Functional immunostimulatory lactic acid bacteria

Abstract

To provide lactic acid bacteria with immunostimulatory capabilities.SOLUTION: Provided are lactic acid bacteria with the capability to activate dendritic cells in Peyer's patches. Oral administration of lactic acid bacteria and / or compositions disclosed herein leads to enhanced rates of activation marker positivity for dendritic cells in intestinal Peyer's patches and elevated rates of cytokine production, thereby ensuring a stimulating effect on the intestinal immune system.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to lactic acid bacteria or a composition comprising lactic acid bacteria. In particular, the present disclosure relates to lactic acid bacteria having the ability to activate dendritic cells, or a composition comprising the lactic acid bacteria, and a food or drink, food additive, feed, supplement, or medicine containing the same. [Background technology]

[0002] There have been several reports of findings that the presence and diversity of intestinal microbiota can change immune status. Meanwhile, in recent years, the use of cancer immunotherapy (IO: immuno-oncology) agents, including immune checkpoint inhibitors, has rapidly increased. In addition, active research is being conducted on the interaction between IO agents and intestinal microbiota. In other words, it is believed that the type and composition of intestinal microbiota also affect the effectiveness of cancer immunotherapy in the host, humans and mammals. Therefore, there is a demand for intestinal bacterial strains that can be used in combination with immune checkpoint inhibitors. Summary of the Invention [Means for solving the problem]

[0003] The present inventors have found that certain types of lactic acid bacteria improve the ability to activate host dendritic cells compared to a standard strain (ATCC) or a control group. Based on this finding, the present disclosure provides lactic acid bacteria capable of activating dendritic cells, compositions containing the lactic acid bacteria, and foods and beverages, food additives, feed, supplements, or medicines containing the same.

[0004] Thus, the present disclosure provides: (Item 1) Lactic acid bacteria that have the ability to activate dendritic cells in Peyer's patches. (Item 2a) Regarding the lactic acid bacteria, the Peyer's patch dendritic cell activation index is IL-6 + / CD45 + : greater than 1.43, MHC class II+ / CD45 + : greater than 1.82, CD80 + / CD45 + : greater than 1.41, and CD86 + / CD45 + : greater than 1.22, The lactic acid bacterium described in the above item, having at least one property selected from the group consisting of: (Item 2b) The Peyer's patch dendritic cell activation index is IL-6 + / CD45 + :2 or more, MHC class II + / CD45 + :2 or more, CD80 + / CD45 + :2 or more, and CD86 + / CD45 + :2 or more, The lactic acid bacterium according to any one of the above items, having at least one property selected from the group consisting of: (Item 2c) The Peyer's patch dendritic cell activation index is IL-6 + / CD45 + : 2.5 or higher, MHC class II + / CD45 + : 2.5 or higher, CD80 + / CD45 + : 2.5 or higher, and CD86 + / CD45 + : 2.5 or higher, The lactic acid bacterium according to any one of the above items, having at least one property selected from the group consisting of: (Item 2d) The Peyer's patch dendritic cell activation index is IL-6 + / CD45 + : 2.5 or higher, MHC class II + / CD45 + :4 or more, CD80 + / CD45 + : 2.5 or higher, and CD86 + / CD45 + : 2.5 or higher, The lactic acid bacterium according to any one of the above items, having at least one property selected from the group consisting of: (Item 2e) The Peyer's patch dendritic cell activation index is IL-6 + / CD45 + : 3 or more, MHC class II + / CD45 + :4 or more, CD80 + / CD45 + :3 or more, and CD86 + / CD45 + : 2.5 or higher, The lactic acid bacterium according to any one of the above items, having at least one property selected from the group consisting of: (Item 2f) The lactic acid bacterium according to any one of the preceding items, wherein the Peyer's patch dendritic cell activation indicator has at least two of the above properties. (item 2g) The lactic acid bacterium according to any one of the above items, wherein the Peyer's patch dendritic cell activation indicator is at least IL-6 and CD80 positive. (Item 2h) The lactic acid bacterium according to any one of the preceding items, wherein the Peyer's patch dendritic cell activation indicator has at least three of the above properties. (Item 2i) The lactic acid bacterium according to any one of the preceding items, wherein the Peyer's patch dendritic cell activation index has four of the above properties. (Item 2) The lactic acid bacterium according to any one of the preceding items, wherein the activation ability is evaluated based on the presence or absence of expression of a cell surface marker and / or a cytokine indicative of activation in the dendritic cells. (Item 3) The lactic acid bacterium according to any one of the above items, wherein the activation ability is evaluated based on the positive rate of cell surface markers and / or cytokines indicative of activation in the dendritic cells. (Item 4) The lactic acid bacterium according to any one of the above items, wherein the activation ability is evaluated based on the presence or absence of expression in the dendritic cells of at least one cell surface marker and / or cytokine selected from the group consisting of IL-6, IL-12, MHC class I, MHC class II, CD80, CD86, CD8, IFN-γ, IFN-α, TNF-α, CD40, IL-1β, IL-23, IL-27, and CCR7. (Item 5) The lactic acid bacterium according to any one of the preceding items, wherein the activation ability is evaluated by at least one positivity rate selected from the group consisting of IL-6 positivity rate, IL-12 positivity rate, MHC class I positivity rate, MHC class II positivity rate, CD80 positivity rate, CD86 positivity rate, CD8 positivity rate, IFN-γ positivity rate, IFN-α positivity rate, TNF-α positivity rate, CD40 positivity rate, IL-1β positivity rate, IL-23 positivity rate, IL-27 positivity rate, and CCR7 positivity rate in the dendritic cells. (Item 6) The activation ability is determined by the expression of CD45 in the dendritic cells. + CD11c + IL-6 + / CD45 + , CD45 + CD11c + IFN-γ + / CD45 + , CD45 + CD11c + MHC class II + / CD45 + , CD45 + CD11c + CD80 + / CD45+ , CD45 + CD11c + CD86 + / CD45 + , and CD45 + CD11c + CD8a + / CD45 + The lactic acid bacterium according to any one of the preceding items, which increases any one of the parameters selected from the group consisting of above a reference value. (Item 6a) The activation ability is determined by evaluating the CD45 activation ability of the dendritic cells using evaluation system A. + CD11c + IL-6 + / CD45 + , CD45 + CD11c + IFN-γ + / CD45 + , CD45 + CD11c + MHC class II + / CD45 + , CD45 + CD11c + CD80 + / CD45 + , CD45 + CD11c + CD86 + / CD45 + , and CD45 + CD11c + CD8a + / CD45 + The lactic acid bacterium according to any one of the preceding items, which increases any one of the parameters selected from the group consisting of above a reference value. (Item 7) The activation ability is determined by the expression of CD45 in the dendritic cells. + CD11c + IL-6 + / CD45 + , CD45 + CD11c + IFN-γ + / CD45 + , CD45 + CD11c +MHC class II + / CD45 + , CD45 + CD11c + CD80 + / CD45 + , CD45 + CD11c + CD86 + / CD45 + , and CD45 + CD11c + CD8a + / CD45 + The lactic acid bacterium according to any one of the preceding items, which increases any one of the parameters selected from the group consisting of at least about three times higher than the reference value. (Item 7a) The activation ability is determined by evaluating the CD45 activation ability of the dendritic cells using evaluation system A. + CD11c + IL-6 + / CD45 + , CD45 + CD11c + IFN-γ + / CD45 + , CD45 + CD11c + MHC class II + / CD45 + , CD45 + CD11c + CD80 + / CD45 + , CD45 + CD11c + CD86 + / CD45 + , and CD45 + CD11c + CD8a + / CD45 + The lactic acid bacterium according to any one of the preceding items, which increases any one of the parameters selected from the group consisting of at least about three times higher than the reference value. (Item 8) The activation ability is determined by the expression of CD45 in the dendritic cells. + CD11c + IL-6 + / CD45+ , CD45 + CD11c + IFN-γ + / CD45 + , CD45 + CD11c + MHC class II + / CD45 + , CD45 + CD11c + CD80 + / CD45 + , CD45 + CD11c + CD86 + / CD45 + , and CD45 + CD11c + CD8a + / CD45 + The lactic acid bacterium according to any one of the preceding items, which increases any one of the parameters selected from the group consisting of at least about 4-fold compared to the reference value. (Item 8a) The activation ability is determined by evaluating the CD45 activation ability of the dendritic cells using evaluation system A. + CD11c + IL-6 + / CD45 + , CD45 + CD11c + IFN-γ + / CD45 + , CD45 + CD11c + MHC class II + / CD45 + , CD45 + CD11c + CD80 + / CD45 + , CD45 + CD11c + CD86 + / CD45 + , and CD45 + CD11c + CD8a + / CD45 +The lactic acid bacterium according to any one of the preceding items, which increases any one of the parameters selected from the group consisting of at least about 4-fold compared to the reference value. (Item 9) The activation ability is determined by the expression of CD45 in the dendritic cells. + CD11c + IL-6 + / CD45 + , CD45 + CD11c + IFN-γ + / CD45 + , CD45 + CD11c + MHC class II + / CD45 + , CD45 + CD11c + CD80 + / CD45 + , CD45 + CD11c + CD86 + / CD45 + , and CD45 + CD11c + CD8a + / CD45 + The lactic acid bacterium according to any one of the preceding items, which increases at least three of the parameters selected from the group consisting of at least about three times higher than the reference value. (Item 9a) The lactic acid bacterium according to any one of the preceding items, wherein the evaluation system A comprises the following steps: 1) (Preparation of administered bacteria) The target strain is cultured, stained with carboxyfluorescein succinimidyl ester (CFSE), and then washed with PBS to prepare the administered bacteria solution. 2) (Collection of small intestinal Peyer's patches) 1.0 x 10 9 Each bacterial solution was administered orally to C57BL / 6 mice at colony forming unit (CFU) / 100 μL, and the mice were dissected about 1 hour later to collect Peyer's patches in the small intestine. 3) (Antibody staining of cells in small intestinal Peyer's patches) Cells from small intestinal Peyer's patches are separated and suspended in cell culture medium, a protein transport inhibitor is added and cultured for approximately 15 hours, then the cells are centrifugally washed with PBS and dead cells are stained, followed by centrifugation and washing with staining buffer (1% fetal bovine serum, 0.09% NaN3 in PBS) and blocking. Cell surface markers are then stained with various labeled antibodies. Next, the cells are fixed and permeabilized with a cell fixation and permeabilization solution, washed with a cell membrane permeabilization buffer, and intracellular markers are stained with anti-cytokine antibodies. The cells are then washed with a cell membrane permeabilization buffer and measured and analyzed using a flow cytometer. (Item 10) The lactic acid bacteria according to any one of the preceding items, wherein the activation ability is greater than that of a reference strain of lactic acid bacteria. (Item 11) The lactic acid bacteria described in any one of the above items, wherein the lactic acid bacteria type strain includes E. faecalis ATCC 700802. (Item 12) The lactic acid bacterium according to any one of the above items, wherein the dendritic cells are present between the follicle-associated epithelium (FAE) and the follicle. (Item 13) The lactic acid bacteria according to any one of the above items, further comprising the ability to activate at least one other cell selected from the group consisting of macrophages, B cells, and T cells. (Item 14) The lactic acid bacterium according to any one of the preceding items, which is selected from the group consisting of the genera Enterococcus, Bifidobacterium, Bacteroides, Lactobacillus, and Ackermansia. (Item 15) The lactic acid bacterium according to any one of the preceding items, which is selected from the group consisting of the genera Enterococcus and Bifidobacterium. (Item 16) A lactic acid bacterium according to any one of the preceding items, which is of the genus Enterococcus. (Item 17) The lactic acid bacterium according to any one of the above items, which is selected from the group consisting of E. faecalis, E. faecium, E. avium, E. casseliflavus, E. gallinarum, and E. hirae. (Item 18) The lactic acid bacterium according to any one of the preceding items, which is Enterococcus faecalis. (Item 19) The lactic acid bacterium according to any one of the above items, wherein the Enterococcus faecalis includes KU-EF-001 strain (NITE BP-03655), KU-EF-002 strain (NITE BP-03656), KU-EF-003 strain (NITE BP-03657), and KU-EF-004 strain (NITE BP-03658). (Item 20) A composition comprising the lactic acid bacteria described in any one of the above items. (Item 21) A composition according to any one of the preceding items for stimulating the immunity of a subject. (Item 22) A composition according to any one of the preceding items for activating dendritic cells in Peyer's patches. (Item 23) A composition according to any one of the preceding items, comprising Enterococcus faecalis. (Item 24) The composition of any one of the preceding items for use in combination with an immunomodulatory agent or a radiological agent. (Item 25) A composition according to any one of the preceding items for enhancing the effect of an immunomodulatory or radiological agent. (Item 26) The composition according to any one of the preceding items, wherein the immunomodulatory agent is at least one selected from an immune checkpoint inhibitor, a CAR-T cell therapy, a bispecific antibody drug, a cancer vaccine, a costimulatory molecule agonist, an immune activator, and a small molecule inhibitor. (Item 27) The composition of any one of the preceding items, wherein the immune checkpoint inhibitor is selected from the group consisting of agents against molecules selected from the group consisting of CTLA-4, PD-1, LAG-3, BTLA, KIR, TIM-3, PD-L1, PD-L2, B7-H3, B7-H4, HVEM, GAL9, CD160, VISTA, BTNL2, TIGIT, PVR, BTN1A1, BTN2A2, BTN3A2, and CSF-1R, and any combination thereof. (Item 28) The composition according to any one of the preceding items, which is a food or drink, a food additive, a feed, a supplement, or a medicine. (Item 29) A pharmaceutical composition comprising a composition according to any one of the preceding items and an immunomodulatory agent.

