Composition for the proliferation and / or activation of CD4-CD8-T cells

Abstract

This invention provides a method for enabling CD4 ‑ CD8 ‑ Novel compositions for T cell proliferation and / or activation. This invention relates to a composition for inducing CD4 cell proliferation and / or activation. ‑ CD8 ‑ Compositions for T cell proliferation and / or activation, comprising spore-forming bacteria or their cultures as active ingredients.

Description

Technical Field

[0001] This invention relates to a method for enabling CD4 - CD8 - Composition for T cell proliferation and / or activation. Background Technology

[0002] T cells are a type of lymphocyte that express T cell receptors on their cell surface. Most T cells express either CD4 or CD8. However, some T cells do not express either CD4 or CD8. - CD8 - (Double-negative) T cells.

[0003] CD4 was reported - CD8 - The association between T cells and anti-tumor efficacy. Non-patent literature 1 reports CD4... - CD8 - T cells play a vital role in immune homeostasis in healthy individuals, functioning as Treg cells, cytotoxic T cells, or Th cells. They influence both the innate and adaptive immune systems in well-defined pathologies closely related to inflammatory diseases, autoimmune diseases, tumorigenesis, and tumorigenesis. Additionally, Non-Patent Literature 2 reports CD4... - CD8 - T cells may become a potential target for cancer immunotherapy.

[0004] Existing technical documents

[0005] Non-patent literature

[0006] Non-patent literature 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, ImmuneDisorders and Cancer. Front. Immunol. 13:816005. doi: 10.3389 / fimmu.2022.816005

[0007] Non-patent literature 2: Okamura K, Nagayama S, Tate T, Kiyotani K, and YusukeNakamura (2020) The potential target of double negative T cells in cancerimmunotherapy, Journal of Clinical Oncology 38:15_suppl, e15180 Summary of the Invention

[0008] The purpose of this invention is to provide a method for enabling CD4 - CD8 - Novel compositions for T cell proliferation and / or activation.

[0009] The inventors have surprisingly discovered that spore-forming bacteria can induce CD4+ spore formation. - CD8 - T cell proliferation and / or activation. Based on this insight, the present invention was completed.

[0010] That is, according to one aspect of the present invention, a method for making CD4 - CD8 - Compositions for T cell proliferation and / or activation, comprising spore-forming bacteria or their cultures as active ingredients. Attached Figure Description

[0011] Figure 1 This section outlines the experiment and its results for Experiment Example 1. A: Schematic diagram of the experiment; B: Scatter plot showing the results of flow cytometry using Miya BM (registered trademark) suspension; C: Chart showing the results of flow cytometry for Experiment Example 1.

[0012] Figure 2 The results of Experiment Example 2 are shown below. A: A scatter plot showing the results of flow cytometry using a heat-free spore suspension; B: A graph showing the results of flow cytometry.

[0013] Figure 3 The results of Experiment Example 3 are shown below. A: Scatter plot showing the results of flow cytometry; B: Chart showing the results of flow cytometry.

[0014] Figure 4 This section summarizes the experiment and its results for Example 4. A: Schematic diagram of the experiment; B: Scatter plot showing the results of flow cytometry; C: Chart showing the results of flow cytometry.

[0015] Figure 5This section summarizes the experiment and its results for Example 5. A: Schematic diagram of the experiment; B: Scatter plot showing the results of flow cytometry. Detailed Implementation

[0016] The following describes one embodiment of the present invention. The present invention is not limited to the following embodiment.

[0017] In this specification, the range "X~Y" means "above X and below Y". Furthermore, unless otherwise specified, the operation and physical property measurements are performed at room temperature (20~25°C) and relative humidity 40~50%RH. In this specification, "A and / or B" means at least one of A and B, including both A and B, or either A or B.

[0018] <used to make CD4 - CD8 - Compositions for T cell proliferation and / or activation >

[0019] One aspect of the present invention is a method for enabling CD4 - CD8 - A composition for T cell proliferation and / or activation, comprising spore-forming bacteria or a culture thereof as an active ingredient. According to one aspect of the invention, a composition for CD4… - CD8 - Novel compositions for T cell proliferation and / or activation.

[0020] This manual will also be used to enable CD4 - CD8 - Compositions for T cell proliferation and / or activation are referred to as "the compositions of the present invention".

[0021] Spore-forming bacteria are bacteria that form spores that exhibit extremely high durability against physical and chemical treatments. Spore formation can be confirmed by methods such as microscopic observation, staining properties based on spore staining, heat resistance based on survival after heating, and resistance to organic solvents or disinfectants based on survival after exposure.

[0022] In the compositions of the present invention, there are no particular limitations on the types of spore-forming bacteria. Examples of spore-forming bacteria include bacteria of the genera *Clostridium*, *Bacillus*, *Amphibacillus*, and *Sporosarcina*.

[0023] In one embodiment, the spore-forming bacterium is selected from at least one bacterium selected from the group consisting of bacteria of the genera Clostridium and Bacillus.

[0024] Examples of bacteria belonging to the genus *Clostridium* include *Clostridium butyricum*, *Clostridium absonum*, *Clostridium acetobutylicum*, *Clostridium beijerinckii*, *Clostridium cadaveris*, *Clostridium clostridiiforme*, *Clostridium cochlearium*, *Clostridium difficile*, *Clostridium innocuum*, *Clostridium novyi*, *Clostridium perfringens*, *Clostridium putrificum*, *Clostridium ramosum*, *Clostridium rectum*, and *Clostridium foetida*. Clostridium scatologenes, Clostridium sordellii, Clostridium sporogenes, Clostridium tertium, Clostridium tyrobutyricum, Clostridium aerotolerans, Clostridium aminophilum, Clostridium aminovalericum, Clostridium celerecrescens, Clostridium cellulosi, Clostridium coccoides, Clostridium hiranonis, Clostridium leptum, Clostridium nexile, Clostridium oroticum, Clostridium polysaccharide-degrading bacteria *Clostridium polysaccharolyticum*, *Clostridium populeti*, *Clostridium scindens*, *Clostridium wedge-shaped*Clostridium sphenoides, Clostridium sporosphaeroides, Clostridium symbiosum, Clostridium xylanolyticum, Clostridium hylemonae, etc.

