Enhancement of the antitumor effect of immune checkpoint inhibitors by administration of Enterobacteria luminocaccia.

Abstract

Provided is a pharmaceutical composition that is capable of enhancing the effect of an immune checkpoint inhibitor on a tumor or cancer in a target.　The provided pharmaceutical composition, which is to be administered in combination with an immune checkpoint inhibitor, comprises cells of an intestinal Ruminococcaceae bacterium, a culture supernatant thereof, a metabolic product thereof and / or a cell extract thereof.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for enhancing the effect of an immune checkpoint inhibitor on tumors or cancer in a subject. In particular, the present invention relates to a pharmaceutical composition comprising the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus, which are administered in combination with an immune checkpoint inhibitor. [Background technology]

[0002] Immune checkpoint inhibitors, which target immunosuppressive mechanisms, have shown remarkable clinical efficacy in multiple types of cancer. Immune checkpoint inhibitors are approved for several cancer types, including malignant melanoma, lung cancer, renal cell carcinoma, head and neck cancer, and gastric cancer. However, monotherapy with immune checkpoint inhibitors still does not provide sufficient therapeutic efficacy.

[0003] Attempts have been made to further activate antitumor immunity and enhance efficacy by simultaneously disabling multiple immunosuppressive mechanisms through the combination of different immunosuppressive therapies or existing approved drugs (Patent Document 1), and several clinical trials have been conducted. However, it is difficult to say that sufficient effects have been obtained so far.

[0004] It is known that an immunosuppressive network, centered on immune checkpoint molecules and regulatory T cells, is established in the tumor microenvironment, inducing immune tolerance (Non-Patent Literature 1). The influence of commensal gut bacteria on these immune responses has been reported repeatedly (Non-Patent Literature 2, Non-Patent Literature 3). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Special Publication 2015-518826 [Non-patent literature]

[0006] [Non-Patent Document 1] Nature Reviews Cancer, 12, 252-264 (2012) [Non-Patent Document 2] Science, 359, 91-97 (2018) [Non-Patent Document 3] Science, 359, 97-103 (2018) [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] An object of the present invention is to provide a pharmaceutical composition capable of enhancing the effect of an immune checkpoint inhibitor against tumors or cancers in a subject. [Means for Solving the Problems]

[0008] The present inventors analyzed the intestinal bacteria of gastric cancer and lung cancer patients treated with an immune checkpoint inhibitor, and found that cases showing efficacy of the immune checkpoint inhibitor had many unclassified genus bacteria of the family Ruminococcaceae. Furthermore, in order to examine the influence of specific bacteria on antitumor immunity, the present inventors isolated and cultured bacteria from the intestinal contents of cases showing efficacy. The isolated bacteria were administered to mice in which the indigenous intestinal bacteria had been reduced by administration of an antibacterial agent, and the combined effect with an immune checkpoint inhibitor was examined. As a result, it was found that Ruminococcaceae YB328 having a 16S rRNA gene having the nucleotide sequence shown in SEQ ID NO: 1 enhances the antitumor effect of the immune checkpoint inhibitor, and thus the present invention was completed.

[0009] That is, the present invention relates to, but is not limited to, the following. [1] A method for isolating Ruminococcaceae intestinal bacteria, comprising: (i) A step of preparing a diluted intestinal contents solution by serially diluting the intestinal contents obtained from a mammal that has been administered an immune checkpoint inhibitor and has obtained a PR (partial response) or better evaluation by post-administration CT imaging evaluation, or has obtained a SD (stable disease) evaluation for six months or more, using an anaerobic diluent; (ii) A step of inoculating a portion of the diluted intestinal contents onto a solid culture medium and culturing it under anaerobic conditions to generate colonies (or multiple colonies) on the solid culture medium that are derived from a single clone of the microorganism contained in the diluted intestinal contents; (iii) A step of confirming whether the bacteria contained in the colony have a 16S rRNA gene that has 95% or more sequence identity with the base sequence shown in Sequence ID No. 1; and (iv) Steps to obtain bacteria that have been confirmed to have a 16S rRNA gene with 95% or more sequence identity with the base sequence shown in Sequence ID No. 1. An isolation method including [details omitted]. [2] The isolation method according to [1], wherein the immune checkpoint inhibitor is an inhibitor against any immune checkpoint molecule selected from the group consisting of PD-1, CTLA-4, TIM-3, BTLA, LAG-3, A2aR, KIR, VISTA, TIGIT, PD-L1, PD-L2, CD80, CD86, GAL-9, HVEM, CD160, MHC class II, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7, or a combination of two or more such inhibitors. [3] The isolation method according to [2], wherein the immune checkpoint inhibitor is selected from an antibody against the immune checkpoint molecule, an antigen-binding fragment of the antibody, and a combination thereof. [4] The isolation method described in [3], wherein the immune checkpoint inhibitor is selected from the group consisting of nivolumab, pembrolizumab, cemiprimab, avelumab, atezolizumab, and durvalumab. [5] The isolation method described in any one of the following paragraphs, [1] to [4], wherein the mammal is human. [6] A method for producing a pharmaceutical composition, comprising the step of compounding the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus isolated by the isolation method described in any one of [1] to [5] to produce a pharmaceutical composition. [7] The manufacturing method according to [6], wherein the pharmaceutical composition is administered in combination with an immune checkpoint inhibitor. [8] A pharmaceutical composition manufactured by the manufacturing method described in [6] or [7]. [9] A pharmaceutical composition comprising the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus, administered in combination with an immune checkpoint inhibitor.

[10] The pharmaceutical composition according to [9], comprising live cells of Enterobacteria luminococcus.

[11] The pharmaceutical composition according to [9] or

[10] , wherein Luminococcus Enterobacteria is a bacterium having a 16S rRNA gene that is 95% or more identical to the base sequence shown in Sequence ID No. 1.

[12] The pharmaceutical composition according to any one of the claims [8] to

[11] , wherein the immune checkpoint inhibitor is an inhibitor of any immune checkpoint molecule selected from the group consisting of PD-1, CTLA-4, TIM-3, BTLA, LAG-3, A2aR, KIR, VISTA, TIGIT, PD-L1, PD-L2, CD80, CD86, GAL-9, HVEM, CD160, MHC class II, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7, or a combination of two or more such inhibitors.

[13] The pharmaceutical composition according to

[12] , wherein the immune checkpoint inhibitor is selected from an antibody against the immune checkpoint molecule, an antigen-binding fragment of the antibody, and a combination thereof.

[14] The pharmaceutical composition according to

[13] , wherein the immune checkpoint inhibitor is selected from the group consisting of nivolumab, pembrolizumab, semiprimab, avelumab, atezolizumab, and durvalumab.

[15] A pharmaceutical composition according to any one of items [8] to

[14] , which is administered orally, enterally, or enema.

[16] A pharmaceutical composition according to any one of [8] to

[15] , wherein an immune checkpoint inhibitor and the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus are administered simultaneously.

[17] The pharmaceutical composition according to

[16] , comprising an immune checkpoint inhibitor and the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus.

[18] A pharmaceutical composition according to any one of [8] to

[15] , wherein an immune checkpoint inhibitor and the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcusse are administered separately.

[19] The pharmaceutical composition according to

[18] , wherein the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus are administered before the administration of an immune checkpoint inhibitor.

[20] The pharmaceutical composition according to

[18] , wherein the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus are administered after administration of an immune checkpoint inhibitor. [twenty one] A pharmaceutical composition according to any one of [8] to

[20] for activating CD8-positive T cells in the subject. [twenty two] A pharmaceutical composition according to any one of items [8] to

[21] for enhancing the immune response against a tumor or cancer in a subject having a tumor or cancer. [twenty three] The pharmaceutical composition according to

[22] , which has a greater effect in enhancing the immune response against tumors or cancer compared to the administration of the immune checkpoint inhibitor alone. [twenty four] A pharmaceutical composition according to any one of items [8] to

[23] for treating tumors or cancer in a subject. [twenty five] The pharmaceutical composition according to

[24] , wherein the treatment eliminates, reduces, or stabilizes a tumor or cancer.

[26] The pharmaceutical composition according to

[25] , which has a greater effect in eliminating, shrinking, or stabilizing tumors or cancer compared to the administration of the aforementioned immune checkpoint inhibitor alone.

[27] A pharmaceutical composition according to any one of items [8] to

[25] for suppressing the recurrence or metastasis of a tumor or cancer in a subject.

[28] The pharmaceutical composition according to

[27] , which has a greater inhibitory effect on tumor or cancer recurrence or metastasis compared to the administration of the immune checkpoint inhibitor alone.

[29] A pharmaceutical composition according to any one of

[22] to

[28] , which is administered in combination with one or more therapies selected from the group consisting of surgical therapy, chemotherapy, and radiotherapy.

[30] Tumors or cancers include malignant pleural mesothelioma, malignant peritoneal mesothelioma, malignant melanoma, malignant lymphoma, brain tumor, glioma, neuroblastoma, thymoma, gastrointestinal stromal tumor, neuroendocrine tumor, testicular tumor, soft tissue sarcoma, nephroblastoma, hepatoblastoma, germ cell tumor, retinoblastoma, osteosarcoma, Ewing's sarcoma, rhabdomyosarcoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, adult T-cell leukemia, multiple myeloma, oropharyngeal cancer, laryngeal cancer, tongue cancer, nasal cavity cancer, paranasal sinus cancer, and thyroid cancer. A pharmaceutical composition according to any one of

[22] to

[29] , selected from the group consisting of cancer, parotid gland cancer, submandibular gland cancer, auditory organ cancer, lung cancer, breast cancer, thymic cancer, esophageal cancer, gastric cancer, colorectal cancer, small intestine cancer, hepatocellular carcinoma, bile duct cancer, gallbladder cancer, pancreatic cancer, renal cell carcinoma, renal pelvis / ureteral cancer, bladder cancer, urachal cancer, adrenal cancer, peritoneal cancer, prostate cancer, cervical cancer, uterine cancer, ovarian cancer, vaginal cancer, vulvar cancer, basal cell carcinoma, squamous cell carcinoma, neuroendocrine cancer, Kaposi's sarcoma, and cancer of unknown primary origin.