[0005] It is contemplated that one or more of the above features may be provided in combinations other than those specifically stated.Further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0006] Furthermore, features and notable actions and effects of the present disclosure other than those described above will become apparent to those skilled in the art by referring to the following description of the preferred embodiments of the present invention and the drawings. Effect of the Invention

[0007] The lactic acid bacteria and / or composition of the present disclosure can be used to stimulate the immunity of a subject. Oral administration of the lactic acid bacteria and / or composition of the present disclosure can increase the activation marker positivity rate and cytokine production rate of dendritic cells in the intestinal Peyer's patches, thereby achieving the effect of stimulating the intestinal immune system.

[0008] Furthermore, by combining administration of an immune checkpoint inhibitor with oral administration of the lactic acid bacteria and / or composition disclosed herein, the antitumor effect can be enhanced, making it possible to apply the present invention to pharmaceuticals. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic diagram showing the administration schedule of each bacterial strain to mice in Example 1. [Diagram 2] Figure 2 is a graph showing the progression of tumor volume when mice subcutaneously implanted with MC38 were orally administered each bacterial strain or PBS, and then intraperitoneally administered immune checkpoint inhibitors (anti-PD-1 antibody and anti-CTLA-4 antibody). [Diagram 3] FIG. 3 is a schematic diagram showing the administration schedule of each bacterial strain to mice in Example 2. [Figure 4] Figure 4 is a graph showing the progression of tumor volume when five types of E. faecalis strains and PBS were orally administered to mice subcutaneously implanted with MC38 cells, and immune checkpoint inhibitors (anti-PD-1 antibody and anti-CTLA-4 antibody) were administered intraperitoneally to the mice. [Diagram 5] FIG. 5 is a schematic diagram showing the administration schedule of each bacterial strain to mice in Example 3. [Figure 6] Figure 6 is a graph showing the changes in tumor volume and survival time in mice that had subcutaneously implanted MC38 cells and were orally administered the KU-EF-004 strain, the E. coli ATCC10798 (K-12) strain, the B. longum ATCC15697 strain, or PBS, and then intraperitoneally administered an anti-CTLA-4 antibody. [Figure 7] FIG. 7 is a schematic diagram showing the administration schedule of each bacterial strain to mice in Example 4. [Figure 8] FIG. 8 is a graph showing the survival time of mice subcutaneously implanted with MC38 cells that were orally administered the KU-EF-004 strain, E. faecalis ATCC700802 strain, or PBS and then intraperitoneally administered an anti-CTLA-4 antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present disclosure will be described below while showing the best mode. Throughout this specification, the expression of the singular form should be understood to include the concept of the plural form, unless otherwise specified. Therefore, the singular article (for example, in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will take precedence.

[0011] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0012] As used herein, "about" means ±10% of the preceding numerical value.

[0013] As used herein, "Peyer's patches" refer to aggregated lymphoid follicles that exist in the jejunum and ileum, in regions where the villi on the luminal side of the intestine are underdeveloped. Peyer's patches contain T cell regions and B cell regions, and antigens are presented to T cells by dendritic cells that capture antigens near the intestinal epithelium, promoting antigen-specific cellular immune responses by T cells and antigen-specific antibody production by B cells.

[0014] As used herein, "dendritic cells" refer to a type of immune cell that functions as an antigen-presenting cell, and primarily presents antigens to T cells to promote cellular immune responses by T cells and antibody production by B cells, forming an antigen-specific immune response. Mature dendritic cells in Peyer's patches express surface markers such as CD45, CD11c, MHC class II, and CD103, and secrete various cytokines (IFN-γ, IL-6, IL-12) to activate various immune responses.

[0015] Dendritic cells are a type of immunocompetent cell named for the dendritic cell processes they have. They are distributed throughout the body, including lymphoid tissues, and are considered to be important cells for immune responses. Dendritic cells that recognize non-self (antigens) such as pathogenic microorganisms and cancers present antigen information to T cells via MHC molecules, triggering adaptive (acquired) immune responses and functioning as antigen-presenting cells. Meanwhile, dendritic cells in the steady state also have the function of inducing and maintaining immune tolerance to prevent excessive immune responses.

[0016] As used herein, "activation ability" refers to the ability to activate any activity or property of a cell, and may be achieved by any mechanism of activation. "Activation ability of dendritic cells" refers to the property of stimulating dendritic cells by dendritic cells capturing and phagocytosing antigens or by specific molecules binding to receptors on the cell surface, thereby promoting the production of dendritic cell surface marker molecules and cytokines, thereby improving the maturation and antigen presentation ability of dendritic cells and inducing an immune response.

[0017] As used herein, the term "positive rate" refers to the percentage of cells expressing a given marker in a cell population. Specifically, the positive rate can be expressed as the percentage of CD11c-positive dendritic cells expressing a target marker and / or cytokine among CD45-positive cells, which is a leukocyte marker.

[0018] As used herein, the term "dendritic cell activation index in Peyer's patches" refers to the rate at which dendritic cells in a reference animal are activated when the bacteria to be measured are inoculated into the reference animal. In this specification, the activation index is calculated by inoculating dendritic cells in the Peyer's patches of the reference animal with a bacterium to be measured, and is calculated by inoculating dendritic cells in the Peyer's patches of the reference animal with a bacterium to be measured. In this specification, the activation index is calculated by inoculating dendritic cells in the Peyer's patches of the reference animal with a bacterium to be measured into the reference animal. 9 The positive rate refers to the multiple of the positive rate in the group administered each bacterial strain, calculated assuming the positive rate in the PBS-administered group to be 1. The positive rate refers to the multiple of the positive rate in the group administered each bacterial strain, calculated in CD45-positive and CD11c-positive dendritic cells in the Peyer's patches about 1 hour after oral administration of colony forming unit (CFU) / 100 μL of bacterial solution, collecting the small intestinal Peyer's patches about 1 hour after culturing and then performing antibody staining.

[0019] As used herein, IL-6 + / CD45 + : MHC class II + / CD45 + , CD80 + / CD45 + , and CD86 + / CD45 + Indices such as the following can be used as a reference: IL-6 + / CD45 + : greater than 1.43, MHC class II + / CD45 + : greater than 1.82, CD80 + / CD45 + : greater than 1.41, and CD86 + / CD45 + : greater than 1.22, These can be used as indicators of dendritic cell activation in Peyer's patches.

[0020] In this specification, the "type strain of lactic acid bacteria" is the E. faecalis ATCC 700802 strain.

[0021] As used herein, the term "immunomodulator" refers to a substance that interacts with a molecule involved in the transmission of a costimulatory signal on an antigen-presenting cell and / or a T cell in T cell activation by an antigen-presenting cell, thereby controlling the transmission of a costimulatory signal, or controls the function of a molecule directly or indirectly involved in the establishment of immune tolerance (immunosuppression) in the immune mechanism. The "immunomodulator" may be a drug selected from, but is not limited to, an antibody, a nucleic acid, a protein, a peptide, and a small molecule compound. The "immunomodulator" may include an immune checkpoint inhibitor, a CAR-T cell drug, a bispecific molecular drug, a cancer vaccine, a costimulatory molecule agonist drug, an immune activator, a small molecule inhibitor, and the like.

[0022] As used herein, the term "immune checkpoint inhibitor" refers to a drug that inhibits the immunosuppressive action of cancer cells or antigen-presenting cells. Examples of immune checkpoint inhibitors include, but are not limited to, drugs against molecules selected from the group consisting of: (1) CTLA-4 (ipilimumab, tremelimumab, etc.); (2) PD-1 (nivolumab, pembrolizumab, AMP-224, AMP-514 (MEDI0680), pidilizumab (CT-011), etc.); (3) LAG-3 (IMP-321, BMS-986016, etc.); (4) BTLA; (5) KIR (IPH2101, etc.); (6) TIM -3; (7) PD-L1 (Durvalumab (MEDI4736), MPDL3280A, BMS-936559, Avelumab (MSB0010718C), etc.); (8) PD-L2; (9) B7-H3 (MGA-271, etc.); (10 )B7-H4;(11)HVEM;(12)GAL9;(13)CD160;(14)VISTA;(15)BTNL2;(16)TIGIT;(17)PVR;(18)BTN1A1;(19)BTN2A2;(20)BTN3A2(Nat Rev Drug Discov. 2013; 12: 130-146; Nikkei Medical Cancer Review 2014; 9; Nat Rev Immunol. 2014; 14: 559-69); and (21) CSF1-R. Preferred examples of immune checkpoint inhibitors include drugs that inhibit the function of CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, VISTA, HVEM, BTLA, CD160, TIGIT, or PVR, and more preferred examples include drugs that inhibit the function of CTLA-4, PD-1, or PD-L1.