[0025] Examples of bacteria belonging to the genus Bacillus include Bacillus cereus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus licheniformis, Bacillus polyfermenticus, Bacillus pumilus, and Bacillus subtilis.

[0026] These bacteria can be obtained from organizations such as the Technical Base for Product Evaluation (NITE), the JCM Laboratory for Microbial Materials Development at RIKEN, the National BioResource Project (NBRP), the American Type Culture Collection (ATCC), and the German National Culture Collection (DSMZ).

[0027] The spore-forming bacteria are preferably bacteria 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, or Clostridium putrificum ATCC25784. Clostridium butyricum MIYAIRI 588 (FERM BP-2789) was deposited as FERM BP-2789 on May 1, 1981, at the Institute of Microbial Industrial Technology, Industrial Technology Research Institute, Ministry of International Trade and Industry (now the Patent Biological Collection Center, Technical Base for Product Evaluation, an independent administrative agency) (Room 120, 2-5-8 Kazusa-Kamazutsu, Kisarazu City, Chiba Prefecture, Japan 292-0818), and as accession number FERM BP-2789 on March 6, 1990, at an international depository based on the Budapest Treaty.

[0028] Spore-forming bacteria can be used alone or in combination of two or more.

[0029] The spore-forming bacteria can be in the form of spores or vegetative bodies. From the viewpoint of further maximizing the effects of the present invention, the spore-forming bacteria are preferably in the form of spores. The spore-forming bacteria included in the composition of the present invention may be only spores, only vegetative bodies, or both spores and vegetative bodies.

[0030] There are no particular restrictions on the method of spore formation; previously known methods can be used depending on the spore-forming fungus used.

[0031] In this invention, the culture of spore-forming bacteria refers to a culture medium in which spore-forming bacteria are cultured, the supernatant obtained by centrifuging the culture medium, the residue containing spore-forming bacteria obtained by centrifuging the culture medium, the purified product derived from the residue, and the dried product of the residue.

[0032] Purified products derived from residues can be obtained from the components that are the target components using methods that are known in the past.

[0033] The dried residue can be obtained by air-drying or vacuum drying the residue containing bacteria obtained by centrifuging the culture medium.

[0034] The culture of the spore-forming bacteria of the present invention can be obtained from the spore-forming bacteria using conventionally known culture methods. For example, the culture medium used in culturing the spore-forming bacteria can be any type of solid or liquid culture medium. Furthermore, as long as the culture medium contains a carbon source that the spore-forming bacteria can assimilate, an appropriate amount of nitrogen source, inorganic salts, and other nutrients, it can be any type of synthetic or natural culture medium. Typically, the culture medium contains a carbon source, a nitrogen source, and inorganic substances.

[0035] The carbon source that can be used in the cultivation of the spore-forming bacteria of the present invention is not particularly limited as long as it is a carbon source that the strain used can assimilate. Specifically, considering the assimilation of the spore-forming bacteria, examples include organic acids such as gluconic acid, decanoic acid, adipic acid, malic acid, citric acid, phenyl acetate, acetic acid, lactic acid, succinic acid, glucuronic acid, and pyruvic acid, as well as their salts; hydrocarbons such as hexadecane; natural products such as wheat and rice; alcohols such as glycerol, methanol, and ethanol; and sugars. The above-mentioned carbon sources are appropriately selected considering the assimilation of the cultured spore-forming bacteria. One or more of the above-mentioned carbon sources can be used.

[0036] Examples of nitrogen sources that can be used in the cultivation of the spore-forming bacteria of this invention include organic nitrogen sources such as meat extracts, fish extracts, peptone, polypeptone, tryptone, yeast extracts, malt extracts, soybean hydrolysate, soybean powder, casein, lactic acid, casein amino acids, glycine, glutamic acid, aspartic acid, and other amino acids, corn steep liquor, and hydrolysates of other animals, plants, and microorganisms; and inorganic nitrogen sources such as ammonium salts (ammonium nitrate, ammonium sulfate, ammonium chloride, etc.), nitrates (sodium nitrate, etc.), nitrites (sodium nitrite, etc.), and urea. The nitrogen sources are appropriately selected considering the assimilation properties of the cultured spore-forming bacteria. One or more of the above nitrogen sources may be used.

[0037] Examples of inorganic substances that can be used in the cultivation of the spore-forming bacteria of the present invention include phosphates, hydrochlorides, sulfates, acetates, carbonates, chlorides, and halides of magnesium, manganese, calcium, sodium, potassium, copper, iron, and zinc. The inorganic substances are appropriately selected considering the assimilation properties of the cultured spore-forming bacteria. Furthermore, one or more of the aforementioned inorganic substances may be used. Additionally, surfactants may be added to the culture medium as needed.

[0038] The culturing of the spore-forming bacteria of the present invention can be carried out by conventional methods. For example, depending on the type of spore-forming bacteria, the bacteria can be cultured under aerobic or anaerobic conditions. In the former case, the culturing of the spore-forming bacteria is carried out by shaking or aeration. Furthermore, the spore-forming bacteria can be cultured continuously or in batches. The culture conditions are appropriately selected based on the composition of the culture medium and the culture method; there are no particular limitations as long as the conditions allow the spore-forming bacteria of the present invention to proliferate, and the conditions can be appropriately selected according to the type of spore-forming bacteria being cultured. The culture temperature is typically 20–42°C, preferably 35–40°C. Furthermore, the pH of the suitable culture medium is not particularly limited, but is preferably 5–11, more preferably 6–10. Moreover, the culture time is not particularly limited and varies depending on the type of spore-forming bacteria being cultured, the amount of culture medium, and the culture conditions. Generally, the culture is carried out to the exponential growth phase in the case of vegetative cells, and for example, 16 hours to 7 days in the case of spores.