[31] A pharmaceutical composition according to any one of [8] to

[20] for increasing the diversity of commensal intestinal bacteria in mammals compared to before administration.

[32] A method for enhancing the immune response against a tumor or cancer, comprising administering to a subject with a tumor or cancer an effective amount of an immune checkpoint inhibitor in combination with an effective amount of a pharmaceutical composition containing the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus.

[33] The method according to

[32] , which has a greater effect in enhancing the immune response against tumors or cancer compared to the administration of the aforementioned immune checkpoint inhibitor alone.

[34] A method for treating a tumor or cancer, comprising administering to a subject with a tumor or cancer an effective amount of an immune checkpoint inhibitor in combination with an effective amount of a pharmaceutical composition containing the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus.

[35] The method according to

[34] , wherein the treatment eliminates, reduces, or stabilizes the tumor or cancer.

[36] The method according to

[35] , which has a greater effect in eliminating, shrinking, or stabilizing tumors or cancers compared to the administration of the aforementioned immune checkpoint inhibitor alone.

[37] A method for suppressing the recurrence or metastasis of a tumor or cancer in a subject having a tumor or cancer, comprising administering to the subject an effective amount of an immune checkpoint inhibitor in combination with an effective amount of a pharmaceutical composition containing the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus.

[38] The method according to

[37] , which has a greater inhibitory effect on tumor or cancer recurrence or metastasis compared to the administration of the aforementioned immune checkpoint inhibitor alone.

[39] The method according to any one of

[32] to

[38] , wherein an immune checkpoint inhibitor is administered simultaneously with the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus.

[40] The method according to any one of

[32] to

[38] , wherein an immune checkpoint inhibitor and the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcusse are administered separately.

[41] The method according to

[40] , wherein cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus are administered before administration of an immune checkpoint inhibitor.

[42] The pharmaceutical composition according to

[40] , wherein the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus are administered after administration of an immune checkpoint inhibitor.

[43] The method described in any one of the items

[32] to

[42] , further combined with one or more therapies selected from the group consisting of surgery, chemotherapy, and radiotherapy.

[44] Tumors or cancers include malignant pleural mesothelioma, malignant peritoneal mesothelioma, malignant melanoma, malignant lymphoma, brain tumor, glioma, neuroblastoma, thymoma, gastrointestinal stromal tumor, neuroendocrine tumor, testicular tumor, soft tissue sarcoma, nephroblastoma, hepatoblastoma, germ cell tumor, retinoblastoma, osteosarcoma, Ewing's sarcoma, rhabdomyosarcoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, adult T-cell leukemia, multiple myeloma, oropharyngeal cancer, laryngeal cancer, tongue cancer, nasal cavity cancer, paranasal sinus cancer, and thyroid cancer. The method according to any one of

[32] to

[43] , selected from the group consisting of basal cell carcinoma, parotid gland carcinoma, submandibular gland carcinoma, auditory gland carcinoma, lung cancer, breast cancer, thymic carcinoma, esophageal cancer, gastric cancer, colorectal cancer, small intestine cancer, hepatocellular carcinoma, bile duct cancer, gallbladder cancer, pancreatic cancer, renal cell carcinoma, renal pelvis and ureteral cancer, bladder cancer, urachal cancer, adrenal gland cancer, peritoneal cancer, prostate cancer, cervical cancer, uterine cancer, ovarian cancer, vaginal cancer, vulvar cancer, basal cell carcinoma, squamous cell carcinoma, neuroendocrine carcinoma, Kaposi's sarcoma, and cancer of unknown primary origin.

[45] A method for increasing the intestinal flora of a mammal compared to before administration, comprising administering an effective amount of a pharmaceutical composition containing the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcusse to the mammal.

[46] A pharmaceutical composition for inducing dendritic cell progenitor cells into type 1 dendritic cells, comprising agonists of multiple TLRs other than TLR4.

[47] The pharmaceutical composition according to

[46] , wherein the plurality of TLRs are TLR1, TLR3, TLR5, TLR7, and TLR9.

[48] The pharmaceutical composition according to

[47] , wherein the plurality of TLRs are TLR5, TLR7, and TLR9.

[49] The pharmaceutical composition according to

[48] , wherein the agonist is a combination of flagellin, R848 (reximod), and CpG-ODN.

[50] The pharmaceutical composition according to any one of

[46] to

[48] , wherein the agonist is the cells of Enterobacteria luminococcus, the culture supernatant, metabolites and / or cell extracts of Enterobacteria luminococcus.

[51] A pharmaceutical composition according to any one of items

[46] to

[50] for treating tumors or cancer in the subject.

[52] A method for inducing dendritic cell progenitor cells into type 1 dendritic cells, comprising contacting the dendritic cell progenitor cells with agonists of multiple TLRs other than TLR4.

[53] The method described in

[52] , performed in vitro.

[54] The method according to

[52] or

[53] , wherein the plurality of TLRs are TLR5, TLR7, and TLR9.

[55] The method according to

[54] , wherein the agonist is a combination of flagellin, R848 (reciquimod), and CpG-ODN.

[56] The method according to any one of

[52] to

[55] , wherein the agonist is the cells of Enterobacteria luminococcus, the culture supernatant, metabolites and / or cell extracts of Enterobacteria luminococcus.

[57] Type 1 dendritic cells induced by any one of the methods described in

[52] to

[56] .

[58] A pharmaceutical composition comprising type 1 dendritic cells as described in

[57] for the treatment of tumors or cancer in the subject.

[59] A pharmaceutical composition comprising type 1 dendritic cells as described in

[57] for enhancing the immune response against a tumor or cancer in a subject having a tumor or cancer. [Effects of the Invention]

[0010] The present invention makes it possible to enhance the effect of immune checkpoint inhibitors on tumors or cancer in a target. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows the results of LEfSe analysis comparing the gut microbiota in patients who responded to and did not respond to immune checkpoint inhibitors. [Figure 2] Figure 2 shows the results of comparing progression-free survival between a patient group with a high median proportion of various bacterial groups in the intestinal flora and a patient group with a low median proportion of various bacterial groups. [Figure 3] Figure 3 shows the results of an investigation into the effects of immune checkpoint inhibitors in mouse tumor models transplanted with intestinal contents from patients who responded to or did not respond to the immune checkpoint inhibitors. [Figure 4] Figure 4 shows the results of an investigation into the effects of immune checkpoint inhibitors in a mouse tumor model treated with antibiotics, in which intestinal contents derived from patients who responded to immune checkpoint inhibitors were transplanted. [Figure 5] Figure 5 shows the phylogenetic analysis results of bacterial 16S rRNA gene sequences in intestinal contents from patients who responded to immune checkpoint inhibitors. [Figure 6] Figure 6 shows the results of investigating the effects of immune checkpoint inhibitors on mouse tumor models treated with antibiotics, in which various bacteria were transplanted. [Figure 7]Figure 7 shows the results of investigating the effects of immune checkpoint inhibitors on mouse tumor models treated with antibiotics, after transplanting either B. vulgatus or ruminococcaceae YB328. [Figure 8] Figure 8 shows the results of flow cytometry measuring dendritic cell maturation markers in dendritic cells co-cultured with ruminococcaceae YB328, B. vulgatus, or a vehicle. [Figure 9] Figure 9 shows the results of measuring the CD8+ T cell activation marker (IFN-γ) when dendritic cells co-cultured with bacteria were co-cultured with CD8+ T cells derived from OT-I mice and stimulated with 1 nM or 100 nM N4 peptide. [Figure 10] Figure 10 shows the results of measuring TCR signaling (ZAP70) and CD28 signaling (Erk) when dendritic cells co-cultured with bacteria were co-cultured with CD8+ T cells derived from OT-I mice and stimulated with N4 peptide or Q4H7 peptide. [Figure 11] Figure 11 shows the effects of different concentrations of N4 peptide stimulation (0nM, 1nM, 10nM, 100nM) on TCR signaling (ZAP70 (pZAP70)), CD28 signaling (Erk (pErk), Akt (pAkt), S6 (pS6)), in two cases: when dendritic cells co-cultured with ruminococcaceae YB328 were co-cultured with CD8+ T cells, and when dendritic cells co-cultured with B. vulgatus were co-cultured with CD8+ T cells. [Figure 12] Figure 12 shows the results of an investigation into the effects of immune checkpoint inhibitors in mouse tumor models treated with antibiotics, where intestinal contents from patients who responded to or did not respond to immune checkpoint inhibitors were transplanted, and either Ruminococcaceae YB328 or B. vulgatus was administered orally. [Figure 13]Figure 13 shows the results of a meta-analysis based on the 16S rRNA gene, comparing the diversity of the bacterial flora in the intestinal contents of mice collected after intestinal contents transplantation and after monobacterial administration. [Figure 14] Figure 14 shows the results of transcriptome analysis performed after co-culturing mouse bone marrow-derived dendritic cells with ruminococcaceae YB328, B. vulgatus, LPS, or vehicle (PBS), followed by RNA extraction from the dendritic cells. [Figure 15] Figure 15 shows the results of FACS analysis of various tissues (near-tumor lymph nodes, lamina propria, and mesenteric lymph nodes) collected from mice that were subcutaneously transplanted with the MC38 cultured cell line and then orally administered with either Ruminococcaceae YB328 or B. vulgatus. [Figure 16] Figure 16 shows the results of FACS analysis of tumors collected from mice that were subcutaneously transplanted with the MC38 cell line and then orally administered with ruminococcaceae YB328 or B. vulgatus. [Figure 17] Figure 17 shows the results of FACS analysis of IRF8 expression after co-culturing bone marrow-derived dendritic cell progenitor cells with FLT3L and ruminococcaceae YB328, B. vulgatus, LPS, or PBS. [Figure 18] Figure 18 shows the results of FACS analysis of p-S6K and p-STAT3 expression after co-culturing mouse bone marrow-derived dendritic cells with ruminococcaceae YB328, B. vulgatus, LPS, or PBS. [Figure 19]Figure 19 shows the induction of dendritic cell progenitor cells into type 1 dendritic cells by TLR complex stimulation. A shows the transcriptome analysis results focusing on various TLRs in dendritic cells stimulated with luminococcusse YB328 and dendritic cells stimulated with B. vulgatus. B shows the results of FACS analysis of the proportion of CD103-positive, CD11b-negative dendritic cells after stimulating bone marrow-derived dendritic cells isolated from MyD88 knockout mice with luminococcusse YB328 or a vehicle. C shows the results of FACS analysis of the proportion of CD103-positive, CD11b-negative dendritic cells after stimulating mouse bone marrow-derived dendritic cells with various mixtures of agonists for TLRs 5, 7, and 9. [Modes for carrying out the invention]

