores (spore concentration: 1 × 10 7The cells were suspended in 100% PBS (2000 cells / mL). Control wells were left unstimulated, and wells were stimulated with anti-CD3-CD28 antibody (2 μL). After pulsing, the cells were cultured for 7 days at 37°C in 2.5% CO2. After culture, the PBMCs were washed and their surfaces were stained with antibodies. The antibodies used were CD3 BV785 (50x dilution, Biolegend), CD4 BV750 (100x dilution, Biolegend), CD8 BV570 (100x dilution, Biolegend), TCRγδ FITC (100x dilution, Milteny Biotec), TCRab PerCP / Cy5.5 (100x dilution, Biolegend), Vg9 APCvio770 (50x dilution, Invitrogen), Vδ1 PE-eFlour610 (50x dilution, Invitrogen), and Vδ2 PE (100x dilution, Milteny Biotec). 7-aminoactinomycin D (7-AAD, Biolegend, Cat No. 420404) was also added to stain dead cells. After addition, the mixture was incubated on ice for 20 minutes. After incubation, PBMCs were fixed with 1% paraformaldehyde (Nacalai Tesque, Cat. No. 09154-85). After fixation, the expression levels of surface molecules were measured by flow cytometry using Cytek Nothrem Lights (Cytec Japan). Data acquired by flow cytometry were analyzed using FlowJo software v10 (Tree Star). The results are shown in Figure 4B and Figure 4C. In Figure 4B, "None" indicates no stimulation, "Anti-CD3 / CD28" indicates anti-CD3-CD28 antibody, "Spore Heat-inact" indicates heat-treated spore suspension, and "Spore" indicates heat-untreated spore suspension. In Figure 4C, "None" indicates no stimulation, "CD3" indicates anti-CD3-CD28 antibody, "CBM588 Heat-inact" indicates a spore suspension with heat treatment, and "CBM588" indicates a spore suspension without heat treatment.

[0089] As shown in Figure 4B and Figure 4C, the groups stimulated with the spore suspension, regardless of heat treatment, showed significantly higher CD4 - CD8 - A significant increase in T cells was observed, and the group stimulated with a suspension of unheated spores expanded Vγ9Vδ2 T cells, which are known to have anti-tumor effects.

[0090] <Test Example 5> 5 mM CTV / 20 μL DMSO from the Cell Trace Violet Cell Proliferation Kit (Thermo Fisher Scientific, Invitrogen, C34557) was diluted 5-fold with PBS to prepare a 1 mM CTV / μL solution. 10 mM CTV / 10 μL was diluted to 3 × 10 7 The cells were suspended in 10 mL of PBS dropwise to a final concentration of 1 μM CTV / μL for labeling. 6 The CTV-labeled PBMCs per well were suspended in 200 μL of RPMI 1640 medium (Thermo Fisher Scientific, Cat. No. 200-02) containing 10% FBS. A spore suspension of strain 588 (spore concentration: 3 × 10 5 cells / mL, 3 × 10 6 cells / mL or 3 x 10 7 cells / mL), a spore suspension of Clostridium sordellii JCM3814 strain (spore concentration: 3 × 10 5 cells / mL or 3 x 10 6 cells / mL), or a suspension of Clostridium putrificum ATCC 25784 spores (spore concentration: 3 × 10 7Spores were added to the wells at a concentration of 1000 spores / mL and suspended in the medium. The spores in the suspension were not heat-treated. A control well was left unstimulated. After pulsing, the wells were cultured at 37°C in 2.5% CO2 for 12 days. After culture, the PBMCs were washed and their surfaces were stained with antibodies. The antibodies used were CD3 BV785 (50x dilution, Biolegend), CD4 BV750 (100x dilution, Biolegend), CD8 BV570 (100x dilution, Biolegend), TCRγδ FITC (100x dilution, Milteny Biotec), TCRab PerCP / Cy5.5 (100x dilution, Biolegend), Vg9 APCvio770 (50x dilution, Invitrogen), Vδ1 PE-eFlour610 (50x dilution, Invitrogen), and Vδ2 PE (100x dilution, Milteny Biotec). 7-aminoactinomycin D (7-AAD, Biolegend, Cat No. 420404) was also added to stain dead cells. After addition, the mixture was incubated on ice for 20 minutes. After incubation, PBMCs were fixed with 1% paraformaldehyde (Nacalai Tesque, Cat. No. 09154-85). After fixation, the expression levels of surface molecules were measured by flow cytometry using Cytek Nothrem Lights (Cytec Japan). Flow cytometry data were analyzed using FlowJo software v10 (Tree Star). The results are shown in Figure 5B. In Figure 5B, "CBM588 Spore" represents the spore suspension of strain 588, "Paeniclostridium sordellii" represents the spore suspension of Clostridium sordellii JCM3814, and "Clostridium putrificum" represents the spore suspension of Clostridium putrificum ATCC25784.

[0091] As shown in Figure 5, the groups stimulated with a spore suspension of Clostridium sordellii JCM3814 strain and Clostridium putrificum ATCC25784 strain proliferated Vγ9Vδ2 T cells, which are known to have antitumor effects, similar to the group stimulated with a spore suspension of 588 strain.

Claims

1. A CD4 containing spores of spore-forming bacteria without heat treatment as an active ingredient. － CD8 － A composition for expanding and / or activating T cells.

2. 2. The composition of claim 1, wherein the spore-forming bacterium is at least one bacterium selected from the group consisting of bacteria of the genus Clostridium and Bacillus.

3. The spore-forming bacteria include Clostridium butyricum, Clostridium absonum, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium cadaveris, Clostridium clostridiiforme, Clostridium cochlearium, and Clostridium difficile. difficile), Clostridium innocuum, Clostridium novyi, Clostridium perfringens, Clostridium putrificum, Clostridium ramosum, Clostridium rectum, Clostridium scatologenes, Clostridium sordellii, Clostridium sporogenes sporogenes), Clostridium tertium, Clostridium tyrobutyricum, Clostridium aerotolerans, Clostridium aminophilum, Clostridium aminovalericum, Clostridium celerecrescens, Clostridium cellulosicellulosi, Clostridium coccoides, Clostridium hiranonis, Clostridium leptum, Clostridium nexile, Clostridium oroticum, Clostridium polysaccharolyticum, Clostridium populeti, Clostridium scindens scindens), Clostridium sphenoides, Clostridium sporosphaeroides, Clostridium symbiosum, Clostridium xylanolyticum, Clostridium hylemonae, Bacillus cereus, Bacillus circulans, Bacillus clausii 2. The composition of claim 1, wherein the bacterium is at least one bacterium selected from the group consisting of Bacillus clausii, Bacillus coagulans, Bacillus licheniformis, Bacillus polyfermenticus, Bacillus pumilus, and Bacillus subtilis.

4. 2. The composition of claim 1, wherein the spore-forming bacterium is at least one bacterium selected from the group consisting of Clostridium butyricum, Clostridium sordellii, and Clostridium putrificum.

5. The CD4 － CD8 － The composition of claim 1 , wherein the T cells comprise γδ T cells.

6. The composition of claim 5, wherein the γδ T cells comprise Vγ9Vδ2 T cells.

7. The composition of claim 1 used in combination with an anti-tumor agent.

8. The composition of claim 1 which is a pharmaceutical composition.

9. The composition according to claim 1, which is a food or beverage composition.

10. a method for detecting CD4+ spores comprising contacting peripheral blood mononuclear cells with spores of a spore-forming bacterium without heat treatment; － CD8 － A method for expanding and / or activating T cells.

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