[0039] As an example of a culture of spore-forming bacteria of the present invention, the method for preparing a culture of Clostridium butyricum MIYAIRI 588 (FERM BP-2789) (hereinafter also referred to as strain 588) will be described.

[0040] The method for preparing the culture medium as a nutrient medium can be the method described in Japanese Patent Application Publication No. 08-252088.

[0041] Specifically, in a medium containing 1.0% peptone, 1.5% yeast extract, and 3% glucose (PYD medium), 588 strains were selected as 10. 6 Inoculate at a rate of / mL, and stir slowly at 27℃ and 20rpm. Using a pH controller, maintain the pH range of 5.5±0.2 with 5% sodium hydroxide aqueous solution while culturing to the early stage of exponential growth. This yields a culture medium containing 588 vegetative cells.

[0042] The method for preparing the spore suspension can be 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).

[0043] Specifically, using ordinary agar medium supplemented with 0.5% glucose and 0.3% calcium carbonate, after culturing at 37°C under anaerobic conditions for 5 days, the spores were suspended in liquids such as the culture medium to obtain a suspension of 588 spores. It should be noted that alternatively, the spores could be suspended in sterile water instead of the culture medium, then washed by centrifugation with sterile water, purified using density gradient centrifugation with 45% urografin (common name: diatrizoate), and then washed by centrifugation with sterile water to obtain a purified spore suspension of 588 spores without vegetative cells.

[0044] In addition, the method described in Japanese Patent Application Publication No. 11-042081 can be used as a method for preparing the spore suspension.

[0045] Specifically, 588 spores were inoculated into 10 ml of CS liquid medium containing 2.0% corn starch, 2.0% amino acid solution, and 0.75% calcium carbonate. The medium was then anaerobically cultured at 35–37°C using the carbon dioxide-replacement steel wool method for 16–24 hours to prepare a pre-culture solution. After the specified incubation period, 0.01–1 ml of this pre-culture solution was inoculated into 10 ml of the main CS liquid medium at 60–100°C. The CS liquid medium was then cooled to 30–40°C with water and anaerobically cultured at 35–37°C using the carbon dioxide-replacement steel wool method for 40–48 hours. This yielded a suspension of 588 spores.

[0046] Next, the obtained culture medium is centrifuged (2,000~6,000g × 10~30 minutes) to separate the "residue containing bacteria obtained from centrifugation of the culture medium". This residue is then dried by air drying 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 (7Pa~66.7kPa), preferably 1~100 Torr (133Pa~13.3kPa), for 1~24 hours, preferably 2~15 hours, to obtain the "dried residue". To obtain the dried residue, spray drying, freeze drying, etc., can be used.

[0047] The spore-forming bacteria or their cultures of the present invention can generate CD4 - CD8 - T cell proliferation and / or activation.

[0048] In this specification, CD4 - CD8 - T cell proliferation can be confirmed by CTV (Cell trace violet) analysis. CD4 - CD8- T cell activation can be confirmed by AIM (Activation-Induced Marker) analysis.

[0049] In this specification, CD4 - CD8 - T cells refer to CD4-negative and CD8-negative T cells. From the viewpoint of further enhancing the effects of the present invention, a preferred embodiment is CD4-negative T cells. - CD8 - T cells include γδT cells, and γδT cells include Vγ9Vδ2T cells.

[0050] The compositions of the present invention comprise an amount (i.e., an effective amount) of the spore-forming fungus or its culture thereof sufficient to achieve the desired effect. The compositions of the present invention may be the spore-forming fungus or its culture itself, or may further comprise other ingredients. Examples of other ingredients include those described in the pharmaceutical and food / drink compositions described later.

[0051] In one embodiment, the composition of the present invention is used in combination with an antitumor agent.

[0052] In this specification, "combination use" refers to simultaneously administering the composition of the present invention and the antitumor agent, or administering the composition of the present invention and the antitumor agent sequentially at prescribed intervals during treatment (combination therapy). The route and means of administration of the composition of the present invention and the antitumor agent may be the same or different.

[0053] As mentioned above, CD4 was reported. - CD8 - The association between T cells and antitumor efficacy. By combining the compositions of the present invention with antitumor agents, it is expected that the efficacy of the antitumor agents can be further enhanced.

[0054] There are no particular limitations on antitumor agents; examples 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, epigenetic-related molecule inhibitors, protein post-translational modification inhibitors, and proteasome inhibitors. Tyrosine kinase inhibitors or immune checkpoint inhibitors are preferred antitumor agents.

[0055] Tyrosine kinase inhibitors are drugs that exhibit anti-tumor effects by inhibiting tyrosine kinases, enzymes involved in various cellular functions such as cell signaling, growth, and division.

[0056] There are no particular limitations on the tyrosine kinase inhibitor; conventionally known tyrosine kinase inhibitors can be used. Preferably, the tyrosine kinase inhibitor is selected from at least one of the group consisting of EGFR tyrosine kinase inhibitors, c-kit receptor tyrosine kinase inhibitors, BCR-ABL tyrosine kinase inhibitors, and multi-kinase inhibitors, and more preferably, multi-kinase inhibitors.

[0057] Specific examples of tyrosine kinase inhibitors include imatinib, dasatinib, nilotinib, afatinib, erlotinib, osimertinib, gefitinib, axitinib, cabozantinib, sunitinib, sorafenib, pazopanib, regorafenib, and lenvatinib.

[0058] Immune checkpoint inhibitors are substances that inhibit the action of immune checkpoints, such as receptors or ligands. Examples include antagonists of inhibitory receptors and agonists of co-stimulatory immune checkpoint receptors.

[0059] The term "antagonist" includes various substances that prevent receptor activation through the binding of receptors to ligands. Examples include substances that bind to receptors and prevent receptor-ligand binding, and substances that bind to ligands and prevent receptor-ligand binding.