[0012] According to one aspect of the present invention, a method for isolating Luminococcus Enterobacteriaceae is provided.

[0013] The present invention provides a method for isolating Luminococcus enterobacteria, (i) A step of preparing a diluted intestinal contents solution by serially diluting the intestinal contents obtained from a mammal that has been administered an immune checkpoint inhibitor and has obtained a PR (partial response) or better evaluation by post-administration CT imaging evaluation, or has obtained a SD (stable disease) evaluation for six months or more, using an anaerobic diluent; (ii) A step of inoculating a portion of the diluted intestinal contents onto a solid culture medium and culturing it under anaerobic conditions to generate colonies (or multiple colonies) on the solid culture medium that are derived from a single clone of the microorganism contained in the diluted intestinal contents; (iii) A step of confirming whether the bacteria contained in the colony have a 16S rRNA gene that has 95% or more sequence identity with the base sequence shown in Sequence ID No. 1; and (iv) Steps to obtain bacteria that have been confirmed to have a 16S rRNA gene with 95% or more sequence identity with the base sequence shown in Sequence ID No. 1. Includes.

[0014] <Method for isolating Luminococcus Enterobacteriaceae> The Luminococcus Enterobacteriaceae in this invention, when administered in combination with an immune checkpoint inhibitor, can enhance the effect of the immune checkpoint inhibitor on tumors or cancer in the target population. The inventors have surprisingly discovered that such Luminococcus Enterobacteriaceae can be isolated from humans who have shown positive responses to immune checkpoint inhibitors.

[0015] <Process for producing a diluted solution of intestinal contents> The present invention provides a method for isolating Luminococcus Enterobacteria, which includes (i) a step of producing a diluted intestinal contents solution by serially diluting the intestinal contents obtained from a mammal that has been administered an immune checkpoint inhibitor and has obtained a PR (partial response) or better evaluation by post-administration CT imaging, or has achieved SD (stable disease) for six months or more, using an anaerobic diluent.

[0016] CR (Complete Response), PR (Partial Response), SD (Stable Disease), and PD (Progressive Disease) are common evaluation criteria in the medical field, indicating the disappearance, reduction, or stabilization of a tumor or cancer. CR means the tumor has completely disappeared; PR means the total size of the tumor has decreased by 30% or more; SD means the tumor size remains unchanged; and PD means the total size of the tumor has increased by 20% or more, and also by 5 mm or more in absolute terms, or a new lesion has appeared. Tumor size can be evaluated by CT imaging. For example, RECIST ver1.1 can be used for CT imaging evaluation.

[0017] The present invention's method for isolating Luminococcus Enterobacteria uses intestinal contents obtained from mammals that have been administered an immune checkpoint inhibitor and have achieved a PR (partial response) or better evaluation by post-administration CT imaging, or have achieved SD (stable disease) for six months or more. Here, a PR (partial response) or better evaluation means PR (partial response) or CR (complete response).

[0018] The present invention provides a method for isolating Luminococcus enterobacteria, which includes the step of serially diluting the above-mentioned intestinal contents using an anaerobic diluent to produce a diluted intestinal contents solution. Any diluent that does not adversely affect the survival of anaerobic bacteria can be used as the anaerobic diluent, but for example, as described in "The World of Intestinal Bacteria" by Tomotari Mitsuoka (1990), Asakura Shoten, the anaerobic diluent (B) in the same book can be used. The dilution ratio can be adjusted as appropriate, but for colony formation on a solid culture medium, for example, 10 -6 ~10 -10 A 10x dilution series can be created within this range, allowing you to select the dilution ratio best suited for colony generation.

[0019] <Colony generation process> The present invention provides a method for isolating Luminococcus enterobacteria, which includes the step of inoculating a portion of the above-mentioned diluted intestinal contents solution onto a solid culture medium and culturing it under anaerobic conditions to generate colonies (or more) derived from a single clone of the microorganism contained in the diluted intestinal contents solution on the solid culture medium.

[0020] As a solid culture medium, for example, EG agar can be used. The standard composition of EG agar is shown below.

[0021] [Table 1]

[0022] Anaerobic conditions refer to an environment in which oxygen is absent and replaced by nitrogen, hydrogen, and carbon dioxide gases as the gas phase. An environment where oxygen is virtually absent can be achieved in a sealed environment such as an anaerobic chamber that can maintain an atmosphere with an oxygen partial pressure low enough to allow the growth of Enterobacteria luminococcus.

[0023] <Process for confirming the base sequence of the bacterial 16S rRNA gene> The present invention provides a method for isolating Luminococcus Enterobacteria, which includes a step of confirming whether the bacteria contained in the generated colonies have a 16S rRNA gene that has 95% or more sequence identity with the base sequence shown in Sequence ID No. 1.

[0024] In the present invention, Luminococcus enterobacteria refers to obligate anaerobic bacteria classified in the phylum Firmicutes, class Clostridium, order Clostridiales, family Ruminococcusceae, and more specifically, bacteria having a 16S rRNA gene that has 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the base sequence shown in Sequence ID No. 1.

[0025] The percentage of identity between two gene sequences can be determined by visual inspection and mathematical calculation, or more preferably, by comparing sequence information using a computer program. A typical and preferred computer program is the Wisconsin Package, version 10.0 program "GAP" from the Genetics Computer Group (GCG; Madison, Wisconsin) (Devereux, et al., 1984, Nucl. Acids Res., 12: 387). Other sequence comparison programs used by those skilled in the art include, for example, the BLASTN program, version 2.2.7, available for use on the website of the U.S. National Library of Medicine: http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / bls.html, or the UW-BLAST2.0 algorithm. Standard default parameter settings for UW-BLAST2.0 are described on the following internet site: http: / / blast.wustl.edu.

[0026] One method for confirming whether the bacteria contained in the generated colonies possess a 16S rRNA gene with 95% or more sequence identity to the sequence shown in Sequence ID No. 1 is to determine the sequence of the 16S rRNA gene of the bacteria forming each colony and compare it with the sequence of Sequence ID No. 1. The bacterial 16S rRNA gene can be amplified by PCR using known primers and sequenced by standard sequencing methods.

[0027] <Process for obtaining bacteria> The present invention provides a method for isolating Luminococcus Enterobacteria, which includes the step of obtaining bacteria that have been confirmed to have a 16S rRNA gene having 95% or more sequence identity with the base sequence shown in Sequence ID No. 1.

[0028] Luminococcusse enterobacteria confirmed to possess a 16S rRNA gene with more than 95% sequence identity to the base sequence shown in Sequence ID No. 1 by the above method can be inoculated from the colonies into a liquid medium and further cultured in large quantities for use. As the liquid medium, for example, a liquid medium that does not contain agar from the above EG agar medium composition (hereinafter sometimes simply referred to as EG medium) can be used.

[0029] <Step to confirm the effect of enhancing the efficacy of immune checkpoint inhibitors against tumors or cancer> The method for isolating Luminococcus Enterobacteria of the present invention may optionally include a step of confirming that the obtained Luminococcus Enterobacteria has the effect of enhancing the effect of immune checkpoint inhibitors against tumors or cancer. For example, the obtained Luminococcus Enterobacteria can be administered in combination with an immune checkpoint inhibitor to mammals such as mice, rats, or humans that have tumors or cancer, and it can be confirmed that the effect of the immune checkpoint inhibitor is enhanced compared to when the immune checkpoint inhibitor is administered alone. Specifically, for example, when the obtained Luminococcus Enterobacteria is administered in combination with an immune checkpoint inhibitor compared to when the immune checkpoint inhibitor is administered alone, if the tumor or cancer disappears, its size is reduced, or its size remains unchanged and stable, then the effect of the immune checkpoint inhibitor can be evaluated as being enhanced.

[0030] <Luminococcasse enteric bacteria for pharmaceutical compositions> According to one aspect of the present invention, a pharmaceutical composition comprising the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus and a method for producing the same are provided.

[0031] The Luminococcus enterobacteria used in the pharmaceutical composition of the present invention are preferably those having a 16S rRNA gene that has 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the base sequence shown in Sequence ID No. 1. Bacteria isolated by the above isolation method can be suitably used as such bacteria.