[0023] Furthermore, the immune checkpoint inhibitor is preferably an antibody against the above-mentioned molecule, for example, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-VISTA antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-CD160 antibody, an anti-TIGIT antibody, or an anti-PVR antibody. The immune checkpoint inhibitor binds to an immune checkpoint molecule or its ligand to inhibit the transmission of an immunosuppressive signal, thereby releasing the suppression of T cell activation by cancer cells, and enabling T cells to attack cancer cells.

[0024] "CAR-T cell therapy" is a medicine for CAR-T therapy, in which chimeric antigen receptor (CAR)-expressing T cells (CAR-T cells) are administered once. CAR-T cells are produced by introducing a gene encoding a CAR against a target antigen into a patient's T cells. Expression of the CAR can enhance the antigen specificity and attack power of T cells. Chimeric antigen receptor (CAR)-introduced T cell therapy is a type of immune cell therapy that uses gene modification technology to give T cells tumor specificity and enhanced function.

[0025] Chimeric antigen receptors (CARs) are receptor molecules that use the variable regions of the heavy and light chains of immunoglobulin in the form of single-chain fragments (scFvs) and are linked to the signaling domain of CD3ζ, which transmits antigenic stimulation into cells. Autologous T cells collected from the patient's peripheral blood can be used as cells to introduce CAR. After stimulating the collected T cells with cytokines or antibodies, the CAR gene is introduced using a viral vector or transposon, and the gene-transduced T cells are expanded by culture to the target cell number for transfusion and then transfused. When the transfused CAR-T binds to tumor cells in the patient's body, the CAR, whose function is activated by the signaling domain, recognizes proteins and glycans on the surface of tumor cells via the scFv. Therefore, CAR-T therapy is not limited to patients with a specific HLA, and is also effective against tumor cells that have escaped immune surveillance by reducing or deleting HLA expression.

[0026] In this specification, the term "bispecific antibody drug" refers to a drug having specificity for two different antigens, and a representative example of an antibody is a bispecific antibody. A bispecific antibody is an antibody in which one antibody molecule has two antigen-binding sites, and each antigen-binding site binds to a different antigen. For example, a bispecific antibody that recognizes a cancer cell antigen and an effector cell antigen is expected to enhance the antitumor effect by effectively contacting these cells.

[0027] In this specification, the term "cancer vaccine" refers to a vaccine preparation that is highly expressed in cancer cells and has strong immunogenicity (the property of an antigen inducing antibody production and cellular immunity) and is used to prevent or treat cancer. Cancer vaccines can be used to induce T lymphocytes that specifically attack cancer cells in the body by administering genes or proteins (cancer antigens) that are specifically expressed in cancer and / or overexpressed compared to normal cells, or peptides that are fragments of such antigens, to prevent or treat cancer. Usually, vaccine preparations present antigens to lymphocytes (cytotoxic T cells, cytotoxic lymphocytes) via human leukocyte antigens (HLA) by dendritic cells, activating them, and the lymphocytes attack cancer cells, thereby providing treatment. To enhance the effectiveness of the vaccine, an auxiliary drug called an adjuvant (immunostimulant) can also be used in combination. Cancer vaccine therapy is a treatment technique that involves administering genes or proteins (cancer antigens) that are expressed specifically in cancer cells (or in excess compared to normal cells), or peptide fragments of these, to induce T lymphocytes in the body that specifically attack cancer cells.

[0028] As used herein, the term "costimulatory molecule agonist agent" refers to a molecular agonist agent having a costimulatory effect, which activates T cells by transmitting auxiliary signals via costimulatory molecules on T cells or antigen-presenting cells, thereby attenuating the immunosuppressive effect of cancer cells or antigen-presenting cells. The costimulatory molecule agonist agent is not particularly limited, but includes agents against molecules selected from the following group: (1) 4-1BB (2) 4-1BB-L; (3) OX40 (4) OX40-L; (5) GITR; (6) CD28; (7) CD40; (8) CD40-L (9) ICOS; (10) ICOS-L; (11) LIGHT; and (12) CD27.

[0029] As used herein, the term "immunostimulator" refers to a drug having an immune activating effect, which directly or indirectly activates immune cells such as T cells and dendritic cells, thereby efficiently stimulating killer T cells in lymph nodes. Examples of immune activators include, but are not limited to, Toll-like receptor (TLR) agonists, stimulator of interferon genes (STING) agonists, cytokines, or drugs against heat shock proteins (HSPs).

[0030] In this specification, "small molecule inhibitor" refers to a drug that has low molecular weight and has some inhibitory effect.Small molecule inhibitors include, but are not limited to, histone deacetylase inhibitors, histone demethylase inhibitors, histone acetyltransferase inhibitors, histone methyltransferase inhibitors, DNA methyltransferase inhibitors, anthracycline antibiotics, platinum drugs, MAPK inhibitors, β-catenin inhibitors, STAT3 inhibitors, NF-kB inhibitors, JAK inhibitors, mTOR inhibitors, IDO inhibitors, COX-2 inhibitors, CXCR4 inhibitors and arginase inhibitors.

[0031] In this specification, the term "radiation agent" is broadly interpreted and refers to any agent used in radiation therapy. Radiation therapy is used in the broadest sense in the field, and is a treatment that damages and kills cancer cells by irradiating them with radiation such as X-rays, electron beams, and gamma rays. Radiation agents are not particularly limited, but include, for example, radioactive substances and radiation sensitizers.

[0032] In this specification, the term "medicine" is interpreted in the broadest sense in the art, includes any medicine, and is used as a concept including not only medicines and quasi-drugs under the Pharmaceutical Affairs Law, but also those applied to animals (veterinary medicines), and is understood to include any drug, composition, etc. intended for the treatment or prevention of a disease, disorder, or condition that requires it. Examples of such applications include applications in the medical field, veterinary science, etc. Typically, a medicine contains a solid or liquid excipient, and may contain additives such as disintegrants, flavoring agents, delayed release agents, lubricants, binders, and colorants as necessary. The form of a pharmaceutical product includes, but is not limited to, tablets, injections, capsules, granules, powders, fine granules, sustained release preparations, etc. The pharmaceutical, microorganism, compound, etc. of the present invention can be made into a pharmaceutical composition by combining with components such as pharmaceutical acceptable general carriers or excipients.

[0033] In this specification, the term "food and drink" has the meaning commonly used in the field and refers to all foods (including beverages) that can be eaten by humans, and one embodiment can be a processed product. For example, the medicine, microorganism, compound, etc. of the present invention can be mixed into processed foods such as confectionery, dairy products, and processed grain products. In addition, the terms "health food" and "functional food" have the meaning commonly used in the industry and refer to a type of food that is distinguished from medicines or general foods. Examples of such foods include, but are not limited to, foods that are taken by subjects for a certain period of time before or with meals.

[0034] In this specification, the term "supplement" does not only mean nutritional supplements for supplementing nutrients, etc., but also means health functional foods and the like that have functions useful for maintaining, restoring, improving, etc. health (for example, anti-obesity effects such as suppressing weight gain and suppressing body fat accumulation, or slimming effects).

[0035] In this specification, the terms "disease," "disorder," and "condition" are used interchangeably and are interpreted in the broadest sense to refer to a state of mind or body disorder or inconvenience in humans or animals, and refer to any condition that is not specifically defined as being unhealthy, such as an illness, disorder, or various symptoms.

[0036] As used herein, "treatment" refers to the act of administering, for example, an active ingredient of the present disclosure to an individual (subject, patient) who has been diagnosed by a physician or equivalent practitioner as having a disease, for the purpose of, for example, alleviating, mitigating, or ameliorating the disease or symptoms, removing causative factors of the disease in the subject, or restoring the subject to a state prior to the onset of the disease.

[0037] In this specification, "prevention" refers to the act of administering the active ingredient of the present disclosure to an individual who has not developed the target disease, for example, with the purpose of preventing the onset of the disease. A vaccine can be said to be a typical example of a medicine for the purpose of prevention. In this disclosure, even if a causative factor of a disease is present in a subject, if the disease has not developed, it is usually not judged to be a disease state, so even in such a state, it can be treated and can be said to be prevention.

[0038] As used herein, the term "growth (activity)", when referring to microorganisms, refers to an increase in the number of individuals, cells, etc. of the microorganism, or its activity.

[0039] (Preferred embodiment) Preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description in this specification. In addition, the following embodiments of the present disclosure can be used alone or in combination.

[0040] In one aspect of the present disclosure, lactic acid bacteria having the ability to activate dendritic cells in Peyer's patches are provided.

[0041] While the main function of the intestine is digestion and absorption, it is also extremely important as an immune organ. Since many pathogenic bacteria enter the body through the mouth and invade the body via the intestines, the intestine has many immune organs arranged as a self-defense system to protect the body from these invaders. Specifically, the intestine has immune organs such as intestinal intraepithelial lymphocytes surrounded by intestinal epithelial cells (absorptive cells) in the intestinal wall, Peyer's patches in the intestinal wall, and the lamina propria mucosa in the lower part of the intestinal wall, all of which are localized as immune system organs.

[0042] Among these, Peyer's patches are important immune organs localized in the intestinal wall, and the intestinal luminal side of Peyer's patches, where antigens such as pathogenic bacteria invade, is covered with a single epithelial layer, and M cells exist at the entrance, through which pathogens are taken in. Major immune cells such as dendritic cells, T cells, and B cells are concentrated in this Peyer's patch region. These intestinal immune cell groups work together to produce immunoglobulin A in response to pathogenic bacterial antigens, and use this to prevent pathogenic bacteria from invading the intestinal tract and entering the body through the intestinal wall.

[0043] In one embodiment, the lactic acid bacteria and / or composition of the present disclosure can obtain an effect of activating the intestinal immune system by increasing the positive rate of a specific cell surface marker and / or increasing the production rate of cytokines in dendritic cells in the intestinal Peyer's patches. Thus, in one embodiment, the ability of the lactic acid bacteria of the present disclosure to activate dendritic cells in Peyer's patches can be evaluated by the presence or absence of expression of cell surface markers and / or cytokines indicative of activation in dendritic cells. In another embodiment, the ability of the lactic acid bacteria of the present disclosure to activate dendritic cells in Peyer's patches can be evaluated by the positive rate of cell surface markers and / or cytokines indicative of activation in dendritic cells.