[0060] Examples of antagonists targeting inhibitory immune checkpoints include antagonistic antibodies that bind to inhibitory immune checkpoint molecules (inhibitory receptors or ligands of such receptors), soluble peptides designed based on inhibitory immune checkpoint ligands that do not activate the receptor, or vectors capable of expressing such peptides.

[0061] There are no particular limitations on the immune checkpoint inhibitors used; conventionally known immune checkpoint inhibitors can be used. Preferably, the immune checkpoint inhibitor is selected from at least one of the group consisting of PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors, and more preferably, a PD-1 inhibitor.

[0062] There are no particular limitations on the specific examples of immune checkpoint inhibitors, but they are selected from at least one of the following groups: nivolumab, ipilimumab, pembrolizumab, cimiprimab, durvalumab, daclizumab, avelumab, and atezolizumab, more preferably from at least one of the following groups: nivolumab, pembrolizumab, and cimiprimab.

[0063] The dosage and method of administration of the compositions and antitumor agents of the present invention can be appropriately determined taking into account the type of tumor, the symptoms, age, sex, weight and condition of the subject.

[0064] In one embodiment, the composition of the present invention is a pharmaceutical composition. As described above, CD4 has been reported. -CD8 - The association between T cells and anti-tumor effects can be used in pharmaceutical applications.

[0065] The pharmaceutical compositions of the present invention contain an amount (i.e., an effective amount) of the spore-forming bacteria or a culture thereof of the present invention sufficient to exert the desired effect.

[0066] The pharmaceutical compositions of the present invention may be the spore-forming bacteria of the present invention or their cultures themselves, or may be combined with additives permitted for formulation, and prepared into oral or non-oral formulations by conventional methods. Examples of additives permitted for formulation include excipients, stabilizers, preservatives, wetting agents, emulsifiers, lubricants, sweeteners, colorants, flavorings, buffers, antioxidants, pH adjusters, binders, thickeners, dispersants, suspending agents, disintegrants, antibacterial agents, and surfactants. Dosage forms are not particularly limited; appropriate formulations are acceptable. Examples include tablets, powders, granules, capsules, pills, sustained-release formulations, solutions, suspensions, emulsions, lotions, injections, drops, external applications, suppositories, and patches.

[0067] In one embodiment, the composition of the present invention is a food or beverage composition.

[0068] The food and beverage products can be prepared by adding conventional additives such as stabilizers to the composition of the present invention, prepared by further mixing various proteins, sugars, fats, trace elements, vitamins, etc., made into liquid, semi-liquid or solid, made into paste, or made by adding the composition of the present invention to ordinary food and beverage products.

[0069] In this specification, "food and drink products" refers to anything other than medicine, and there are no particular restrictions on the form in which it can be ingested orally by mammals or other organisms. Its form can be any of the following: liquid (solution, suspension, emulsion, etc.), semi-liquid, powder, or solid molded product. Therefore, food and drink products can be, for example, in the form of beverages, or in the form of tablets, such as nutritional supplements.

[0070] As food and beverage products, specifically, examples include instant noodles, steamed or boiled foods, canned goods, microwaveable foods, instant soups (such as miso soup), freeze-dried foods, and other ready-to-eat foods; beverages such as soft drinks, fruit juices, vegetable drinks, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, nutritional drinks, and alcoholic beverages; wheat flour products such as bread, pasta, noodles, cake mix, fried chicken mix, and bread flour; and sugar, caramel, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, and crackers. (er) Japanese sweets, desserts and other snacks; sauces, tomato-based seasonings, flavored seasonings, cooking mixes, condiments, salad dressings, soups, curry and Japanese stew seasonings and other condiments; processed oils, butter, margarine, mayonnaise and other oils; dairy products, yogurt, lactic acid bacteria beverages, ice cream, cream and other dairy products; processed aquatic products, such as fish, ham and sausages, and aquatic porridge products; processed livestock products, such as ham and sausages; processed agricultural products, such as canned agricultural products, jams, marmalade, pickles, boiled beans, and grain products; frozen foods; nutritional foods, etc.

[0071] In this specification, "food and beverage products" also includes categories such as health foods, functional foods, foods for specific health purposes, nutritional supplements, foods labeled as reducing disease risk, or foods for patients. Furthermore, when the term "food and beverage products" is used for mammals other than humans, it can be used to mean that it includes animal feed.

[0072] In the food and beverage composition of the present invention, ingredients with other functions may be further added. Additionally, by incorporating the active ingredients of the present invention into everyday foods, health foods, functional foods, or supplements (e.g., foods containing one or more minerals such as calcium and magnesium, or vitamins such as vitamin K), food and beverage compositions that, in addition to the effects of the present invention, also possess functions based on other ingredients can be provided.

[0073] <used to make CD4 - CD8 - Methods for T cell proliferation and / or activation >

[0074] Another aspect of the invention is for enabling CD4 - CD8 - A method for T cell proliferation and / or activation, comprising: contacting peripheral blood mononuclear cells with a spore-forming bacterium or a culture thereof. The spore-forming bacterium or its culture of the present invention can induce CD4+ proliferation in peripheral blood mononuclear cells. - CD8 - T cells proliferate significantly and / or become activated.

[0075] There are no particular restrictions on the preparation and isolation methods of peripheral blood mononuclear cells (PBMCs), and they can be prepared using previously known methods. For example, PBMCs can be isolated from blood samples collected from subjects by using density gradient centrifugation to remove plasma components, red blood cells, platelets, granulocytes, etc.

[0076] There are no particular restrictions on the source of peripheral blood mononuclear cells as long as they are from mammals. Mammals include 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, cattle, horses, sheep, camels, and goats. Primates are preferred, and humans are more preferred.

[0077] In this method, "spore-forming bacteria or their cultures" are the same as those described above, so the description is omitted.