[0032] An example of the Ruminococaceae enterobacteria used in the pharmaceutical composition of the present invention is Ruminococaceae YB328 isolated by the present inventors. Ruminococaceae YB328 has a 16S rRNA gene with the nucleotide sequence shown in Sequence ID No. 1. The applicant has deposited, maintained, and preserved Ruminococaceae YB328 at the RIKEN (National Research and Development Institute). The applicant guarantees that, in cases falling under each item of Article 27-3 of the Enforcement Regulations of the Patent Act of Japan, Ruminococaceae YB328 will be distributed to third parties subject to compliance with each applicable law.

[0033] Ruminococcusae YB328 can be suitably cultured at 37°C in an anaerobic chamber using the above-mentioned EG medium.

[0034] The pharmaceutical composition of the present invention may contain multiple types of Ruminococcusae enterobacteria or a single type of Ruminococcusae enterobacteria. For example, Ruminococcusae YB328 (Ruminococcaceae YB328) can be cited as a Ruminococcusae enterobacteria that can exert its effects as a single type. When a composition containing an extremely wide variety of bacteria, such as the contents of the intestines themselves, is implanted into the human body, there is a risk of adverse reactions such as infections and allergic reactions. However, by using multiple types of Ruminococcusae enterobacteria or a single type of Ruminococcusae enterobacteria of the present invention, it is possible to avoid such risks.

[0035] The present invention provides a method for producing a pharmaceutical composition, comprising the step of incorporating the above-mentioned Luminococcus Enteric Bacteria. The method for producing a pharmaceutical composition of the present invention may also include the step of incorporating Luminococcus Enteric Bacteria isolated by the above-mentioned method for isolating Luminococcus Enteric Bacteria.

[0036] <Cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus> The pharmaceutical composition of the present invention comprises the cells, culture supernatant, metabolites, and / or cell extracts of the above-mentioned Luminococcus Enterobacteriaceae. When using cell cells, live cell cells are preferred. Live cell cells may be in the form of a culture containing a culture medium for Luminococcus Enterobacteriaceae, or in the form of freeze-dried cell cells. The culture supernatant of Luminococcus Enterobacteriaceae used in the present invention refers to the liquid portion remaining after removing bacteria from a bacterial culture by methods such as centrifugation. The metabolites of Luminococcus Enterobacteriaceae used in the present invention can be appropriately purified from the above-mentioned culture or culture supernatant. The cell extract of Luminococcus Enterobacteriaceae used in the present invention refers to an extract obtained by destroying the cell cells by methods such as pulverization, sonication, or dissolution by alkaline treatment, and optionally fractionating and / or purifying the extract. Cell contents, cell membrane components, purified products thereof, or combinations thereof can be used as appropriate.

[0037] <Immune checkpoint inhibitors> The pharmaceutical composition of the present invention can be administered in combination with an immune checkpoint inhibitor. In this specification, an immune checkpoint inhibitor is a drug that inhibits the function of immune checkpoint molecules and releases the suppression of the T cell response. The immune checkpoint inhibitor used in the present invention is preferably a human immune checkpoint inhibitor, and for example, an inhibitor against any immune checkpoint molecule selected from the group consisting of PD-1, CTLA-4, TIM-3, BTLA, LAG-3, A2aR, KIR, VISTA, TIGIT, PD-L1, PD-L2, CD80, CD86, GAL-9, HVEM, CD160, MHC class II, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7, or a combination of two or more of these inhibitors can be suitably used. The immune checkpoint inhibitor used in the present invention is preferably an antibody against the immune checkpoint molecule, an antigen-binding fragment of an antibody, or a combination thereof, and is selected from the group consisting of, for example, nivolumab, pembrolizumab, semiprimab, avelumab, atezolizumab, and durvalumab.

[0038] <Pharmaceutical composition> The form of the pharmaceutical composition of the present invention is not particularly limited, but depending on the purpose, the form can be appropriately selected from tablets, powders, granules, capsules, enteric-coated capsules, suppositories, liquids, suspensions, gels, etc. The pharmaceutical composition of the present invention may use the cells, culture supernatant, metabolites, and / or cell extracts of Enterococcus luminocasse as is, or it may be formulated using the cells, culture supernatant, metabolites, and / or cell extracts of Enterococcus luminocasse in addition to pharmaceutically acceptable carriers, diluents, and / or excipients.

[0039] The method of administering the pharmaceutical composition of the present invention is not particularly limited and can be appropriately determined considering the formulation form, the patient's age and sex, the severity of the disease, etc. For example, oral administration, enteral administration, enema administration, etc., can be suitably used.

[0040] The pharmaceutical composition of the present invention can be administered in combination with an immune checkpoint inhibitor. Specifically, a pharmaceutical composition containing the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcusse can be administered simultaneously with the administration of an immune checkpoint inhibitor, separately from the administration of an immune checkpoint inhibitor, before the administration of an immune checkpoint inhibitor, or after the administration of an immune checkpoint inhibitor. The pharmaceutical composition of the present invention may also be used in the form of a combination preparation in which the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcusse and an immune checkpoint inhibitor are combined in a single formulation.

[0041] The administration regimen for the pharmaceutical composition of the present invention can be appropriately set considering the formulation form, the patient's age and sex, the severity of the disease, etc., but typically, approximately 1 x 10⁶ Luminococcus Enterobacteria per patient per day is administered. 8 pieces~1×10 11 It is preferable to administer it individually, approximately 1 x 10 times per patient. 9 pieces~1×10 10 Individual administration is even more preferable.

[0042] According to another aspect of the present invention, a pharmaceutical composition is provided comprising the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus for activating CD8-positive T cells in a subject.

[0043] As described later, the Luminococcus Enterobacteria of the present invention matures dendritic cells and, compared to other bacteria such as B. vulgatus or vehicles such as PBS or physiological saline, promotes the development of CD8-positive T cells (CD8 + It has the effect of activating T cells. CD8 + T cell activation can be analyzed, for example, by measuring the expression level of IFN-γ protein or the polynucleotide encoding it. The expression levels of the protein or polynucleotide can be analyzed using known methods such as quantitative protein expression analysis such as flow cytometry and Western blotting, and quantitative gene expression analysis such as transcriptome analysis and real-time quantitative PCR. According to another aspect of the present invention, a pharmaceutical composition is provided comprising the cells, culture supernatant, metabolites and / or cell extracts of Enterobacteria luminococcus, administered in combination with an immune checkpoint inhibitor, for enhancing the immune response against tumor or cancer in subjects having a tumor or cancer.

[0044] <tumor or cancer> In this specification, tumors or cancers include, for example, malignant pleural mesothelioma, malignant peritoneal mesothelioma, malignant melanoma, malignant lymphoma, brain tumor, glioma, neuroblastoma, thymoma, gastrointestinal stromal tumor, neuroendocrine tumor, testicular tumor, soft tissue sarcoma, nephroblastoma, hepatoblastoma, germ cell tumor, retinoblastoma, osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, adult T-cell leukemia, multiple myeloma, oral pharyngeal cancer, laryngeal cancer, tongue cancer, nasal cavity cancer, paranasal sinus cancer, thyroid cancer, parotid gland cancer, submandibular gland cancer, auditory organ cancer, lung cancer, breast cancer, thymic cancer, esophageal cancer, gastric cancer, colorectal cancer, small intestine cancer, hepatocellular cancer, bile duct cancer, gallbladder cancer, pancreatic cancer, renal cell cancer, renal pelvis / ureteral cancer, bladder cancer, urachal cancer, adrenal gland cancer, peritoneal cancer, prostate cancer, cervical cancer, uterine body cancer, ovarian cancer, vaginal cancer, vulvar cancer, basal cell cancer, squamous cell cancer, neuroendocrine cancer, Kaposi sarcoma, and cancer of unknown primary origin, but are not limited thereto.

[0045] <Enhancing effect on immune response> By comparing the case where an immune checkpoint inhibitor is administered alone to a subject with the case where the pharmaceutical composition of the present invention is administered in combination with an immune checkpoint inhibitor, based on an index indicating that the immune response is activated, it is possible to evaluate that the immune response against a tumor or cancer is enhanced. As an index indicating that the immune response is activated, for example, CD8, which is a cytotoxic T cell or its progenitor cell + Proliferation and / or activation of T cells, CD8 + CD62L in CD8 - CD44 + Increase in the proportion of cells, CD8 + TNF-α in CD8 + IFN-γ + Increase in the proportion of cells, decrease in the number of regulatory T cells (Treg, CD4 + CD25 + FoxP3 + Cells), decrease in the number of FoxP3 + Ratio of the number of CD4 + Increase in the ratio of the number of cells, increase in the expression of dendritic cell maturation markers (CD80, CD86, MHC class I, etc.), CD8+ Increased expression of T cell activation markers (such as IFN-γ), increased expression of TCR signaling pathways (such as ZAP70), and increased expression of CD28 signaling pathways (such as Erk (pErk), Akt (pAkt), and S6 (pS6)) can be employed, but are not limited to these.

[0046] According to another aspect of the present invention, a pharmaceutical composition is provided comprising cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus, administered in combination with an immune checkpoint inhibitor, for the treatment of a tumor or cancer in a subject.

[0047] <Therapeutic effects of tumors or cancer> Compared to the administration of an immune checkpoint inhibitor alone, if the pharmaceutical composition of the present invention is administered in combination with an immune checkpoint inhibitor and the tumor or cancer disappears, its size decreases, or its size remains unchanged and stable, then the therapeutic effect of administering the pharmaceutical composition of the present invention in combination with an immune checkpoint inhibitor can be evaluated as having demonstrated a therapeutic effect on the tumor or cancer.