[0044] In one embodiment, the cell surface marker and / or cytokine serving as an indicator of the activation is not particularly limited as long as it is a cell surface marker and / or cytokine serving as an indicator of the activation ability of dendritic cells in Peyer's patches, and examples thereof include at least one cell surface marker and / or cytokine selected from the group consisting of IL-6, IL-12, MHC class I, MHC class II, CD80, CD86, CD8, IFN-γ, IFN-α, TNF-α, CD40, IL-1β, IL-23, IL-27, and CCR7. Thus, in one embodiment, the activation ability of the lactic acid bacteria of the present disclosure can be evaluated by at least one positivity rate selected from the group consisting of IL-6 positivity rate, IL-12 positivity rate, MHC class I positivity rate, MHC class II positivity rate, CD80 positivity rate, CD86 positivity rate, CD8 positivity rate, IFN-γ positivity rate, IFN-α positivity rate, TNF-α positivity rate, CD40 positivity rate, IL-1β positivity rate, IL-23 positivity rate, IL-27 positivity rate, and CCR7 positivity rate in dendritic cells. Without being bound by theory, these cell surface markers and / or cytokines are important auxiliary signals for activated dendritic cell action (e.g., important costimulatory molecules for T cells (CD80, CD86, etc.), MHC presenting antigen peptides that induce T cell activation / differentiation, cytokines that enhance / regulate immune responses (IL-6, etc.)), and therefore can be indicators of the activation ability of dendritic cells in Peyer's patches by the lactic acid bacteria of the present disclosure.

[0045] In one embodiment, the ability of the lactic acid bacteria of the present disclosure to activate dendritic cells in Peyer's patches is determined by measuring the CD45 + CD11c + IL-6 + / CD45 + , CD45 + CD11c + IFN-γ + / CD45 + , CD45 + CD11c + MHC class II + / CD45 + , CD45 + CD11c+ CD80 + / CD45 + , CD45 + CD11c + CD86 + / CD45 + , and CD45 + CD11c + CD8a + / CD45 + Any one of the parameters selected from the group consisting of can be increased above a reference value, in which case the activation ability can be evaluated by evaluation system A. The reference value can be the positive rate of the PBS-administered group, in which case the above parameters can be expressed as a multiple of the positive rates in each bacterial strain-administered group, calculated by calculating the positive rates of each parameter and CD45-positive / CD11c-positive dendritic cells in CD45-positive leukocytes for the bacterial strain-administered group, with the positive rate for the PBS-administered group set at 1.

[0046] In one embodiment, the ability of the lactic acid bacteria of the present disclosure to activate dendritic cells in Peyer's patches can increase any one of the above parameters in dendritic cells by at least about 2.0-fold, about 2.3-fold, about 2.5-fold, about 2.7-fold, about 3.0-fold, about 3.3-fold, about 3.5-fold, about 3.7-fold, about 4.0-fold, about 4.3-fold, about 4.5-fold, about 4.7-fold, or about 5.0-fold compared to the baseline value.

[0047] In one embodiment, the ability of the lactic acid bacteria of the present disclosure to activate dendritic cells in Peyer's patches can increase any one, preferably at least two, more preferably at least three, or more preferably all of the above-mentioned parameters in dendritic cells by at least about 2.0-fold, about 2.3-fold, about 2.5-fold, about 2.7-fold, about 3.0-fold, about 3.3-fold, about 3.5-fold, about 3.7-fold, about 4.0-fold, about 4.3-fold, about 4.5-fold, about 4.7-fold, or about 5.0-fold compared to the baseline value.

[0048] In one embodiment, evaluation system A can include the following steps. 1) (Preparation of administration bacteria) The target strain is cultured, stained with carboxyfluorescein succinimidyl ester (CFSE), and then washed with PBS to prepare an administration solution. 2) (Collection of small intestinal Peyer's patches) 1.0 x 10 9 Each bacterial solution was administered by forced intragastric oral administration at colony forming unit (CFU) / 100 μL to C57BL / 6 mice, and the mice were dissected about 1 hour later to collect Peyer's patches of the small intestine. 3) (Antibody staining of cells in small intestinal Peyer's patches) Cells from small intestinal Peyer's patches are separated and suspended in cell culture medium, a protein transport inhibitor is added and the cells are cultured for approximately 15 hours, then the cells are washed by centrifugation with PBS, dead cells are stained, and the cells are washed by centrifugation with staining buffer (1% fetal bovine serum, 0.09% NaN3 in PBS) and blocked. Cell surface markers are then stained with various labeled antibodies. Next, the cells are fixed and permeabilized with a cell fixation / permeabilization solution, washed with a cell membrane permeabilization buffer, and intracellular markers are stained with anti-cytokine antibodies. The cells are washed with a cell membrane permeabilization buffer and measured and analyzed using a flow cytometer.

[0049] In one embodiment, the lactic acid bacteria of the present disclosure have a Peyer's patch dendritic cell activation index of: IL-6 + / CD45 + : greater than 1.43, MHC class II + / CD45 + : greater than 1.82, CD80 + / CD45 + : greater than 1.41, and CD86 + / CD45 + : greater than 1.22, In this case, as an indicator of dendritic cell activation in Peyer's patches, 1.0 × 10 9In case that small intestinal Peyer's patches are collected about 1 hour after oral administration of colony forming unit (CFU) / 100 μL of bacterial solution and cultured for about 15 hours and then antibody staining is performed, the positivity rates of each of the following markers in CD45-positive leukocytes in CD45-positive and CD11c-positive dendritic cells in Peyer's patches can be calculated, and the positivity rate in the PBS-administered group can be set to 1, and the multiple of the positivity rate in each bacterial strain-administered group can be used.

[0050] In another embodiment, the lactic acid bacteria of the present disclosure has an activation index of Peyer's patch dendritic cells of IL-6 + / CD45 + :2 or more, MHC class II + / CD45 + :2 or more, CD80 + / CD45 + :2 or more, and CD86 + / CD45 + :2 or more, The polymer may have at least one property selected from the group consisting of:

[0051] In another embodiment, the lactic acid bacteria of the present disclosure has an activation index of Peyer's patch dendritic cells of IL-6 + / CD45 + : 2.5 or higher, MHC class II + / CD45 + : 2.5 or higher, CD80 + / CD45 + : 2.5 or higher, and CD86 + / CD45 + : 2.5 or higher, The polymer may have at least one property selected from the group consisting of:

[0052] In another embodiment, the lactic acid bacteria of the present disclosure has an activation index of Peyer's patch dendritic cells of IL-6+ / CD45 + : 2.5 or higher, MHC class II + / CD45 + :4 or more, CD80 + / CD45 + : 2.5 or higher, and CD86 + / CD45 + : 2.5 or higher, The polymer may have at least one property selected from the group consisting of:

[0053] In another embodiment, the lactic acid bacteria of the present disclosure has an activation index of Peyer's patch dendritic cells of IL-6 + / CD45 + : 3 or more, MHC class II + / CD45 + :4 or more, CD80 + / CD45 + :3 or more, and CD86 + / CD45 + : 2.5 or higher, The polymer may have at least one property selected from the group consisting of:

[0054] In still another embodiment, the lactic acid bacteria of the present disclosure can have an indicator of activation of dendritic cells within Peyer's patches that has at least two, preferably at least three, and more preferably all of the above properties, for example, the lactic acid bacteria of the present disclosure can have an indicator of activation of dendritic cells within Peyer's patches that is at least positive for IL-6 and CD80.

[0055] In one embodiment, the ability of the lactic acid bacteria of the present disclosure to activate dendritic cells in Peyer's patches may be greater than that of a reference strain of lactic acid bacteria, and an example of the reference strain of lactic acid bacteria may be E. faecalis ATCC 700802.

[0056] Peyer's patches are aggregated lymphoid follicles that contain a number of lymphoid follicles, and many of the individual lymphoid follicles have activated germinal centers. Many B cells gather in Peyer's patches, which function as sites for the production of antibodies against non-self substances, and lymphoid follicles in which B cells have accumulated are covered with dome-shaped follicle-associated epithelium (FAE). Thus, in one embodiment, dendritic cells activated by the lactic acid bacteria of the present disclosure can exist between the follicle-associated epithelium (FAE) and the follicle. Without being bound by theory, it is known that dendritic cells present between the follicle-associated epithelium (FAE) and the follicle are responsible for immune responses to non-self antigens on the intestinal mucosa, and are believed to be activated by the lactic acid bacteria of the present disclosure.

[0057] In one embodiment of the present disclosure, the lactic acid bacteria of the present disclosure may have the ability to activate at least one other cell selected from the group consisting of macrophages, B cells, and T cells. Without being bound by theory, it is believed that the lactic acid bacteria of the present disclosure can activate the TLR of macrophages by peptidoglycan in the cell wall of lactic acid bacteria, and that B cells and T cells are known to be stimulated by activated dendritic cells, and therefore, each of them can be activated by the lactic acid bacteria of the present disclosure.

[0058] In one embodiment of the present disclosure, the lactic acid bacteria contained in the composition of the present disclosure may include acid bacteria of the genera Enterococcus, Bifidobacterium, Bacteroides, Lactobacillus, and Ackermansia, preferably acid bacteria of the genera Enterococcus and Bifidobacterium, and more preferably acid bacteria of the genus Enterococcus.

[0059] Bacteria of the genus Enterococcus include, but are not limited to, Enterococcus alcedinis, Enterococcus aquimarinus, Enterococcus asini, Enterococcus avium, Enterococcus bulliens, Enterococcus caccae, Enterococcus camelliae, Enterococcus canintestini, Enterococcus canis, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus columbae, Enterococcus devriesei, Enterococcus diestrammenae, Enterococcus dispar, Enterococcus durans, Enterococcus eurekensis, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus gilvus, Enterococcus haemoperoxidus, Enterococcus hermanniensis, Enterococcus hirae, Enterococcus italicus, Enterococcus lactis, Enterococcus lemanii, Enterococcus malodoratus, Enterococcus moraviensis, Enterococcus mundtii, Enterococcus olivae, Enterococcus pallens, Enterococcus phoeniculicola, Enterococcus plantarum, Enterococcus pseudoavium, Enterococcus quebecensis, Enterococcus raffinosus, Enterococcus ratti, Enterococcus rivorum, Enterococcus rotai, Enterococcus saccharolyticus、Enterococcus silesiacus、Enterococcussolitarius, Enterococcus sulfureus, Enterococcus termitis, Enterococcus thailandicus, Enterococcus ureasiticus, Enterococcus ureilyticus, Enterococcus viikkiensis, Enterococcus villorum, Enterococcus xiangfangensis, etc. In one embodiment, preferred examples of bacteria of the genus Enterococcus include E. faecalis, E. faecium, E. avium, E. casseliflavus, E. gallinarum, and E. hirae, and more preferred examples include Enterococcus faecalis.