[0078] There are no particular limitations on the method of contacting peripheral blood mononuclear cells with spore-forming bacteria or their cultures. For example, methods such as culturing in a medium containing peripheral blood mononuclear cells and spore-forming bacteria or their cultures can be cited. When culturing in a medium containing peripheral blood mononuclear cells and spore-forming bacteria or their cultures, the culture conditions such as culture temperature and culture time can be determined according to known culture conditions for peripheral blood mononuclear cells.

[0079] In this method, blood samples or T cells isolated from peripheral blood mononuclear cells can be used instead of peripheral blood mononuclear cells. T cell isolation can be performed using methods already known.

[0080] One implementation of this method is used to enable CD4 - CD8 - Methods for T cell proliferation and / or activation include contacting T cells with spore-forming bacteria or their cultures.

[0081] Another aspect of the invention is for enabling CD4 - CD8 - Methods for T cell proliferation and / or activation include administering an effective amount of a spore-forming bacterium or a culture thereof to a subject.

[0082] In this method, "spore-forming bacteria or their cultures" is the same as described above, so the description is omitted. "Subject" is the same as "subject derived from peripheral blood mononuclear cells" described above, so the description is omitted.

[0083] In this approach, "effective quantity" refers to the amount of CD4 that makes CD4 effective. - CD8 -The amount of active ingredient (i.e., spore-forming bacteria or their cultures) required for T cell proliferation and / or activation.

[0084] In this method, spore-forming bacteria or their cultures can be administered orally, intravenously, intramuscularly, intramuscularly, intraperitoneally, transdermally (e.g., as a topical application), or by inhalation. Spore-forming bacteria or their cultures can be prepared into oral or non-oral formulations according to conventional methods, in combination with permitted additives for formulation. As for permitted additives for formulation, the same matters are described above, and therefore, descriptions are omitted.

[0085] The following are examples of embodiments of the present invention.

[0086] [1] A method for enabling CD4 - CD8 - Compositions for T cell proliferation and / or activation, comprising spore-forming bacteria or their cultures as active ingredients.

[0087] [2] According to the composition of [1], wherein the spore-forming bacterium is at least one bacterium selected from the group consisting of bacteria of the genera Clostridium and Bacillus.

[0088] [3] According to the composition described in [1] or [2], wherein the spore-forming bacterium is selected from Clostridium butyricum, Clostridium absonum, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium cadaveris, Clostridium clostridiiforme, Clostridium cochlearium, Clostridium difficile, Clostridium innocuum, Clostridium novyi, Clostridium perfringens, Clostridium putrificum, Clostridium ramosum, Clostridium rectum, and Clostridium scutellarioides. Clostridium scatologenes, Clostridium sordellii, Clostridium sporogenes, Clostridium tertium, Clostridium tyrobutyricum, Clostridium aerorotolerans, Clostridium aminophilum, Clostridium aminovalericum, Clostridium celerecrescens, Clostridium cellulosi, Clostridium coccoides, Clostridium hiranonis, Clostridium leptum, Clostridium nexile, Clostridium oroticum, Clostridium polysaccharolyticum, Clostridium populeti, Clostridium lysateThe bacteria are defined as at least one species from the group consisting of *Clostridium scindens*, *Clostridium sphenoides*, *Clostridium sporosphaeroides*, *Clostridium symbiosum*, *Clostridium xylanolyticum*, *Clostridium hylemonae*, *Bacillus cereus*, *Bacillus circulans*, *Bacillus clausii*, *Bacillus coagulans*, *Bacillus licheniformis*, *Bacillus polyfermenticus*, *Bacillus pumilus*, and *Bacillus subtilis*.

[0089] [4] According to the composition of [3], wherein the spore-forming bacterium is at least one bacterium selected from the group consisting of Clostridium butyricum, Clostridium sordellii and Clostridium putrificum.

[0090] [5] The composition according to any one of [1] to [4], wherein the spore-forming fungus is in the form of a spore.

[0091] [6] The composition according to any one of [1] to [5], wherein the CD4 - CD8 - T cells include γδT cells.

[0092] [7] The composition according to [6], wherein the γδT cells comprise Vγ9Vδ2T cells.

[0093] [8] The composition according to any one of [1] to [7] is used in combination with an antitumor agent.

[0094] [9] The composition according to any one of [1] to [8] is a pharmaceutical composition.

[0095]

[10] The composition according to any one of [1] to [7] is a food and beverage composition.

[0096]

[11] A method for enabling CD4 - CD8- Methods for T cell proliferation and / or activation include: contacting peripheral blood mononuclear cells with spore-forming bacteria or their cultures.

[0097]

[12] A method for enabling CD4 - CD8 - Methods for T cell proliferation and / or activation include administering an effective amount of a spore-forming bacterium or a culture thereof to a subject.

[0098]

[13] A spore-forming bacterium or a culture thereof, used to induce CD4 - CD8 - T cell proliferation and / or activation.

[0099] Example

[0100] The present invention will now be described in detail using examples. However, the present invention is not limited to these examples.

[0101] All the following experiments were conducted with the approval of the Ethics Committee of Kumamoto University. Human cell samples were obtained from peripheral blood mononuclear cells (PBMCs) of healthy individuals through heparinized blood collection. All blood samples were collected after obtaining informed consent and confirming the blood provider's intent. Patient samples were processed and analyzed using a code devoid of personal information.