[0048] As indicators of tumor or cancer disappearance, reduction, or stabilization, the commonly used evaluation criteria in this industry, such as CR (Complete Response), PR (Partial Response), SD (Stable Disease), and PD (Progressive Disease), may be adopted. CR refers to a state where the tumor has completely disappeared; PR refers to a state where the total size of the tumor has decreased by 30% or more; SD refers to a state where the size of the tumor remains unchanged; and PD refers to a state where the total size of the tumor has increased by 20% or more, and also by 5 mm or more in absolute terms, or a state where new lesions have appeared. In this specification, a CR, PR, or SD evaluation is considered to indicate a therapeutic effect on the tumor or cancer.

[0049] According to another aspect of the present invention, a pharmaceutical composition is provided comprising cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus, administered in combination with an immune checkpoint inhibitor, for the purpose of suppressing the recurrence or metastasis of a tumor or cancer in a subject.

[0050] <Effect of suppressing recurrence or metastasis of tumors or cancer> In this specification, recurrence of a tumor or cancer means the reappearance of a tumor or cancer in the vicinity of the treated tumor or cancer within one month, six months, one year, three years, five years, or ten years after treatment for the tumor or cancer. In this specification, metastasis of a tumor or cancer means the development of a tumor or cancer in a location distant from the treated tumor or cancer within one month, six months, one year, three years, five years, or ten years after treatment for the tumor or cancer.

[0051] Compared to the administration of an immune checkpoint inhibitor alone, if the pharmaceutical composition of the present invention is administered in combination with an immune checkpoint inhibitor, and if there is no recurrence or metastasis of the tumor or cancer within 1 month, 6 months, 1 year, 3 years, 5 years, or 10 years after treatment for the tumor or cancer, or if the timing of its occurrence is delayed, or if the number of occurrences is reduced, then it can be evaluated that the administration of the pharmaceutical composition of the present invention in combination with an immune checkpoint inhibitor has an inhibitory effect on suppressing the recurrence or metastasis of the tumor or cancer.

[0052] The pharmaceutical composition of the present invention may be administered in combination with one or more therapies selected from the group consisting of surgical therapy, chemotherapy, and radiotherapy.

[0053] <Surgical treatment, chemotherapy, and radiotherapy> Surgical therapies that can be combined with the pharmaceutical composition of the present invention include, but are not limited to, open surgery and endoscopic surgery aimed at the removal of tumors or cancerous lesions, removal of organs containing tumors or cancer, and dissection of lymph nodes surrounding tumors or cancer. Chemotherapy that can be combined with the pharmaceutical composition of the present invention refers to treatment with drugs that inhibit the proliferation or growth of tumors or cancer cells or promote their death, and includes, but is not limited to, hormone therapy and molecular targeted therapy. Radiotherapy that can be combined with the pharmaceutical composition of the present invention refers to radiation therapy aimed at killing tumors or cancer cells, shrinking tumors or cancer, preventing recurrence or metastasis of tumors or cancer, and alleviating the symptoms of tumors or cancer, and includes, but is not limited to, external beam radiation and internal beam radiation.

[0054] <Methods to enhance the immune response, methods for treating tumors or cancer, methods to suppress the recurrence or metastasis of tumors or cancer> According to another aspect of the present invention, a method is provided for enhancing the immune response to a tumor or cancer, comprising administering to a subject having a tumor or cancer an effective amount of an immune checkpoint inhibitor in combination with an effective amount of a pharmaceutical composition comprising cells, culture supernatant, metabolites and / or cell extracts of Enterobacteria luminococcus.

[0055] According to another aspect of the present invention, a method for treating a tumor or cancer is provided, comprising administering to a subject having a tumor or cancer an effective amount of an immune checkpoint inhibitor in combination with an effective amount of a pharmaceutical composition comprising cells, culture supernatant, metabolites and / or cell extracts of Enterobacteria luminococcus.

[0056] According to another aspect of the present invention, a method is provided for suppressing the recurrence or metastasis of a tumor or cancer in a subject having a tumor or cancer, comprising administering to the subject an effective amount of an immune checkpoint inhibitor in combination with an effective amount of a pharmaceutical composition comprising cells, culture supernatant, metabolites and / or cell extracts of Enterobacteria luminococcus.

[0057] The above method may be further combined with one or more therapies selected from the group consisting of surgical therapy, chemotherapy, and radiotherapy.

[0058] <Pharmaceutical composition or method for increasing the diversity of intestinal commensal bacteria> The cells, culture supernatant, metabolites, and / or cell extracts of the above-mentioned Enterobacteria luminococcusse can increase the diversity of the commensal gut flora of a mammal when administered to the mammal, compared to before administration. Therefore, in one aspect, the present invention relates to a pharmaceutical composition comprising the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcusse for increasing the diversity of the commensal gut flora of a mammal compared to before administration. In another aspect, the present invention also relates to a method for increasing the commensal gut flora of a mammal compared to before administration, comprising administering to the mammal an effective amount of a pharmaceutical composition comprising the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcusse.

[0059] The diversity of intestinal flora can be measured using, for example, the Shannon-Wiener index (hereinafter sometimes referred to as the Shannon index). The Shannon index is expressed by the following formula:

[0060]

number

[0061] The pharmaceutical composition comprising the cells, culture supernatant, metabolites, and / or cell extracts of the Luminococcus Enterobacteriaceae of the present invention, when administered to mammals, can increase the Shannon Index H' in the intestinal commensal bacteria after administration compared to the Shannon Index H' in the intestinal commensal bacteria before administration, preferably by a significant amount. It has been found that in cases of non-response with immune checkpoint inhibitors, the diversity of intestinal commensal bacteria is low. The Luminococcus Enterobacteriaceae of the present invention has a high effect in increasing the diversity of intestinal commensal bacteria compared to other bacteria. Although not bound by any particular theory, it is possible that increasing the diversity of intestinal commensal bacteria contributes to one of the mechanisms by which the Luminococcus Enterobacteriaceae of the present invention elicits an antitumor immune response.

[0062] The pharmaceutical composition of the present invention for increasing the diversity of commensal bacteria in the intestines of mammals may be administered in combination with an immune checkpoint inhibitor.

[0063] <Pharmaceutical composition and method for inducing dendritic cell progenitor cells into type 1 dendritic cells> As described later, the inventors have found that Luminococcus enterobacteria can induce dendritic cell progenitor cells into type 1 dendritic cells. Furthermore, they have found that simultaneous stimulation of multiple TLRs other than TLR4 is important for the induction of dendritic cell progenitor cells into type 1 dendritic cells.

[0064] Therefore, the present invention relates to a pharmaceutical composition for inducing dendritic cell progenitor cells into type 1 dendritic cells, and also to a pharmaceutical composition comprising agonists of multiple TLRs other than TLR4.

[0065] Dendritic cell progenitor cells are those in which the expression of dendritic cell maturation markers is lower than that of mature dendritic cells. Examples of dendritic cell maturation markers include, but are not limited to, CD80, CD86, and MHC class I. In this invention, dendritic cell progenitor cells are preferably bone marrow-derived dendritic cell residual cells. Dendritic cell progenitor cells differentiate in response to various stimuli and ultimately differentiate into type 1 dendritic cells (also called standard type 1 dendritic cells, cDC1), type 2 dendritic cells (also called standard type 2 dendritic cells, cDC2), or plasmacytoid dendritic cells (pDC). In this invention, type 1 dendritic cells refer to CD103-positive, CD11b-negative dendritic cells.

[0066] The pharmaceutical composition for inducing dendritic cell progenitor cells into type 1 dendritic cells of the present invention comprises agonists of multiple TLRs other than TLR4. These multiple TLRs other than TLR4 include, but are not limited to, multiple TLRs selected from the group consisting of TLR1, TLR2, TLR3, TLR5, TLR6, TLR7, TLR8, and TLR9. Examples of TLR1 / TLR2 agonists include Pam3CSK4, examples of TLR2 agonists include histones, examples of TLR2 / TLR6 agonists include thymosan and MALP-2, examples of TLR3 agonists include Poly(I) / Poly(C), examples of TLR4 agonists include LPS, examples of TLR5 agonists include flagelline, examples of TLR7 agonists include R837 (imiquimod), examples of TLR7 / 8 agonists include R848 (reciquimod), and examples of TLR9 agonists include CpG oligodeoxynucleotides (CpG-ODN) such as ODN1826, but are not limited to these. Cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus may be used as agonists included in the pharmaceutical composition for inducing dendritic cell progenitor cells to type 1 dendritic cells according to the present invention.

[0067] The pharmaceutical composition for inducing dendritic cell progenitor cells to type 1 dendritic cells of the present invention can be used to treat tumors or cancer in subjects. Furthermore, the pharmaceutical composition for inducing dendritic cell progenitor cells to type 1 dendritic cells of the present invention can be used to enhance the immune response against tumors or cancer in subjects having tumors or cancer.

[0068] The present invention also relates to a method for inducing dendritic cell progenitor cells into type 1 dendritic cells, the method comprising contacting the dendritic cell progenitor cells with agonists of a plurality of TLRs other than TLR4.

[0069] The method for inducing dendritic cell progenitor cells into type 1 dendritic cells according to the present invention may be performed in vivo or in vitro.

[0070] The origin of the dendritic cell precursor cells used in the method for inducing type 1 dendritic cells of the present invention is not limited, but is preferably human-derived.

[0071] The present invention further relates to type 1 dendritic cells induced by the method for inducing the above-mentioned dendritic cell precursor cells into type 1 dendritic cells.