[0060] The design of the skeletal muscle and the skeletal muscle of the B.S. actinocoloniiform、B. adolescentis、B. aerophilum、B. aesculapii、B. angulatum、B. animalis、 subsp. animalis、subsp. lactis、B. aquikefiri、B. asteroids、B. avesanii、B. biavatii、B. bifidum、B. bohemicum、B. both、B. boum、B. brief、B. callitrichos、B. catenulatum、B. choerinum、B. commune、B. coryneform、B. crudilactis、B. cuniculi、B. denticolens、B. dentium、B. eulemuris、B. faecale、B. gallicum、B. gallinarum、B. even、B. indicum、B. inopinatum、B. kashiwanohense、B. lemurum、B. longum、subsp. longum、subsp. infantis、subsp. I am、B. magnum、B. merycicum、B. minimum、B. Mongolian、B. moukalabense、B. myosotis、B. parvulorum、B. pseudocatenulatum、B. pseudolongum、subsp. pseudolongum、subsp. globosum、B. psychraerophilum、B. pullorum、B. ramosum、B. reuteri、B. ruminale、B. ruminantium、B. saeculare、B. saguini、B. scardovii、B. simiae、B. stellenboschense、B. subtle、B. thermacidophilum、subsp. thermacidophilum、subsp. porcinum、B. thermophilum、B. tissieri、またはB. tsurumiense is a friend.

[0061] Bacteria of the genus Bacteroides include, but are not limited to, Bacteroides acidifaciens, Bacteroides caccae, Bacteroides caecicola, Bacteroides caecigallinarum, Bacteroides caecimuris, Bacteroides ellulosilyticus, Bacteroides clarus, Bacteroides coprosuis, Bacteroides eggerthii, Bacteroides faecalis, Bacteroides faecichinchillae, Bacteroides faecis, Bacteroides finegoldii, Bacteroides fluxus, Bacteroides fragilis, Bacteroides galacturonicus, Bacteroides gallinaceum, Bacteroides gallinarum, Bacteroides graminisolvens, Bacteroides helcogenes, Bacteroides intestinalis, Bacteroides koreensis, Bacteroides kribbi, Bacteroides luti, Bacteroides nordii, Bacteroides oleiciplenus, Bacteroides ovatus, Bacteroides pectinophilus, Bacteroides polypragmatus, Bacteroides propionicifaciens, Bacteroides pyogenes, Bacteroides It can include reticulotermitis, Bacteroides rodentium, Bacteroides salyersiae, Bacteroides stercorirosori, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, or Bacteroides xylanisolvens, etc.

[0062] Examples of bacteria belonging to the genus Lactobacillus, but not limited to these, include L. acetotolerans, L. acidifarinae, L. acidipiscis, L. acidophilus, L. agilis, L. algidus, L. alimentarius, L. amylolyticus, L. amylophilus, L. amylotrophicus, L. amylovorus, L. animalis, L. antri, L. apodemi, L. aviarius, L. bifermentans, L. brevis, L. buchneri, L. camelliae, L. casei, L. catenaformis, L. ceti, L. coleohominis, L. collinoides, L. composti, L. concavus, L. coryniformis, L. crispatus, L. crustorum, L. curvatus, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis, L. dextrinicus, L. diolivorans, L. equi, L. equigenerosi, L. farraginis, L. farciminis, L. fermentum, L. fornicalis, L. fructivorans, L. frumenti, L. fuchuensis, L. gallinarum, L. gasseri, L. gastricus, L. ghanensis, L. graminis, L. hammesii, L. hamsteri, L. harbinensis, L. hayakitensis, L. helveticus, L. hilgardii, L. homohiochii, L. iners, L. ingluviei, L. intestinalis, L. jensenii, L. johnsonii, L. kalixensis, L. kefiranofaciens, L. kefiri, L. kimchii, L. kitasatonis, L. kunkeei, L. leichmannii, L.lindneri、L. malefermentans、L. mali、L. manihotivorans、L. mindensis、L. mucosae、L. murinus、L. nagelii、L. namurensis、L. nantensis、L. oligofermentans、L. oris、L. panis、L. pantheris、L. parabrevis、L. parabuchneri、L. paracasei、L. paracollinoids、L. parafarraginis、L. parakefiri、L. paralimentaryus、L. paraplantarum、L. pentosus、L. perolens、L. plantarum、L. pontis、L. protectus、L. psittaci、L. rennini、L. reuteri、L. rhamnosus、L. rimae、L. rogosae、L. rossiae、L. ruminis、L. saerimneri、L. tiger、L. salivarius、L. sanfranciscensis、L. satsumensis、L. secaliphilus、L. sharpeae、L. siliginis、L. spicheri、L. suebicus、L. thailandensis、L. ultunensis、L. vaccinostercus、L. vaginalis、L. versmoldensis、L. vini、L. vitulinus、L. zeae、L. zymae is strong.

[0063] The preparation of the Akkermansia muciniphila and Akkermansia glycaniphila.

[0064] In one embodiment, the lactic acid bacteria contained in the composition of the present disclosure is Enterococcus faecalis, and preferably includes the KU-EF-001 strain (NITE BP-03655), the KU-EF-002 strain (NITE BP-03656), the KU-EF-003 strain (NITE BP-03657), and the KU-EF-004 strain (NITE BP-03658).

[0065] In one aspect of the present disclosure, a composition comprising the lactic acid bacteria of the present disclosure is provided. As shown in the Examples, the lactic acid bacteria of the present disclosure can stimulate the immunity of a subject, and therefore the composition comprising the lactic acid bacteria of the present disclosure can be used to stimulate the immunity of a subject.

[0066] In one embodiment, the lactic acid bacteria of the present disclosure can activate dendritic cells in Peyer's patches, and therefore a composition containing the lactic acid bacteria of the present disclosure can be intended for activating dendritic cells in Peyer's patches.

[0067] It is known that lipoteichoic acid and peptidoglycan in the cell wall of lactic acid bacteria stimulate TLR on dendritic cells, and that activation of dendritic cells stimulates immune cells such as T lymphocytes between intestinal epithelial cells and B lymphocytes in the lamina propria of the mucosa. It is also known that short-chain fatty acids (such as lactic acid and acetic acid) produced by lactic acid bacteria improve the intestinal flora and intestinal environment. Therefore, by ingesting a composition containing the lactic acid bacteria of the present disclosure, the intestinal immune system of a subject can be activated. Therefore, in one embodiment of the present disclosure, the composition containing the lactic acid bacteria of the present disclosure can be used in combination with an immunomodulator or a radiation agent, and / or can be used to enhance the action thereof.

[0068] In one embodiment, the immunomodulatory agent may include, but is not limited to, immune checkpoint inhibitors, CAR-T cell therapy drugs, bispecific antibody drugs, cancer vaccines, costimulatory molecule agonist drugs, immune activators, and small molecule inhibitors. Any commonly available immune checkpoint inhibitor may be used as the immune checkpoint inhibitor, and examples of immune checkpoint inhibitors include agents against molecules selected from the group consisting of CTLA-4, PD-1, LAG-3, BTLA, KIR, TIM-3, PD-L1, PD-L2, B7-H3, B7-H4, HVEM, GAL9, CD160, VISTA, BTNL2, TIGIT, PVR, BTN1A1, BTN2A2, BTN3A2, and CSF-1R, and any combination thereof.

[0069] In one embodiment, the lactic acid bacteria contained in the composition of the present disclosure may be live bacteria, and in this case, they may be added directly to food or feed as a liquid, tablet, powder, or the like. They may also be directly ingested as a liquid or tablet. In one embodiment, the number of live bacteria contained in the composition of the present disclosure is not particularly limited, and may be a very small amount of at least 1 CFU / g, at least about 10 CFU / g, at least about 1×10 2 CFU / g, at least about 1 × 10 3 CFU / g, at least about 1 × 10 4 CFU / g, at least about 1 × 10 5 CFU / g, at least about 1 × 10 6 CFU / g, at least about 1 × 10 7 CFU / g, at least about 1 × 10 8 CFU / g, at least about 1 × 10 9 CFU / g, at least about 1 × 10 10 CFU / g, at least about 1 × 10 12 CFU / g, at least about 1 × 10 14 CFU / g, at least about 1 × 10 16 CFU / g, or at least about 1 × 10 18The viable cell count in the composition of the present disclosure can be appropriately changed depending on the form of the composition and the form of food or feed to which the composition is added.

[0070] In one embodiment, the composition of the present disclosure can be used as a food or drink (including foods for specified health uses and foods with functional claims), food additives, feed, supplements, or medicines. When the composition of the present disclosure is used as a food or drink, it is possible to add an explanation such as "recommended for ____ people" or "useful for ____" to the label.

[0071] <Administration Method> When it is preferable to administer the compound for the purpose of treating or preventing a disease, it is administered in any form of preparation. The route of administration is not particularly limited, and includes oral administration and parenteral administration. Examples of preparations suitable for oral administration include, for example, tablets, granules, fine granules, powders, syrups, solutions, capsules, or suspensions. Examples of preparations suitable for parenteral administration include, for example, injections, drips, inhalants, sprays, suppositories, transdermal absorbents, transmucosal absorbents, etc.

[0072] For the preparation of liquid preparations for oral administration, additives for preparations such as water, sugars such as sucrose, sorbitol, fructose, etc., glycols such as polyethylene glycol, propylene glycol, etc., oils such as sesame oil, olive oil, soybean oil, etc., preservatives such as p-hydroxybenzoic acid esters, etc. can be used. For the preparation of solid preparations such as capsules, tablets, powders, or granules, excipients such as lactose, glucose, sucrose, mannitol, etc., disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, binders such as polyvinyl alcohol, hydroxypropyl cellulose, gelatin, etc., surfactants such as fatty acid esters, etc., and plasticizers such as glycerin can be used.

[0073] Among the preparations for parenteral administration, preparations for intravascular administration such as injections and drip infusions can be prepared using an aqueous medium that is preferably isotonic with human blood. For example, injections can be prepared as solutions, suspensions or dispersions using an aqueous medium selected from a salt solution, a glucose solution, or a mixture of a salt solution and a glucose solution, together with appropriate auxiliaries according to conventional methods. Suppositories for enteral administration can be prepared using a carrier such as cocoa butter, hydrogenated oils and fats, or hydrogenated fatty acids. Among the preparations for parenteral administration, sprays can be prepared using a carrier that does not irritate the human oral and respiratory mucosa and can disperse the active ingredient bacteria as fine particles to promote absorption. Examples of such carriers include lactose and glycerin. Depending on the properties of the transformed bifidobacteria and the carrier used, the preparations can be prepared in the form of aerosols or dry powders. For the production of preparations for parenteral administration, one or more additives for preparations selected from, for example, diluents, flavors, preservatives, excipients, disintegrants, lubricants, binders, surfactants, plasticizers, etc. can be used.