[0102] <Experimental Example 1>

[0103] Using PBMCs from healthy individuals, this study investigated whether stimulation with Miya BM (registered trademark) tablets (manufactured by Miya Risan Pharmaceutical Co., Ltd., Japan) induced the activity of specific T cell subsets. Miya BM (registered trademark) tablets contain Clostridium butyricum MIYAIRI 588 (FERM BP-2789) (strain 588) as the active ingredient. Specifically, AIM (Activation-Induced Marker) analysis was performed, using flow cytometry to analyze the activation marker (CD137). 1×10⁻⁶ PBMCs were used. 6PBMCs / wells were suspended in 100 µL of RPMI 1640 medium (Thermo Fisher Scientific, Cat No: 200-02) containing 10% FBS. One Miya BM (trademarked) tablet was dissolved in 3 mL of pure water for suspension. 3 µL, 10 µL, and 30 µL of Miya BM (trademarked) suspensions were added to the wells, respectively. Wells without Miya BM (trademarked) tablet stimulation were placed as negative controls. After pulse stimulation, the wells were incubated at 37°C and 2.5% CO2 for 24 hours. After incubation, the PBMCs were washed and stained with antibodies. As antibodies, CD3 FITC (UCHT1, 100-fold dilution, Biolegend), CD8 APCcy7 (RPA-T8, 100-fold dilution, Biolegend), CD14 PerCP / Cy5.5 (HCD14, 100-fold dilution, Biolegend), CD19 PerCP / Cy5.5 (HIB19, 10-fold dilution, Biolegend), CD25 PEcy7 (M-A251, 50-fold dilution, Biolegend), and CD137 APC (4B4-1, 50-fold dilution, Biolegend) were used. Additionally, 7-aminoactinomycin D (7-AAD, Biolegend, Cat No: 420404) was added for staining dead cells. After addition, the cells were incubated on ice for 20 minutes. After incubation, PBMCs were fixed with 1% paraformaldehyde (Nacalai Tesque Co., Ltd., Cat No: 09154-85). Following fixation, the expression levels of surface molecules were determined by flow cytometry using FACS Canto II (BD Biosciences). Data obtained by flow cytometry were analyzed using FACS Diva v9.0 (BD Biosciences) and FlowJo software v10 (Tree Star). The results are presented below. Figure 1 B and Figure 1 C. Figure 1 B indicates the result when using Miya BM (registered trademark) suspension (10µL). Figure 1 C represents the results when using Miya BM (registered trademark) suspension (10µL) (CBM588) and the control group (No stimu).

[0104] like Figure 1 B and Figure 1 As shown in C, stimulation with Miya BM (registered trademark) tablets resulted in [a certain effect] on CD4. -CD8 - Significant activation was observed in T cells, and CD4 activation was also confirmed compared to no stimulation. - CD8 - A significant increase in activation markers of T cell subsets.

[0105] <Experimental Example 2>

[0106] Culture media or suspensions of 588 strains, which are components of Miya BM, were prepared. As culture media or suspensions of the 588 strains, suspensions of untreated spores, suspensions of treated spores, culture media of untreated vegetative cells, and culture media of treated vegetative cells were prepared. Heat treatment was performed at 90°C for 90 minutes for spores and at 70°C for 30 minutes for vegetative cells. The spores were added to PBMCs from the same healthy human source as in Experimental Example 1, and AIM analysis was performed to verify whether the same subpopulation activation as in Experimental Example 1 was obtained. The 588 spores were prepared as follows: Using ordinary agar medium supplemented with 0.5% glucose and 0.3% calcium carbonate, the spores were cultured at 37°C under anaerobic conditions for 5 days, followed by centrifugation with sterile water. Purification was then performed using density gradient centrifugation with 45% urethane (common name: diatrizoate). Further centrifugation with sterile water was performed to prepare a suspension of 588 spores. In the following experimental cases, 588 spores were also formed.

[0107] Specifically, AIM (Activation-Induced Marker) analysis was performed using flow cytometry, with the activation markers (CD69 OX40 CD137) as indicators. For 1×10 6 Prepare 100 µL of RPMI 1640 medium (Thermo Fisher Scientific, Cat No: 200-02) containing 10% FBS for each well of PBMCs / well. Add 1 µL and 3 µL of the culture medium containing 588 strains to each well. The concentration of spores or vegetative cells in the culture medium is 1 × 10⁻⁶. 7Cells / mL. As a negative control, wells without stimulation were set up. After pulse, the cells were cultured at 37°C and 2.5% CO2 for 24 hours. After culture, PBMCs were washed and stained with the same antibody as in Example 1 and OX40 (Ber-ACT35, 50-fold dilution, Biolegend). Additionally, 7-aminoactinomycin D (7-AAD, Biolegend, Cat No: 420404) was added for staining 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 determined by flow cytometry using FACS Canto II (BD Biosciences). Data obtained from flow cytometry were analyzed using FACS Divav9.0 (manufactured by BD Biosciences) and FlowJo software v10 (manufactured by Tree Star). The results are presented below. Figure 2 A and Figure 2 B. It should be noted that, Figure 2 A represents the result when using a suspension of heat-free spores (3 µL). Figure 2 In B, "No stimu" represents the control, "G" represents the suspension of spores without heat treatment, "G inact" represents the suspension of spores with heat treatment, "E" represents the culture medium of vegetative bodies without heat treatment, and "E inact" represents the culture medium of vegetative bodies with heat treatment.

[0108] like Figure 2 As shown in A, when stimulated with a suspension (3 µL) of heat-free spores, CD4 - CD8 - Significant activation was confirmed in T cells. For example... Figure 2 As shown in Figure B, the group stimulated with the culture medium of 588 strains showed significantly higher levels of CD4 compared to the unstimulated group. - CD8 - T cell activation showed a response consistent with that of Miya BM (registered trademark) tablets. Furthermore, compared to the group stimulated with culture medium containing 588 strains, the group stimulated with a suspension of spores showed more confirmed CD4 activity than the group stimulated with vegetative cells. - CD8 - T cell activation. Regarding the effect of heat treatment on CD4... - CD8 - The effect of T cell activation was observed in the spore suspension; compared to the heat-treated group, the untreated group showed greater activation of CD4+. - CD8- T cell activation. In the trophoblast culture medium, no difference in CD4 activation was observed between the untreated and heat-treated groups. - CD8 - The effect of T cell activation.