[0072] Type 1 dendritic cells induced by the method of inducing dendritic cell progenitor cells to type 1 dendritic cells according to the present invention can be used to treat tumors or cancer in subjects. Furthermore, type 1 dendritic cells induced by the method of inducing dendritic cell progenitor cells to type 1 dendritic cells according to the present invention can be used to enhance the immune response against tumors or cancer in subjects having tumors or cancer.

[0073] Accordingly, the present invention also relates to a pharmaceutical composition for treating tumors or cancer in a subject, comprising type 1 dendritic cells induced by a method for inducing dendritic cell progenitor cells into type 1 dendritic cells. Furthermore, the present invention also relates to a pharmaceutical composition for enhancing the immune response against tumors or cancer, comprising type 1 dendritic cells induced by a method for inducing dendritic cell progenitor cells into type 1 dendritic cells.

[0074] The following describes some examples, but this does not limit the scope of the present invention. Those skilled in the art can modify and adapt the present invention in various ways, and these modifications are also included within the scope of the present invention. [Examples]

[0075] <Metagenomic analysis of intestinal commensal bacteria in patients who responded to and did not respond to immune checkpoint inhibitors> Metagenomic analysis of gut microbiota was performed using intestinal contents samples from human patients (43 with gastric cancer and 18 with lung cancer) who received the immune checkpoint inhibitors nivolumab or pembrolizumab. Successful cases were defined as those who achieved a partial response (PR) or better according to RECIST ver1.1 evaluation using CT images, or those who achieved stable disease (SD) for six months or more. Non-responsive cases were defined as all other cases.

[0076] Figure 1 shows the results of LEfSe analysis comparing the intestinal commensal bacteria of successful and non-successful cases. This revealed that the Ruminococcus enteric flora was frequently observed in successful cases, and further identified Ruminococcus species and unclassified Ruminococcus species belonging to the Ruminococciaceae family.

[0077] Next, we investigated the correlation between the differences in the proportion of each bacterial group in the gut microbiota and the progression-free survival (PFS) of patients. Specifically, we compared progression-free survival between a patient group with a high median proportion of the Ruminococcus enteric bacteria in the gut microbiota (high Ruminococcus enteric bacteria group) and a patient group with a low median proportion of the Ruminococcus enteric bacteria in the gut microbiota (low Ruminococcus enteric bacteria group). Similarly, for the unclassified Ruminococcus bacteria group, the Ruminococcus bacteria group, and the Bacteroides bacteria group, we compared progression-free survival between patient groups with a high median proportion of each bacterial group in the gut microbiota and patient groups with a low median proportion of each bacterial group. The results are shown in Figure 2. High-Ruminococcus Enterobacteriaceae, high-Ruminococcus Unclassified Bacteriaceae, and high-Ruminococcus Bacteriaceae tended to have longer progression-free survival periods, while high-Bacteroides Bacteriaceae tended to have shorter progression-free survival periods.

[0078] <Examples of successful responses with immune checkpoint inhibitors: Transplantation of intestinal contents into mice> Suspensions were prepared by suspending intestinal contents from the above-mentioned successful and unsuccessful cases in an isotonic solution. These suspensions were administered to sterile BALB / cAJcl mice to investigate the effect of an immune checkpoint inhibitor (an anti-PD-1 antibody, Ultra-LEAF Purified anti-mouse CD279 (PD-1) (RMP1-14), purchased from BioLegend). The mice were divided into four groups: a group of mice treated with an immune checkpoint inhibitor and a group of mice not treated with an immune checkpoint inhibitor, as well as a group of mice that did not respond to an immune checkpoint inhibitor and a group of mice not treated with an immune checkpoint inhibitor. Fourteen days after administration of intestinal contents, MC38 cultured cells were transplanted subcutaneously into the mice to create an immune-responsive mouse tumor model. Five, eight, and eleven days after MC38 cultured cell transplantation, the treatment group received an immune checkpoint inhibitor, while the untreated group received intraperitoneal administration of PBS. Subsequently, tumor size and survival time were observed and compared for each mouse group. The tumor volume was calculated based on the measured tumor diameter using the following formula.

[0079]

number

[0080] The results are shown in Figure 3. The group of successful intestinal content transplantation patients treated with immune checkpoint inhibitors (successful treatment group) showed a significant reduction in tumor volume and extended survival compared to the group of successful intestinal content transplantation patients not treated with immune checkpoint inhibitors (successful treatment group), the group of non-successful intestinal content transplantation patients treated with immune checkpoint inhibitors (non-successful treatment group), and the group of non-successful intestinal content transplantation patients not treated with immune checkpoint inhibitors (non-successful treatment group). In other words, successful intestinal content transplantation was shown to enhance the effect of immune checkpoint inhibitors on tumors, that is, to enhance the immune response against tumors. Furthermore, the group of successful patients not treated showed a reduction in tumor volume and extended survival compared to the group of non-successful patients not treated. Therefore, successful intestinal content transplantation has an antitumor effect on its own, but when combined with immune checkpoint inhibitor treatment, it showed a synergistic antitumor effect.

[0081] <Transplantation of intestinal contents from patients who responded to immune checkpoint inhibitors into antibiotic-treated mice> The above intestinal contents transplantation experiment was performed using SPF mice that had been administered antibiotics beforehand.

[0082] Intestinal contents from the above-mentioned successful and unsuccessful cases were suspended in an isotonic solution to prepare suspensions. These suspensions were administered to sterile BALB / cAJcl mice that had been given antibiotics (ampicillin, vancomycin, neomycin, metronidazole) for 6 days, and the effect on the efficacy of an immune checkpoint inhibitor (Ultra-LEAF Purified anti-mouse CD279 (PD-1) (RMP1-14), an anti-PD-1 antibody purchased from BioLegend) was investigated. Mice were divided into four groups: an immune checkpoint inhibitor treatment group (responding intestinal content transplant + anti-PD-1 antibody (Anti PD-1 mAb) +) and a non-treatment group (responding intestinal content transplant + isotype control +) among the responding intestinal content transplant group; and an immune checkpoint inhibitor treatment group (non-responding intestinal content transplant + anti-PD-1 antibody +) and a non-treatment group (non-responding intestinal content transplant + isotype control +) among the non-responding intestinal content transplant group. Fourteen days after administration of intestinal content, MC38 cultured cells were transplanted subcutaneously into the mice to create an immune-responsive mouse tumor model. Five, eight, and eleven days after MC38 cultured cell transplantation, the treatment group received immune checkpoint inhibitors, while the untreated group received intraperitoneal administration of an isotype control antibody (Ultra-LEAF Purified Rat IgG2a, κ isotype Ctrl (RTK2758), purchased from BioLegend). Fourteen days later, the mice were euthanized, and lymphocytes were isolated from the recovered tumors. Flow cytometry was used to analyze tumor-infiltrating T cells. The results are shown in Figure 4. CD8 of the treatment group in response cases. + CD62L in cells - CD44 + The proportion of cell fractions was shown to be significantly larger in all groups compared to the response group (untreated), the non-response group (treated), and the non-response group (untreated). Furthermore, the CD8 of the response group was significantly higher. + TNF-α in cells + IFN-γ + The proportion of cell fractions was also shown to be significantly larger in the responding group (untreated), the non-responding group (treated), and the non-responding group (untreated) compared to all of them. In other words, the responding group showed a higher CD8 fraction compared to the responding group (untreated), the non-responding group (treated), and the non-responding group (untreated). +The proportion of effector cells in the cells was significantly higher, and CD8 + The study demonstrated a high level of cell-produced cytokines. This indicates that successful intestinal content transplantation has a significant immune response-enhancing effect even when performed alone, and that it has a synergistic immune response-enhancing effect when combined with immune checkpoint inhibitors.

[0083] <Examples of successful responses with immune checkpoint inhibitors: Isolation of Luminococcus enterobacteria from intestinal contents> The intestinal contents from the above successful cases were diluted in the anaerobic dilution (B) described in "The World of Intestinal Bacteria" by Tomotari Mitsuoka (1990), Asakura Shoten, to prepare a diluted intestinal contents solution. 10 -7 ~, 10 -8 , and 10 -9 Diluted intestinal contents were inoculated onto EG agar plates and cultured in an anaerobic chamber at 37°C for 3 to 4 days to generate colonies.

[0084] The 16S rRNA gene sequence was determined for each bacterial colony generated, and phylogenetic analysis was performed according to standard methods. The results are shown in Figure 5. Bacteria possessing the 16S rRNA gene with the nucleotide sequence shown in Sequence ID No. 1 were named Ruminococccaceae YB328.

[0085] <Transplantation of various bacteria into tumor mice treated with immune checkpoint inhibitors> Sterile BALB / cAJcl mice were administered antibiotics (ampicillin, vancomycin, neomycin, metronidazole) for 6 days, and MC38 cultured cells were subcutaneously transplanted into them. Immunotherapy checkpoint inhibitors (anti-PD-1 antibodies) were administered at 5, 8, and 11 days. The control group received isotype controls. The monobacterial transplantation group received oral administration of each bacterium (Akkermansia muchiniphilliam, Eggerhella lenta, Clostridum colicanis, Bacteroides vulgatus (B. vulgatus), Ersipelatoctostridum ransam, Ruminococccaceae YB328) simultaneously with the administration of either an immune checkpoint inhibitor or isotype control. Subsequently, tumor diameters were measured for each mouse, and the calculated tumor volumes are plotted in Figure 6. The group treated with Ruminococaceae YB328 showed a significant reduction in tumor volume compared to the group treated with immune checkpoint inhibitors alone, and also compared to the group transplanted with other bacteria. In other words, Ruminococaceae YB328, an enterobacterium of Ruminococaceae derived from the intestinal contents of successful patients, was shown to exhibit a synergistic antitumor effect when combined with immune checkpoint inhibitors.