[0074] <Agents and additives> In one embodiment of the present disclosure, the composition of the present disclosure may further include additives such as formulation additives, excipients, fillers, binders, thickeners, emulsifiers, colorants, fragrances, food additives, and seasonings depending on the dosage form. Examples of such additives and excipients include vitamins such as ascorbic acid, biotin, calcium pantothenate, carotene, niacin, pyridoxine hydrochloride, riboflavin, sodium pantothenate, thiamine hydrochloride, tocopherol, vitamin A, vitamin B12, and vitamin D; sodium phosphates such as sodium metaphosphate, sodium phosphate, sodium pyrophosphate, and trisodium phosphate; preservatives such as calcium sorbate, benzoic acid, paraoxybenzoic acid esters, and sodium benzoate; gum arabic, tragacanth, sodium alginate, methylcellulose, carboxymethylcellulose, calcium alginate, calcium silicate, mannitol, sorbitol, lactose, soluble starch, amino acids, glucose, fructose, sucrose, honey, fatty acid esters, and silicon dioxide.

[0075] In one embodiment of the present disclosure, the composition of the present disclosure may be in the form of, for example, powder, granule, pellet, tablet, capsule, oil, and liquid.

[0076] In one embodiment of the present disclosure, the composition of the present disclosure may be included in food and beverage products, quasi-drug products, and medicines. Among these, examples of food and beverage products include various processed foods such as vegetable processed foods, seaweed processed foods, and meat processed products; soups, snacks, breads, noodles, ice creams, tea, juices, coffee, health foods, and dairy products such as yogurt and cheese.

[0077] In one aspect of the present disclosure, a pharmaceutical composition is provided that includes a composition comprising the lactic acid bacteria of the present disclosure and an immunomodulatory agent, in which the lactic acid bacteria or compositions of the present disclosure described elsewhere herein can be used.

[0078] (General technology) The molecular biological, biochemical and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al. (1995). PCR Strategies, Academic Press; Ausubel, FM (1999).These methods are described in Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Special Edition of Experimental Medicine: "Gene Introduction & Expression Analysis Experimental Methods" Yodosha, 1997, etc., and the relevant parts (possibly in their entirety) of these are incorporated herein by reference.

[0079] Regarding DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, gene synthesis and fragment synthesis services such as those of GeneArt, GenScript, Integrated DNA Technologies (IDT), etc. can be used. In addition, for example, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (I996). Bioconjugate Techniques, Academic Press, etc., the relevant portions of which are incorporated herein by reference.

[0080] In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when "within the range of" two values ​​is specified, the range includes the two values ​​themselves. All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each was specifically set forth.

[0081] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described in this specification, but is limited only by the scope of the claims. EXAMPLES

[0082] method Bacterial strains and media The following five bacterial strains were purchased from the American Type Culture Collection (ATCC) and used as the main bacterial species that constitute the human intestinal flora. ·Escherichia coli ATCC 10798 (K-12) ·Enterococcus faecalis ATCC 700802 ·Enterococcus hirae ATCC 9790 ·Bifidobacterium longum ATCC 15697 ·Lactobacillus acidophilus ATCC 4536

[0083] E. coli ATCC 10798 (K-12) was cultured aerobically at 37°C in Luria-Bertani (LB) medium (Nacalai Tesque, Kyoto, Japan). E. faecalis ATCC700802 and E. hirae ATCC9790 were cultured aerobically at 37°C in Brain Heart Infusion (BHI) medium (Merck, Darmstadt, Germany). B. longum ATCC 15697 was cultured anaerobically at 37°C in Gifu Anaerobic Medium (GAM) medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan). L. acidophilus ATCC 4536 was cultured anaerobically at 37°C in DE MAN, ROGOSA and SHARPE (MRS) medium (Merck).

[0084] In addition, the following E. faecalis strains were isolated from clinical specimens at Kobe University Hospital. These strains were also cultured aerobically at 37°C using Brain Heart Infusion (BHI) medium (Merck, Darmstadt, Germany) in the same manner as above. All of these strains are vancomycin-sensitive. KU-EF-001 strain (NITE BP-03655): E. faecalis 22811 (derived from a urine sample) KU-EF-002 strain (NITE BP-03656): E. faecalis 22837 (from balloon urine) KU-EF-003 strain (NITE BP-03657): E. faecalis 22850 (derived from venous blood) KU-EF-004 strain (NITE BP-03658): E. faecalis 22869 (urinary origin)

[0085] Preparation of the administration solution Each bacterial strain was cultured as described above, and after washing three times with phosphate-buffered saline (PBS), a bacterial suspension of appropriate concentration was prepared in PBS. Each bacterial suspension was frozen and stored at -80°C until use.

[0086] (Example 1: Selection of bacterial species) Seven-week-old female C57BL / 6 mice were used. 5 Mouse colon cancer cell line MC38 cells were subcutaneously inoculated into the right flank of mice under anesthesia, and the day of tumor inoculation was designated as day 0. On day 7, mice were randomly assigned to each of the following oral administration groups (n=3). PBS, E. coli ATCC 10798 (K-12) strain, E. faecalis ATCC 700802 strain, E. hirae ATCC 9790 strain, B. longum ATCC 15697 strain, and L. acidophilus ATCC 4536 strain were inoculated at 1.0×10 cells / mL under anesthesia using a probe. 9Colony forming units (CFU) / 100 μl PBS was administered by intragastric forced oral administration. Anti-programmed death 1 (PD-1) antibody, anti-programmed death ligand 1 (PD-L1) antibody, anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody (all Bio X Cell, Lebanon, NH) and anti-PD-1 antibody × anti-CTLA-4 antibody (1:1) were each administered intraperitoneally at 200 μg / 100 μL. Oral administration was started on the 7th day after tumor implantation, and was performed once a day, 5 times a week for 4 consecutive weeks. Intraperitoneal administration was performed twice a week from the 11th day, for a total of 5 times (Figure 1). Tumor diameters were measured daily, and the volumes were calculated using the following formula: tumor volume (mm 3 ) = Diameter (mm) x Short diameter (mm) 2 ×1 / 2. The endpoint was a tumor diameter of 20 mm or more.

[0087] Result 1 Figure 2 shows the change in tumor volume when five types of bacterial strains and PBS were orally administered to mice with subcutaneously implanted MC38, and immune checkpoint inhibitors (anti-PD-1 antibody and anti-CTLA-4 antibody) were administered intraperitoneally to these mice. Because the endpoint was a tumor diameter of 20 mm or more, calculation of the average tumor volume was discontinued for treatment groups that included individuals with tumor diameters of 20 mm or more. Analysis of the relationship between oral administration of each bacterial species and the antitumor effect of immune checkpoint inhibitors (anti-PD-1 antibody and anti-CTLA-4 antibody) showed a significant reduction in tumor volume when oral administration of E.faecalis strains and B.longum strains was combined with administration of anti-PD-1 antibody and anti-CTLA-4 antibody. No obvious side effects were observed during the administration period.

[0088] These results suggest that E. faecalis and B. longum strains improve the cancer therapeutic effects of anti-PD-1 and anti-CTLA-4 antibodies.

[0089] (Example 2: Comparison of 3 E faecalis strains + 1 strain (ATCC strain)) Seven-week-old female C57BL / 6 mice were used. 5 Mice were subcutaneously inoculated with 1.0 × 10 MC38 cells into the right flank under anesthesia, and the day of tumor inoculation was designated as day 0. On day 8, mice were randomly assigned to the following oral administration groups (n = 4). PBS, E. faecalis ATCC 700802 strain, KU-EF-001 strain, KU-EF-002 strain, KU-EF-003 strain, and KU-EF-004 strain were each inoculated with 1.0 × 10 MC38 cells into the right flank under anesthesia using a probe. 9 CFU / 100μl PBS was administered by intragastric forced oral administration. Anti-PD-1 antibody (Bio X Cell) and anti-CTLA-4 antibody (Bio X Cell) were administered intraperitoneally at 200μg / 100μL for each of the oral administration groups. Oral administration was started on the 8th day, once a day, 5 times a week for 4 consecutive weeks. Intraperitoneal administration was started on the 12th day, twice a week for a total of 5 times (Figure 3). Tumor diameter was measured daily, and the volume was calculated using the following formula: Tumor volume (mm 3 ) = Diameter (mm) x Short diameter (mm) 2 ×1 / 2. The endpoint is a tumor diameter of 20 mm or more and a tumor volume of 500 mm 3 It was decided.

[0090] Result 2 Figure 4 shows the progression of tumor volume when five types of E.faecalis strains and PBS were orally administered to mice with subcutaneously implanted MC38, and immune checkpoint inhibitors (anti-PD-1 antibody and anti-CTLA-4 antibody) were intraperitoneally administered to them. Since the endpoint was a tumor diameter of 20 mm or more, calculation of the average tumor volume was discontinued for treatment groups that included individuals with tumor diameters of 20 mm or more. Analysis of the relationship between oral administration of each bacterial strain and the antitumor effect of various immune checkpoint inhibitors showed the most significant reduction in tumor volume when oral administration of the KU-EF-004 strain was combined with the combined administration of anti-PD-1 antibody and anti-CTLA-4 antibody. Survival time was most extended when oral administration of the KU-EF-002 strain was combined with the intraperitoneal administration of anti-PD-1 antibody and anti-CTLA-4 antibody. The combination of oral administration of KU-EF-004 and intraperitoneal administration of anti-PD-1 and anti-CTLA-4 antibodies showed an extension of survival time, second only to the KU-EF-002 group. These results suggest that the KU-EF-004 and KU-EF-002 strains enhance the host's sensitivity to immune checkpoint inhibitors.

[0091] (Example 3: Comparison of KU-EF-004 strain with other Enterobacteriaceae strains) Seven-week-old female C57BL / 6J mice were used. 5 MC38 was subcutaneously inoculated into the right flank of mice under anesthesia, and the day of tumor inoculation was designated as day 0. On day 6, mice were randomly assigned to the following oral administration groups (n=5). PBS, KU-EF-004 strain, E. coli ATCC10798 (K-12) strain, and B. longum ATCC15697 strain were administered at 1.0×10 sucrose under anesthesia using a probe. 9 CFU / 100 μl PBS was administered by forced intragastric oral administration. 10 μg / 100 μL of anti-CTLA-4 antibody (InvivoGen, San Diego, CA) was administered intraperitoneally to each of the oral administration groups. Oral administration was started on the 7th day after tumor implantation, and was performed once a day, 5 times a week for 5 consecutive weeks. Intraperitoneal administration was performed once a week from the 10th day, for a total of 4 times (Figure 5). Tumor diameter was measured daily, and the volume was calculated using the following formula: Tumor volume (mm 3) = Diameter (mm) x Short diameter (mm) 2 ×1 / 2. The endpoint is a tumor diameter of 20 mm or more, or a tumor volume of 500 mm 3 It was decided.