[0109] <Experimental Example 3>

[0110] We attempted to identify immune subsets activated by stimulation of 588 spores using PBMCs derived from healthy individuals, with cell proliferation as the indicator. Specifically, we performed CTV (Cell Trace Violet) analysis, labeling PBMCs with CTV pigment, and used flow cytometry to detect decreases in CTV pigment concentration corresponding to cell division, thereby evaluating cell proliferation.

[0111] Using the Cell Trace Violet Cell Proliferation Kit (Thermo Fisher Scientific, Invitrogen, C34557), a 1 mM CTV / µL solution was prepared by diluting 5 mM CTV / 20 µL DMSO 5-fold with PBS. 10 mM CTV / 10 µL was then added dropwise to a final volume of 1 × 10⁻⁶. 7 Resuspend PBMCs in 10 mL PBS and label to a final concentration of 1 µM CTV / µL. Add 1 × 10 6 CTV-labeled PBMCs / well were suspended in 200 µL of RPMI 1640 medium (Thermo Fisher Scientific, Cat No: 200-02) containing 10% FBS. A suspension of heat-untreated spores was added (spore concentration: 1 × 10⁻⁶). 7PBMCs were suspended in suspension (cells / mL). As a control, wells without stimulation and wells with stimulation from anti-CD3-CD28 antibody (2 µL) were set up. After pulse, the cells were cultured at 37°C and 2.5% CO2 for 7 days. After culture, PBMCs were washed and the surface was stained with antibodies. The antibodies used were CD3 FITC (UCHT1, 100-fold dilution, Biolegend), CD8 APCcy7 (RPA-T8, 100-fold dilution, Biolegend), CD14 PerCP / Cy5.5 (HCD14, 100-fold dilution, Biolegend), and CD19 PerCP / Cy5.5 (HIB19, 10-fold dilution, Biolegend). In addition, 7-aminoactinomycin D (7-AAD, Biolegend, Cat No: 420404) was added for staining dead cells. After addition, the cells were incubated on ice for 20 minutes. After incubation, PBMCs were fixed with 1% paraformaldehyde (Nacalai Tesque Co., Ltd., Cat No: 09154-85). Following fixation, the expression levels of surface molecules were determined by flow cytometry using Cytek Notrhern Lights (Cytek Japan). Data obtained from flow cytometry were analyzed using FlowJo software v10 (Tree Star). The results are presented below. Figure 3 A and Figure 3 B. In Figure 3 In A, "None" indicates no stimulation, "Anti-CD3 / CD28" indicates anti-CD3-CD28 antibody, and "spores" indicates a suspension of spores without heat treatment. Figure 3 In B, “None” means no stimulation, “CD3” means anti-CD3-CD28 antibody, and “spore” means a suspension of spores without heat treatment.

[0112] like Figure 3 A and Figure 3 As shown in Figure B, compared with the unstimulated group and the group stimulated with anti-CD3-CD28 antibody, CD4 was confirmed in the group stimulated with a suspension of heat-free spores. - CD8 - Significant proliferation of T cells.

[0113] <Experimental Example 4>

[0114] Using PBMCs from healthy individuals, an attempt was made to determine the CD4-forming components that significantly proliferated upon spore stimulation. - CD8 - Subgroups of T cells.

[0115] Using the Cell Trace Violet Cell Proliferation Kit (Thermo Fisher Scientific, Invitrogen, C34557), a 1 mM CTV / µL solution was prepared by diluting 5 mM CTV / 20 µL DMSO 5-fold with PBS. 10 mM CTV / 10 µL was then added dropwise to a final volume of 1 × 10⁻⁶. 7 Resuspend PBMCs in 10 mL PBS and label to a final concentration of 1 µM CTV / µL. Add 1 × 10 6 CTV-labeled PBMCs / well were suspended in 200 µL of RPMI 1640 medium (Thermo Fisher Scientific, Cat No: 200-02) containing 10% FBS. A suspension of heat-untreated spores was added (spore concentration: 1 × 10⁻⁶). 7 (cells / mL) or a suspension of heat-treated spores (spore concentration: 1×10⁻⁶ cells / mL) 7PBMCs were suspended in suspension (cells / mL). As a control, wells with no irritation and wells with irritation from anti-CD3-CD28 antibody (2 µL) were prepared. After pulse, the cells were cultured at 37°C and 2.5% CO2 for 7 days. After culture, the PBMCs were washed and stained with antibody. The antibodies used were CD3 BV785 (50-fold dilution, Biolegend), CD4 BV750 (100-fold dilution, Biolegend), CD8 BV570 (100-fold dilution, Biolegend), TCRγδ FITC (100-fold dilution, Milteny Biotec), TCRab PerCP / Cy5.5 (100-fold dilution, Biolegend), Vg9 APCvio770 (50-fold dilution, Invtrrogen), and Vδ1 PE-eFlour610 (50-fold dilution, Invtrrogen) and Vδ2 PE (100-fold dilution, Milteny Biotec). Additionally, 7-aminoactinomycin D (7-AAD, Biolegend, Cat No: 420404) was added for staining dead cells. After addition, the cells were incubated on ice for 20 minutes. After incubation, PBMCs were fixed with 1% paraformaldehyde (Nacalai Tesque Co., Ltd., Cat No: 09154-85). Following fixation, the expression levels of surface molecules were determined by flow cytometry using Cytek Notrhern Lights (Cytek Japan). Data obtained by flow cytometry were analyzed using FlowJo software v10 (Tree Star). The results are presented below. Figure 4 B and Figure 4 C. In Figure 4 In section B, "None" indicates no stimulation, "Anti-CD3 / CD28" indicates anti-CD3-CD28 antibody, "Spore Heat-inact" indicates a suspension of heat-treated spores, and "Spore" indicates a suspension of untreated spores. Figure 4 In the C, "None" indicates no stimulation, "CD3" indicates anti-CD3-CD28 antibody, "CBM588 Heat-inact" indicates a suspension of heat-treated spores, and "CBM588" indicates a suspension of untreated spores.