[0086] Similar experiments were conducted with B. vulgatus and Ruminococcaceae YB328. Mice were euthanized 14 days after subcutaneous transplantation of MC38 cultured cells, and lymphocytes were isolated from the recovered tumors. Tumor-infiltrating T cells were analyzed using flow cytometry. The results are shown in Figure 7. CD8 in the Ruminococcaceae YB328-treated group. + CD62L in cells - CD44 +The proportion of cell fractions was shown to be significantly higher compared to the B. vulgatus administration group and the immune checkpoint inhibitor alone treatment group. Furthermore, in the Ruminococcaceae YB328 administration group, TNF-α + in CD8 + IFN-γ + The proportion of cell fractions was also shown to be significantly higher compared to the B. vulgatus administration group and the immune checkpoint inhibitor alone treatment group. That is, in the Ruminococcaceae YB328 administration group, the proportion of effector cells in CD8 + cells was significantly higher, and it was shown that there was more cytokine produced by CD8 + cells. This indicates that Ruminococcaceae YB328 has a synergistic effect of enhancing the immune response when combined with an immune checkpoint inhibitor.

[0087] <Effect of Ruminococcaceae intestinal bacteria on T cell activation> After co-culturing mouse bone marrow-derived dendritic cells (synonymous with dendritic cell progenitor cells) with Ruminococcaceae YB328 or B. vulgatus or vehicle, flow cytometry was used to measure dendritic cell maturation markers (CD80, CD86, MHC class I). The results are shown in Figure 8. For all measured dendritic cell maturation markers, a significant increase in expression was confirmed when co-cultured with Ruminococcaceae YB328 compared to co-culturing with B. vulgatus or vehicle. That is, it was shown that Ruminococcaceae YB328 has a significantly higher dendritic cell maturation effect compared to B. vulgatus or vehicle.

[0088] Next, the dendritic cells after the above co-culture were collected, and CD8 from OT-I mice+ T cells were co-cultured and stimulated with either N4 peptide (1nM, 10nM, or 100nM) or Q4H7 peptide (1nM, 10nM, or 100nM), known OVA antigen peptides with different affinities to the TCR. It is known that Q4H7 peptide has a lower affinity for the TCR than N4 peptide. Subsequently, CD8 was analyzed using ELISA. + We measured the T cell activation marker (IFN-γ) and the TCR signal (ZAP70 (pZAP70)) and CD28 signal (Erk (pErk), Akt (pAkt), S6 (pS6)) using flow cytometry.

[0089] CD8 when stimulated with 1 nM or 100 nM N4 peptide + Figure 9 shows the results of measuring the T cell activation marker (IFN-γ). Dendritic cells co-cultured with ruminococcaceae YB328 and CD8 + When co-cultured with T cells, dendritic cells co-cultured with B. vulgatus and CD8 + Compared to dendritic cells co-cultured with T cells, dendritic cells showed significantly higher IFN-γ production in response to N4 peptide stimulation. In other words, dendritic cells co-cultured with Ruminococcaceae YB328 showed significantly higher CD8 production compared to dendritic cells co-cultured with B. vulgatus. + It was shown to have a T-cell activating effect. Surprisingly, this effect was also demonstrated at a low concentration of 1 nM of N4 peptide. This suggests that Ruminococccae YB328 has a high immune response enhancing effect.

[0090] Figure 10 shows the measurement results of the TCR signal (ZAP70) and CD28 signal (Erk) when stimulated with N4 peptide or Q4H7 peptide. Dendritic cells co-cultured with luminococcusae YB328 and CD8 + When co-cultured with T cells, dendritic cells co-cultured with B. vulgatus and CD8+ When co-cultured with T cells or with normal dendritic cells and CD8 + Compared to dendritic cells co-cultured with T cells, dendritic cells co-cultured with ruminococcaceae YB328 showed significantly higher TCR signaling (ZAP70) and CD28 signaling (Erk) production in response to N4 peptide stimulation or Q4H7 peptide stimulation. In other words, dendritic cells co-cultured with ruminococcaceae YB328 showed significantly higher activation effects on both TCR and CD28 signaling compared to dendritic cells co-cultured with B. vulgatus. Surprisingly, this effect was also demonstrated by the Q4H7 peptide, which is usually thought to have a low TCR affinity and therefore contribute little to T cell activation.

[0091] Furthermore, the effects of different concentrations of N4 peptide stimulation (0nM, 1nM, 10nM, 100nM) on TCR signaling (ZAP70 (pZAP70)) and CD28 signaling (Erk (pErk), Akt (pAkt), S6 (pS6)) were investigated in dendritic cells co-cultured with luminococcusae YB328 and CD8 + When co-cultured with T cells, and when dendritic cells co-cultured with B. vulgatus and CD8 + Figure 11 shows the results of each investigation when co-cultured with T cells. For all signals, dendritic cells and CD8 co-cultured with ruminococcaceae YB328 + When co-cultured with T cells, dendritic cells co-cultured with B. vulgatus and CD8 + Compared to co-culture with T cells, significantly higher production was observed in response to all concentrations of N4 peptide stimulation examined. In other words, dendritic cells co-cultured with Ruminococcaceae YB328 showed significantly higher activation effects on both TCR and CD28 signaling, even at low concentrations of N4 peptide stimulation, compared to dendritic cells co-cultured with B. vulgatus.

[0092] From the above, it was shown that Ruminococaceae YB328 can mature dendritic cells, which are antigen-presenting cells, and that this results in a high activation effect on the two pathways necessary for T cell activation (TCR and CD28), even when using low concentrations of antigen stimulation or antigen stimulation with low affinity. This suggests that in vivo, Ruminococaceae enterobacteria such as Ruminococaceae YB328 have a high immune response enhancing effect.

[0093] <Effects of Enterobacteria luminococcus in mice with intestinal contents transplanted into tumors> Sterile BALB / cAJcl mice, administered antibiotics (ampicillin, vancomycin, neomycin, metronidazole) for 6 days, were then transplanted with suspensions of intestinal contents from mice that responded to the above-mentioned immune checkpoint inhibitors and those that did not, followed by transplantation of commensal intestinal bacteria. Subsequently, MC38 cultured cells were subcutaneously transplanted into the mice, and at 5, 8, and 11 days later, an immune checkpoint inhibitor (an anti-PD-1 antibody, Ultra-LEAF Purified anti-mouse CD279 (PD-1) (RMP1-14), purchased from BioLegend) was administered intraperitoneally, followed by oral administration of either Ruminococcaceae YB328 or B. vulgatus monocytoplasm. The tumor diameter was then measured for each mouse, and the tumor volume calculated based on these measurements is plotted in Figure 12. Furthermore, a meta-analysis based on the 16S rRNA gene was performed on the intestinal contents of mice collected after intestinal contents transplantation and after monomicrobial administration, and the diversity of the bacterial flora in each intestinal contents was compared, as shown in Figure 13.

[0094] In Figure 12, tumor growth was significantly suppressed in mice treated with ruminococcaceae YB328 monotherapy, regardless of whether they received intestinal contents from responding or non-responding mice. On the other hand, tumor growth was not suppressed in mice treated with B. vulgatus monotherapy, regardless of whether they received intestinal contents from responding or non-responding mice.

[0095] Figure 13 shows that the diversity of the gut microbiota significantly increased after administration of ruminococcaceae YB328 monocytoplasm compared to before administration. It is known that in cases of non-response with immune checkpoint inhibitors, the diversity of gut commensal bacteria is low. This experiment revealed that the ruminococcaceae enteroplasm of the present invention has a high effect in increasing the diversity of gut commensal bacteria compared to other bacteria. Although not bound by any particular theory, it is possible that increasing the diversity of gut commensal bacteria contributes to one of the mechanisms by which the ruminococcaceae enteroplasm of the present invention elicits an antitumor immune response.

[0096] <Effects of Enterobacteria luminococcus on dendritic cell differentiation (in vitro)> Mouse bone marrow-derived dendritic cells were co-cultured with luminococcusae YB328, B. vulgatus, LPS, or vehicle (PBS). RNA was then extracted from the dendritic cells and transcriptome analysis was performed. The results showed that dendritic cells stimulated with luminococcusae YB328 expressed genes characteristic of type 1 dendritic cells (cDC1), such as batf3, Irf8, and FLT3, more highly than dendritic cells stimulated with B. vulgatus, LPS, or vehicle (PBS) (Figure 14).

[0097] <Effects of Enterobacteria luminococcus on dendritic cell differentiation (in vivo) Part 1> Sterile BALB / cAJcl mice were administered antibiotics (ampicillin, vancomycin, neomycin, metronidazole) for 6 days, then subcutaneously transplanted with MC38 cultured cells. Five days later, they were orally administered either Ruminococcaceae YB328 or B. vulgatus, and eight days later, tissue samples (lymph nodes near the tumor, lamina propria, mesenteric lymph nodes) were collected.

[0098] FACS analysis revealed that the proportion of CD103-positive migratory dendritic cells in tumor-proximal lymph nodes and CD103-positive CD11b-negative dendritic cells in the lamina propria was significantly higher in the luminococcusse YB328-treated group. Furthermore, CCR7 expression was found to be higher in the mesenteric lymph nodes in the luminococcusse YB328-treated group (Figure 15). This suggests that YB328 administration induces dendritic cells with high migratory ability and a high proportion of cDC1 cells in vivo.