[0092] Result 3 Figure 6 shows the progression of tumor volume and survival time when three types of bacteria strains and PBS were orally administered to mice with subcutaneously implanted MC38, and anti-CTLA-4 antibody was administered intraperitoneally to them. Since the endpoint was a tumor diameter of 20 mm or more, calculation of the average tumor volume was discontinued for treatment groups that included individuals with tumor diameters of 20 mm or more. Analysis of the relationship between oral administration of each bacterial species and the anti-CTLA-4 antibody showed the most significant reduction in tumor volume when oral administration of the KU-EF-004 strain was combined with anti-CTLA-4 antibody administration. Survival time was significantly extended when oral administration of the KU-EF-004 strain was combined with intraperitoneal administration of anti-CTLA-4 antibody (p=0.0088). These results suggest that the KU-EF-004 strain enhances the host's sensitivity to anti-CTLA-4 antibody compared to the E. coli ATCC10798 (K-12) strain and the B. longum ATCC15697 strain.

[0093] (Example 4: Comparison of KU-EF-004 strain and E. faecalis ATCC 700802 strain) Seven-week-old female C57BL / 6J mice were used. 5 MC38 was subcutaneously inoculated into the right flank of mice under anesthesia, and the day of tumor inoculation was designated as day 0. On day 6, mice were randomly assigned to the following oral administration groups (n=5). PBS, KU-EF-004 strain, and E. faecalis ATCC 700802 strain were inoculated at 1.0 × 10 cells / mL using a probe under anesthesia. 9CFU / 100 μl PBS was administered orally by intragastric force. 10 μg / 100 μL of anti-CTLA-4 antibody (InvivoGen) was administered intraperitoneally to each of the oral administration groups. Oral administration was started on the 6th day after tumor implantation, and was carried out once a day, 5 times a week for 5 consecutive weeks. Intraperitoneal administration was carried out once a week from the 10th day, for a total of 4 times (Figure 7). Tumor diameter was measured daily, and the volume was calculated using the following formula: Tumor volume (mm 3 ) = Diameter (mm) x Short diameter (mm) 2 ×1 / 2. The endpoint is a tumor diameter of 20 mm or more, or a tumor volume of 500 mm 3 It was decided.

[0094] Result 4 Mice with subcutaneously implanted MC38 were orally administered with KU-EF-004, E.faecalis ATCC700802 or PBS, and the survival time when anti-CTLA-4 antibody was administered intraperitoneally is shown in Figure 8. The endpoint was set to a tumor diameter of 20 mm or more, so the calculation of the average tumor volume was discontinued for treatment groups that included individuals with tumor diameters of 20 mm or more. Analysis of the relationship between oral administration of each bacterial strain and survival time of anti-CTLA-4 antibody showed that the survival time was significantly extended when oral administration of KU-EF-004 strain was combined with intraperitoneal administration of anti-CTLA-4 antibody (p=0.0031). These results suggest that KU-EF-004 strain enhances the host's sensitivity to anti-CTLA-4 antibody compared to E.faecalis ATCC700802 strain.

[0095] (Example 5: Uptake into dendritic cells in Peyer's patches by oral administration of KU-EF-004 strain to mice) E. coli ATCC 10798 (K-12), KU-EF-004, and B. longum ATCC 15697 were cultured and collected by centrifugation. Each cell was stained with 10 μg / mL carboxyfluorescein succinimidyl ester (CFSE) at 37°C for 30 minutes, then washed with PBS to prepare the bacterial solution. 9CFU / 100μL of each bacterial solution was administered intragastrically to 7-week-old female C57BL / 6N mice (n=3). Mice were dissected 1 hour after administration to collect the small intestinal Peyer's patches. The Peyer's patches were homogenized to prepare single cell suspensions, and then incubated with 10μg / mL anti-CD16 / 32 antibody (BioLegend, San Diego, CA) diluted in staining buffer (1% fetal bovine serum, 0.09% NaN3in PBS) for 20 minutes on ice for blocking. The cells were washed by centrifugation with staining buffer, and incubated with 2μg / mL PE-labeled anti-CD11c antibody (BioLegend) for 30 minutes on ice in the dark to stain dendritic cells. After washing with staining buffer, measurements were performed using a Guava flow cytometer (Luminex, Austin, Tx). Analysis was performed using the accompanying InCyte software (Luminex).

[0096] Result 5 It was confirmed that CFSE-labeled KU-EF-004 was taken up by cells (dendritic cells) positive for CD11c, a dendritic cell marker, in the Peyer's patches of the small intestine one hour after oral administration of the strain.

[0097] Example 6: Activation of dendritic cells in the small intestinal Peyer's patches by oral administration of E. faecalis strains to mice KU-EF-001, KU-EF-002, KU-EF-003, and KU-EF-004 strains, and Enterococcus faecalis ATCC 700802 strain were cultured and collected by centrifugation. Each bacterial cell was stained with 10 μg / mL carboxyfluorescein succinimidyl ester (CFSE) at 37°C for 30 minutes, then washed with PBS to prepare the bacterial solution. 9 Each bacterial solution was administered orally at 100 μL / CFU to female C57BL / 6N mice (n=3). One hour after administration, the mice were dissected and the small intestinal Peyer's patches were collected.

[0098] After homogenizing the Peyer's patches to prepare a single-cell suspension, the cells were suspended in RPMI-1640 (10% fetal bovine serum, 10 mM HEPES, 1 mM non-essential amino acids, 1 mM sodium pyruvate, 50 μM 2-mercaptomethanol, 100 IU / ml penicillin, 100 μg / ml streptomycin) and cultured at 37°C and 5% CO2 for 15 hours. Golgistop (BD Bioscience) was added 3 hours after the start of culture. After the culture was completed, the cells were collected and washed by centrifugation with PBS. The cells were stained with Zombie NIR (BioLegend, San Diego, CA) diluted 1000-fold with PBS for 15 minutes at room temperature. After washing by centrifugation with staining buffer (1% fetal bovine serum, 0.09% NaN3 in PBS), cells were blocked by incubation with 10 μg / mL anti-CD16 / 32 antibody (BioLegend, San Diego, CA) diluted in staining buffer for 20 min on ice. Cells were washed by centrifugation with staining buffer, and dendritic cells were stained with Brilliant Violet (BV) 510-conjugated anti-CD45 antibody and PE-conjugated anti-CD11c antibody. In addition, cells were stained for cell surface markers with BV421-conjugated anti-MHC class II antibody, BV650-conjugated anti-CD80 antibody, PE-Dazzle594-conjugated anti-CD86 antibody, BV570-conjugated CD8a antibody, and BV605-conjugated IFN-γ antibody (all BioLegend) for 30 min on ice in the dark. The cells were then fixed and permeabilized by incubating in BD Fixation / Permeabilization Solution (BD Bioscience, Franklin Lakes, NJ) for 20 minutes on ice in the dark, and then washed twice with 1x BD Perm / Wash buffer (BD Bioscience) by centrifugation. The cells were then stained for intracellular marker (IL-6) with APC-labeled anti-interleukin 6 (IL-6) antibody (BioLegend) for 30 minutes on ice in the dark. The cells were then washed twice with 1x BD Perm / Wash buffer, resuspended in staining buffer, and filtered through a 30 μm mesh. The samples were then analyzed by MACSQuant.(登録商標) The measurements were performed using an Analyzer 16 (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and the accompanying MACSQuantify 商標 The analysis was performed using software, and dendritic cells were defined as CD45 positive and CD11c positive, and the expression of surface markers (MHC class II, CD80, CD86, and CD8a) and production of intracellular markers (IL-6, IFN-γ) were analyzed.

[0099] Result 6 CFSE-stained KU-EF-001, KU-EF-002, KU-EF-003, and KU-EF-004 strains were orally administered to mice, and the small intestinal Peyer's patches were collected 1 hour after administration, cultured for 15 hours, and stained. The percentages of dendritic cells expressing IL-6 and IFN-γ, which are cytokines that activate cellular immunity, MHC class II, CD80, and CD86, which are dendritic cell maturation markers, and CD8a, which is a surface marker involved in Th1 induction, relative to the total CD45-positive leukocytes (positive rate) were calculated, and compared with the control group (PBS-administered group), the positive rates were higher in all of the KU-EF-001, KU-EF-002, KU-EF-003, and KU-EF-004 strains than in the control group (PBS-administered group). Furthermore, the positive rates in the groups administered KU-EF-001, KU-EF-002, and KU-EF-004 strains were all higher than the positive rate in the group administered E. faecalis ATCC 700802 strain (Table 1).

[0100] These results suggest that oral administration of lactobacillus strains, including the KU-EF-004 strain, increases the activity of dendritic cells in the Peyer's patches of the small intestine.

[0101] [Table 1]

[0102] (Example 7: Combination with cancer immunotherapy) A composition containing the lactic acid bacteria of the present disclosure is used in combination with cancer immunotherapy. A composition containing the lactic acid bacteria of the present disclosure is used in combination with cancer immunotherapy for patients receiving immune checkpoint inhibitors (anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, etc.), cell therapy (CAR-T cell therapy, TCR-T cell therapy, NK cell therapy, NKT cell therapy, γδT cell therapy, etc.), or other cancer immunotherapy (BCG bladder instillation therapy, cytokine therapy (IL-2, IL-12, IFN-γ, etc.), etc.).

[0103] (Example 8: Example of use as a single agent) The composition containing the lactic acid bacteria of the present disclosure is used as a single agent to confirm the cancer prevention effect, etc. The composition containing the lactic acid bacteria of the present disclosure is, for example, contained in food or supplements and applied to a subject.

[0104] Example 9: Administration of lactic acid bacteria to various infection models The composition containing the lactic acid bacteria of the present disclosure is used alone or in combination with other immunostimulants to confirm the preventive effect against infectious diseases, etc. Examples of infectious disease models include the following.

[0105] Salmonella infection model The lactic acid bacteria of the present disclosure were cultured and centrifuged to prepare 1×10 9 A composition containing CFU is administered to mice by forced oral administration into the stomach. The administration is continued for several weeks. After the final oral administration, the mice are orally inoculated with a lethal dose of Salmonella enterica serovar Typhimurium to infect them. The effectiveness of the lactic acid bacteria in preventing Salmonella infection is confirmed based on changes in body weight and survival rate after inoculation with Salmonella typhimurium, as well as quantitative results of the number of bacteria in the infected organs.

[0106] Influenza A infection model The lactic acid bacteria of the present disclosure were cultured and centrifuged to prepare 1×10 9A composition containing CFU will be administered to mice by forced intragastric oral administration over a period of several weeks. After the final oral administration, the mice will be infected by intranasal inoculation with a lethal dose of influenza A virus (A / PR8 / 34 (H1N1)). The effectiveness of the lactic acid bacteria in preventing influenza A infection will be confirmed based on weight change and survival rate after influenza A virus inoculation, as well as quantitative results of virus counts in infected organs.

[0107] Clostridium difficile infection model The lactic acid bacteria of the present disclosure were cultured and centrifuged to prepare 1×10 9 A composition containing CFU is administered to mice by forced oral administration into the stomach. The administration is continued for several weeks. After the final oral administration, the mice are orally inoculated with a lethal dose of Clostridium difficile to infect them. The preventive effect of the lactic acid bacteria against Clostridium difficile infection is confirmed based on weight change and survival rate after inoculation with Clostridium difficile, as well as quantitative results of the number of bacteria in the infected organs.