[0116] like Figure 4 B and Figure 4 As shown in C, CD4 was confirmed in all groups stimulated with spore suspension, regardless of whether heat treatment was performed, compared to the control group. - CD8 -The number of T cells increased significantly. It was found that stimulation with a suspension of heat-free spores led to the proliferation of Vγ9Vδ2 T cells, which are known to have antitumor effects.

[0117] <Experimental Example 5>

[0118] Using the Cell Trace Violet Cell Proliferation Kit (Thermo Fisher Scientific, Invitrogen, C34557), a 1 mM CTV / µL solution was prepared by diluting 5 mM CTV / 20 µL DMSO 5-fold with PBS. 10 mM CTV / 10 µL was then added dropwise to a final volume of 3 × 10⁻⁶. 7 Resuspend PBMCs in 10 mL PBS and label to a final concentration of 1 µM CTV / µL. Add 3 × 10 6 CTV-labeled PBMCs / well were suspended in 200 µL of RPMI 1640 medium (Thermo Fisher Scientific, Cat No: 200-02) containing 10% FBS. A suspension of 588 spores was then added (spore concentration: 3 × 10⁻⁶). 5 cells / mL, 3×10 6 1 cell / mL or 3×10 7 A suspension of spores of Clostridium sordellii strain JCM3814 (spore concentration: 3 × 10⁻⁶ cells / mL). 5 1 cell / mL or 3×10 6 (cells / mL), or a suspension of spores of Clostridium putrificum strain ATCC25784 (spore concentration: 3 × 10⁻⁶ cells / mL). 7The PBMCs were suspended in a suspension (cells / mL). The spores in the added suspension were not heat-treated. As a control, non-irritating wells were set up. After pulse treatment, the PBMCs were cultured for 12 days at 37°C and 2.5% CO2. After culture, the PBMCs were washed and their surfaces stained with antibodies. The antibodies used were CD3 BV785 (50-fold dilution, Biolegend), CD4 BV750 (100-fold dilution, Biolegend), CD8 BV570 (100-fold dilution, Biolegend), TCRγδ FITC (100-fold dilution, Milteny Biotec), TCRab PerCP / Cy5.5 (100-fold dilution, Biolegend), Vg9APCvio770 (50-fold dilution, Invtrrogen), and Vδ1 PE-eFlour610 (50-fold dilution, Invtrrogen) and Vδ2 PE (100-fold dilution, Milteny Biotec). Additionally, 7-aminoactinomycin D (7-AAD, Biolegend, Cat No: 420404) was added for staining dead cells. After addition, the cells were incubated on ice for 20 minutes. After incubation, PBMCs were fixed with 1% paraformaldehyde (Nacalai Tesque Co., Ltd., Cat No: 09154-85). Following fixation, the expression levels of surface molecules were determined by flow cytometry using Cytek Notrhern Lights (Cytek Japan). Data obtained from flow cytometry were analyzed using FlowJo software v10 (Tree Star). The results are presented below. Figure 5 B. In Figure 5 In section B, “CBM588 Spore” indicates a suspension of 588 spores, “Paeniclostridium sordelli” indicates a suspension of Clostridium sordellii JCM3814 spores, and “Clostridium putrificum” indicates a suspension of Clostridium putrificum ATCC25784 spores.

[0119] like Figure 5As shown, the groups stimulated with suspensions of Clostridium sordellii JCM3814 spores and Clostridium putrificum ATCC25784 spores, as well as the group stimulated with suspensions of 588 spores, all exhibited the same antitumor effect on Vγ9Vδ2T cells.

Claims

1. A device for enabling CD4 - CD8 - Compositions for T cell proliferation and / or activation, comprising spore-forming bacteria or their cultures as active ingredients.

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

3. The composition according to claim 1, wherein, The spore-forming bacteria are selected from *Clostridium butyricum*, *Clostridium absonum*, *Clostridium acetobutylicum*, *Clostridium beijerinckii*, *Clostridium cadaveris*, *Clostridium clostridiiforme*, *Clostridium cochlearium*, *Clostridium difficile*, *Clostridium innocuum*, *Clostridium novyi*, *Clostridium perfringens*, *Clostridium putrificum*, *Clostridium ramosum*, *Clostridium rectum*, *Clostridium scatologenes*, and *Clostridium soxella*. Clostridium sporogenes, Clostridium tertium, Clostridium tyrobutyricum, Clostridium aerorotolerans, Clostridium aminophilum, Clostridium aminovalericum, Clostridium celerecrescens, Clostridium cellulosi, Clostridium coccoides, Clostridium hiranonis, Clostridium leptum, Clostridium nexile, Clostridium oroticum, Clostridium polysaccharolyticum, Clostridium populeti, Clostridium lysate scindens), Clostridiumsphenoides, ClostridiumAt least one bacterium from the group consisting of *Bacillus sporosphaeroides*, *Clostridium 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 according to claim 1, wherein, The spore-forming bacterium is selected from at least one bacterium in the group consisting of Clostridium butyricum, Clostridium sordellii, and Clostridium putrificum.

5. The composition according to claim 1, wherein, The spore-forming bacteria are in the form of spores.

6. The composition according to claim 1, wherein, The CD4 - CD8 - T cells include γδT cells.

7. The composition according to claim 6, wherein, The γδT cells comprise Vγ9Vδ2T cells.

8. The composition according to claim 1, used in combination with an antitumor agent.

9. The composition according to claim 1, wherein it is a pharmaceutical composition.

10. The composition according to claim 1, wherein it is a food or beverage composition.

11. A method for enabling CD4 - CD8 - Methods for T cell proliferation and / or activation include: Contact peripheral blood mononuclear cells with spore-forming bacteria or their cultures.

12. A device for enabling CD4 - CD8 - Methods for T cell proliferation and / or activation include: The subject is given an effective amount of spore-forming bacteria or its culture.

13. A spore-forming bacterium or a culture thereof, used to induce CD4+ spore formation. - CD8 - T cell proliferation and / or activation.

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