[0099] <Effects of Enterobacteria luminococcus on dendritic cell differentiation (in vivo) Part 2> Sterile BALB / cAJcl mice, administered antibiotics (ampicillin, vancomycin, neomycin, metronidazole) for 6 days, were subcutaneously transplanted with MC38 cultured cells. Immunotherapy checkpoint inhibitors (anti-PD-1 antibody, RMP1-14, BioLegend, USA) or isotype control antibodies (RTK2758, BioLegend, USA) were administered intravenously twice at 3-day intervals. Luminococcusae YB328, B. vulgatus, LPS, or vehicle (PBS) were orally administered 5, 8, and 11 days after subcutaneous transplantation of MC38 cultured cells, and tumors were collected 13 days later.

[0100] FACS analysis revealed that the proportion of CD103-positive dendritic cells within the tumor was significantly higher in the group treated with luminococcusse TB328 (Figure 16).

[0101] <Mechanism of dendritic cell differentiation induction by Enterobacteria luminococcus> Dendritic cell progenitor cells (bone marrow-derived dendritic cells) were isolated from mouse bone marrow cells and co-cultured with FLT3 ligand (FLT3L) necessary for differentiation into cDC1, Ruminococcaceae YB328 or B. vulgatus or LPS or vehicle (PBS), and then FACS analysis was performed on the expression of IRF8. High expression of IRF8 was observed in all cases when high-concentration (100 ng / ml) FLT3L was administered, but expression of IRF8 was maintained only in the Ruminococcaceae YBS328-administered group when low-concentration (1 ng / ml) FLT3L was administered (Figure 17). From this, it was suggested that Ruminococcaceae YB328 might induce the differentiation of cDC1 independently of FLT3L.

[0102] Furthermore, after bone marrow-derived dendritic cells were co-cultured with Ruminococcaceae YB328 or B. vulgatus or LPS or vehicle (PBS) for 4 hours, FACS analysis was performed on the expression of p-S6 kinase (p-S6K) and p-STAT3. It was found that the expression of the quantitative molecules of p-S6K and p-STAT3 was high only in the Ruminococcaceae YB328-administered group (Figure 18).

[0103] FLT3L is thought to be involved in the differentiation of dendritic cells by activating the PI3K-mTOR pathway. From the above results, it is thought that Ruminococcaceae YB328 is involved in the induction of differentiation into cDC1 by activating the PI3K-mTOR pathway instead of FLT3L.

[0104] <Induction of dendritic cell progenitor cells into type 1 dendritic cells by TLR complex stimulation> As described above, it has become clear that Ruminococcaceae YB328 is involved in the expression of various molecules related to the differentiation of dendritic cells. Therefore, in order to clarify the upstream of the differentiation induction pathway of cDC1 by Ruminococcaceae YB328, we focused on TLR and examined as follows.

[0105] First, in the transcriptome analysis shown in Figure 14, we focused on TLRs and found that dendritic cell progenitor cells stimulated with luminococcusse YB328 showed higher expression of various TLRs compared to dendritic cell progenitor cells stimulated with B. vulugatus (Figure 19, A). In particular, TLR1, TLR3, TLR5, TLR7, and TLR9 showed high expression.

[0106] Furthermore, MyD88 knockout mouse (MyD88 - / - Bone marrow-derived dendritic cells collected from () were stimulated with luminococcusse YB328, and then subjected to FACS analysis. As a result, CD103-positive, CD11b-negative dendritic cells were not induced, which was similar to the result obtained when stimulated with the vehicle (Figure 19, B). This suggests that the differentiation of CD103-positive dendritic cells induced by luminococcusse YB328 is dependent on MyD88-TLR signaling, indicating that TLR signaling plays an important role.

[0107] In the transcriptome analysis described above, TLR5, 7, and 9 showed particularly high expression in the luminococcusse YB328 group. Therefore, when mouse bone marrow-derived dendritic cells (intact dendritic cell progenitor cells that have not been stimulated by bacteria, etc.) were treated with various mixtures of agonists for TLR5, 7, and 9 (flagelin, R848 (leximod), and ODN-1826, respectively), CD103-positive CD11b-negative dendritic cells, i.e., type 1 dendritic cells, were significantly induced in dendritic cells treated with all three agonists for TLR5, 7, and 9 compared to those treated with LPS or a control (Figure 19, C). Furthermore, this experiment also found that adding LPS, an agonist for TLR4, to the mixture of agonists for TLR5, 7, and 9 significantly reduced the induction of CD103-positive CD11b-negative dendritic cells, i.e., type 1 dendritic cells (Figure 19, C). This suggests that stimulating multiple TLRs other than TLR4 simultaneously induces type 1 dendritic cells from dendritic cell progenitor cells. Furthermore, it is suggested that luminococcusse YB328 stimulates multiple TLRs involved in the induction of these type 1 dendritic cells.

Claims

1. A pharmaceutical composition comprising Luminococcus Enterobacteria, wherein the Luminococcus Enterobacteria has a 16S rRNA gene having 99% or more sequence identity with the base sequence shown in Sequence ID No. 1, and induces cDC1 differentiation independently of FLT3L.

2. A pharmaceutical composition comprising bacterial cells of Luminococcus enterobacteriaceae having a 16S rRNA gene having 99% or more sequence identity with the base sequence shown in Sequence ID No. 1, wherein the pharmaceutical composition is administered in combination with an immune checkpoint inhibitor.

3. The pharmaceutical composition according to claim 2, wherein the bacterial cells of Luminococcus entericitis are live cells.

4. The pharmaceutical composition according to claim 2 or 3, wherein the immune checkpoint inhibitor is an inhibitor of any immune checkpoint molecule selected from the group consisting of PD-1, CTLA-4, TIM-3, BTLA, LAG-3, A2aR, KIR, VISTA, TIGIT, PD-L1, PD-L2, CD80, CD86, GAL-9, HVEM, CD160, MHC class II, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7, or a combination of two or more such inhibitors.

5. The pharmaceutical composition according to claim 4, wherein the immune checkpoint inhibitor is selected from an antibody against the immune checkpoint molecule, an antigen-binding fragment of the antibody, and a combination thereof.

6. A pharmaceutical composition according to any one of claims 1 to 5, which is administered by oral administration, enteral administration, or enema administration.

7. A pharmaceutical composition according to any one of claims 2 to 6, wherein an immune checkpoint inhibitor and the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus are administered simultaneously.

8. The pharmaceutical composition according to claim 7, comprising an immune checkpoint inhibitor and the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus.

9. The pharmaceutical composition according to any one of claims 2 to 6, wherein an immune checkpoint inhibitor and the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus are administered separately.

10. The pharmaceutical composition according to claim 9, wherein the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus are administered before the administration of an immune checkpoint inhibitor.

11. The pharmaceutical composition according to claim 9, wherein, after administration of an immune checkpoint inhibitor, the cells, culture supernatant, metabolites, and / or cell extracts of Enterobacteria luminococcus are administered.

12. A pharmaceutical composition according to any one of claims 1 to 11 for activating CD8-positive T cells in a target area.

13. A pharmaceutical composition according to any one of claims 1 to 11, for enhancing the immune response against a tumor or cancer in a subject having a tumor or cancer.

14. The pharmaceutical composition according to claim 13, which has a greater effect in enhancing the immune response against tumors or cancer compared to the case of administering the immune checkpoint inhibitor alone.

15. A pharmaceutical composition according to any one of claims 1 to 14 for treating tumors or cancer in a subject.

16. The pharmaceutical composition according to claim 15, wherein the treatment eliminates, reduces, or stabilizes a tumor or cancer.

17. The pharmaceutical composition according to claim 16, which has a greater effect in eliminating, shrinking, or stabilizing tumors or cancer compared to the case of administering the immune checkpoint inhibitor alone.

18. A pharmaceutical composition according to any one of claims 1 to 14 for suppressing the recurrence or metastasis of a tumor or cancer in a target.

19. The pharmaceutical composition according to claim 18, which has a greater inhibitory effect on tumor or cancer recurrence or metastasis compared to the case of administration of the immune checkpoint inhibitor alone.

20. A pharmaceutical composition according to any one of claims 13 to 19, which is administered in combination with one or more therapies selected from the group consisting of surgical therapy, chemotherapy, and radiotherapy.

21. A pharmaceutical composition according to any one of claims 1 to 11 for increasing the diversity of commensal bacteria in the intestines of mammals compared to before administration.

22. The pharmaceutical composition according to any one of claims 1 to 21, wherein the ruminococcaceae enteric bacteria is ruminococcaceae YB328.

23. Luminococcusse enterobacteria, wherein the Luminococcusse enterobacteria possess a 16S rRNA gene having 99% or more sequence identity with the base sequence shown in Sequence ID No. 1, and induces cDC1 differentiation independently of FLT3L.

24. Luminococcusse enterobacteria, wherein the Luminococcusse enterobacteria possess a 16S rRNA gene having 99% or more sequence identity with the base sequence shown in Sequence ID No. 1, induces cDC1 differentiation independently of FLT3L, and further possesses at least one of the following characteristics (1) to (3): (1) It can be suitably cultured in an anaerobic chamber at 37°C using EG medium; (2) It can mature dendritic cells; and (3) It has the ability to increase the diversity of intestinal flora.

25. A bacterial cell of Luminococcusse enterobacteria, wherein the Luminococcusse enterobacteria has a 16S rRNA gene that has 99% or more sequence identity with the base sequence shown in Sequence ID No. 1, and induces cDC1 differentiation independently of FLT3L.

26. The luminococcus enteric bacteria or bacterial cells according to any one of claims 23 to 25, wherein the luminococcus enteric bacteria is luminococcus YB328 (Ruminococcaceae YB328).

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