[0108] Staphylococcus aureus infection model The lactic acid bacteria of the present disclosure were cultured and centrifuged to prepare 1×10 9 A composition containing CFU is administered to mice by forced oral administration into the stomach. The administration is continued for several weeks. After the final oral administration, the mice are subcutaneously inoculated with Staphylococcus aureus to infect them. The preventive effect of the lactic acid bacteria against Staphylococcus aureus infection is confirmed based on weight change and survival rate after inoculation of Staphylococcus aureus, as well as quantitative results of bacterial counts in infected organs.

[0109] (Note) Although the present disclosure has been illustrated using the preferred embodiments thereof, it is understood that the present disclosure should be interpreted in scope only by the claims. It is understood that the patents, patent applications, and other documents cited in this specification are incorporated by reference into this specification in the same manner as if the contents themselves were specifically set forth herein. [Industrial Applicability]

[0110] The lactic acid bacteria disclosed herein may be used in functional foods and functional feed (for pets, livestock, etc.), supplements, and pharmaceuticals that have the functions and effects exhibited by the lactic acid bacteria disclosed herein, and have high applicability in many industries. [Accession number]

[0111] Enterococcus faecalis KU-EF-001 strain (NITE BP-03655) JPEG2024025452000002.jpg220153

[0112] Enterococcus faecalis KU-EF-002 strain (NITE BP-03656) JPEG2024025452000003.jpg213153

[0113] Enterococcus faecalis KU-EF-003 strain (NITE BP-03657) JPEG2024025452000004.jpg219153

[0114] Enterococcus faecalis KU-EF-004 strain (NITE BP-03658) JPEG2024025452000005.jpg215153

Claims

1. Lactic acid bacteria that have the ability to activate dendritic cells in Peyer's patches.

2. Regarding the lactic acid bacteria, the Peyer's patch dendritic cell activation index is IL-6 ＋ / CD45 ＋ : greater than 1.43, MHC class II ＋ / CD45 ＋ : greater than 1.82, CD80 ＋ / CD45 ＋ : greater than 1.41, and CD86 ＋ / CD45 ＋ : greater than 1.22, The lactic acid bacterium according to claim 1, having at least one property selected from the group consisting of:

3. The Peyer's patch dendritic cell activation index is IL-6 ＋ / CD45 ＋ : 2 or more, MHC class II ＋ / CD45 ＋ : 2 or more, CD80 ＋ / CD45 ＋ : 2 or more, and CD86 ＋ / CD45 ＋ : 2 or more, The lactic acid bacterium according to claim 2, having at least one property selected from the group consisting of:

4. The Peyer's patch dendritic cell activation index is IL-6 ＋ / CD45 ＋ : 2.5 or above, MHC class II ＋ / CD45 ＋ : 2.5 or above, CD80 ＋ / CD45 ＋ : 2.5 or more, and CD86 ＋ / CD45 ＋ : 2.5 or above, The lactic acid bacterium according to claim 2, having at least one property selected from the group consisting of:

5. The Peyer's patch dendritic cell activation index is IL-6 ＋ / CD45 ＋ : 2.5 or above, MHC class II ＋ / CD45 ＋ : 4 or above, CD80 ＋ / CD45 ＋ : 2.5 or more, and CD86 ＋ / CD45 ＋ : 2.5 or above, The lactic acid bacterium according to claim 2, having at least one property selected from the group consisting of:

6. The Peyer's patch dendritic cell activation index is IL-6 ＋ / CD45 ＋ : 3 or more, MHC class II ＋ / CD45 ＋ : 4 or above, CD80 ＋ / CD45 ＋ : 3 or more, and CD86 ＋ / CD45 ＋ : 2.5 or above, The lactic acid bacterium according to claim 2, having at least one property selected from the group consisting of:

7. The lactic acid bacterium according to any one of claims 2 to 6, wherein the indicator of activation of dendritic cells in Peyer's patches has at least two of the above properties.

8. The lactic acid bacterium according to any one of claims 2 to 6, wherein the Peyer's patch dendritic cell activation indicator is at least IL-6 and CD80 positive.

9. The lactic acid bacterium according to any one of claims 2 to 6, wherein the indicator of activation of dendritic cells in Peyer's patches has at least three of the above properties.

10. The lactic acid bacterium according to any one of claims 2 to 6, wherein the indicator of activation of dendritic cells in Peyer's patches has four of the above properties.

11. The lactic acid bacterium according to any one of claims 1 to 6, wherein the activation ability is evaluated by at least one positivity rate selected from the group consisting of IL-6 positivity rate, IL-12 positivity rate, MHC class I positivity rate, MHC class II positivity rate, CD80 positivity rate, CD86 positivity rate, CD8 positivity rate, IFN-γ positivity rate, IFN-α positivity rate, TNF-α positivity rate, CD40 positivity rate, IL-1β positivity rate, IL-23 positivity rate, IL-27 positivity rate, and CCR7 positivity rate in the dendritic cells.

12. The activation ability is determined by the expression of CD45 in the dendritic cells. ＋ CD11c ＋ IL-6 ＋ / CD45 ＋ , CD45 ＋ CD11c ＋ IFN-γ ＋ / CD45 ＋ , CD45 ＋ CD11c ＋ MHC class II ＋ / CD45 ＋ , CD45 ＋ CD11c ＋ CD80 ＋ / CD45 ＋ , CD45 ＋ CD11c ＋ CD86 ＋ / CD45 ＋ , and CD45 ＋ CD11c ＋ CD8a ＋ / CD45 ＋ The lactic acid bacterium according to any one of claims 1 to 6, which increases any one of the parameters selected from the group consisting of:

13. The activation ability is determined by the expression of CD45 in the dendritic cells. ＋ CD11c ＋ IL-6 ＋ / CD45 ＋ , CD45 ＋ CD11c ＋ IFN-γ ＋ / CD45 ＋ , CD45 ＋ CD11c ＋ MHC class II ＋ / CD45 ＋ , CD45 ＋ CD11c ＋ CD80 ＋ / CD45 ＋ , CD45 ＋ CD11c ＋ CD86 ＋ / CD45 ＋ , and CD45 ＋ CD11c ＋ CD8a ＋ / CD45 ＋ The lactic acid bacterium according to any one of claims 1 to 6, which increases any one of the parameters selected from the group consisting of at least about three times the reference value.

14. The activation ability is determined by the expression of CD45 in the dendritic cells. ＋ CD11c ＋ IL-6 ＋ / CD45 ＋ , CD45 ＋ CD11c ＋ IFN-γ ＋ / CD45 ＋ , CD45 ＋ CD11c ＋ MHC class II ＋ / CD45 ＋ , CD45 ＋ CD11c ＋ CD80 ＋ / CD45 ＋ , CD45 ＋ CD11c ＋ CD86 ＋ / CD45 ＋ , and CD45 ＋ CD11c ＋ CD8a ＋ / CD45 ＋ The lactic acid bacterium according to any one of claims 1 to 6, which increases any one of the parameters selected from the group consisting of at least about four times the reference value.

15. The activation ability is determined by the expression of CD45 in the dendritic cells. ＋ CD11c ＋ IL-6 ＋ / CD45 ＋ , CD45 ＋ CD11c ＋ IFN-γ ＋ / CD45 ＋ , CD45 ＋ CD11c ＋ MHC class II ＋ / CD45 ＋ , CD45 ＋ CD11c ＋ CD80 ＋ / CD45 ＋ , CD45 ＋ CD11c ＋ CD86 ＋ / CD45 ＋ , and CD45 ＋ CD11c ＋ CD8a ＋ / CD45 ＋ The lactic acid bacterium according to any one of claims 1 to 6, which increases at least three of the parameters selected from the group consisting of:

16. The lactic acid bacterium according to any one of claims 1 to 6, wherein the activation ability is greater than that of a lactic acid bacterium type strain.

17. The lactic acid bacterium according to claim 16, wherein the lactic acid bacterium type strain comprises E. faecalis ATCC 700802.

18. The lactic acid bacterium according to any one of claims 1 to 6, wherein the dendritic cells are present between the follicle-associated epithelium (FAE) and the follicle.

19. The lactic acid bacteria according to any one of claims 1 to 6, further comprising the ability to activate at least one other cell selected from the group consisting of macrophages, B cells, and T cells.

20. The lactic acid bacterium according to any one of claims 1 to 6, which is selected from the group consisting of the genera Enterococcus, Bifidobacterium, Bacteroides, Lactobacillus, and Ackermansia.

21. The lactic acid bacterium according to any one of claims 1 to 6, which is selected from the group consisting of the genera Enterococcus and Bifidobacterium.

22. The lactic acid bacterium according to any one of claims 1 to 6, which belongs to the genus Enterococcus.

23. The lactic acid bacterium according to any one of claims 1 to 6, which is selected from the group consisting of E. faecalis, E. faecium, E. avium, E. casseliflavus, E. gallinarum, and E. hirae.

24. The lactic acid bacterium according to any one of claims 1 to 6, which is Enterococcus faecalis.

25. The Enterococcus faecalis strain includes KU-EF-001 strain (NITE BP-03655), KU-EF-002 strain (NITE BP-03656), KU-EF-003 strain (NITE BP-03657), and KU-EF-004 strain (NITE BP-03658). The lactic acid bacterium according to claim 24.

26. A composition comprising the lactic acid bacteria according to any one of claims 1 to 6.

27. The composition according to claim 26 for stimulating the immunity of a subject.

28. The composition of claim 26 for activating dendritic cells in Peyer's patches.

29. 29. The composition of claim 28, comprising Enterococcus faecalis.

30. 27. The composition of claim 26 for use in combination with an immunomodulatory agent or a radiological agent.

31. 27. The composition of claim 26 for enhancing the effect of an immunomodulatory agent or a radiological agent.

32. The composition of claim 30, wherein the immunomodulatory agent is at least one selected from an immune checkpoint inhibitor, a CAR-T cell therapy, a bispecific antibody drug, a cancer vaccine, a costimulatory molecule agonist, an immune activator, and a small molecule inhibitor.

33. 33. The composition of claim 32, wherein the immune checkpoint inhibitor is selected from the group consisting of agents against a molecule selected from the group consisting of CTLA-4, PD-1, LAG-3, BTLA, KIR, TIM-3, PD-L1, PD-L2, B7-H3, B7-H4, HVEM, GAL9, CD160, VISTA, BTNL2, TIGIT, PVR, BTN1A1, BTN2A2, BTN3A2, and CSF-1R, and any combination thereof.

34. The composition of claim 26, which is a food or drink, a food additive, a feed, a supplement, or a medicine.

35. 27. A pharmaceutical composition comprising the composition of claim 26 and an immunomodulatory agent.

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