Biomarker comprising lactobacillus salivarius or bacteroides flavius for responsive prediction and prognosis of immune anticancer drug treatment, and method for responsiveness prediction and prognosis of immune anticancer drug treatment using same

Abstract

The present invention. relates to a biomarker comprising Lactobacillus salivarius or Bacteroides flavius for responsiveness prediction and prognosis of immune anticancer drug treatment and a method for responsiveness prediction and prognosis of immune anticancer drug treatment using same. In the present invention, Lactobacillus salivarius and Bacteroides plebeius strains were selected as strains that inhibit the therapeutic effect of immune anticancer agents in a cohort of patients treated with immune anticancer agents who have received fecal microorganism transplantation. By detecting the strain, the present invention has the effects of predicting immune anticancer drug treatment responsiveness or prognosis after immune anticancer drug treatment and securing the efficiency of existing fecal microorganism transplantation. In addition, the selected strain is a strain present in the human intestine and has excellent safety due to low therapeutic side effects, and thus is expected to be advantageously used for treatment of autoimmune diseases and the like through immune activity inhibition.

Description

Biomarkers for predicting immunotherapy response and prognosis including Lactobacillus salivarius or Bacteroides plebius and methods for predicting immunotherapy response and prognosis using the same

[0001] The present invention relates to a biomarker for predicting immunotherapy response and prognosis including Lactobacillus salivarius or Bacteroides plebius, and a method for predicting immunotherapy response and prognosis using the same.

[0002] This application claims priority to Korean Patent Application No. 10-2023-0070783, filed June 1, 2023, the entire disclosure of which is incorporated herein by reference.

[0003]

[0004] Traditional cancer treatments have focused on removing as many cancer cells as possible from the patient through surgery, radiation therapy, and chemotherapy. However, surgery and radiation therapy are only effective when the cancer is relatively early and has not metastasized, effectively eliminating cancer cells. While chemotherapy is widely used, it is not effective for most solid tumors and has numerous side effects. To address these issues, immunotherapy using immunotherapy agents has recently emerged.

[0005] Immunotherapy, or immunotherapy, is a treatment that activates the body's immune system to fight cancer cells. It offers a new paradigm in cancer treatment, with high cure rates expected in patients who respond. However, the actual success rate of immunotherapy, such as anti-PD-1 / anti-PD-L1, is only 15-20%, indicating a low responsiveness to immunotherapy. This response varies significantly depending on the type and stage of the cancer and the patient's condition. Furthermore, the increased activity and secretion of immune cells following the use of immunotherapy can also affect normal cells, potentially leading to adverse effects.

[0006] Accordingly, considering the diversity of therapeutic responses to immunotherapy, research on companion diagnostics using biomarkers that can predict therapeutic responses to immunotherapy is actively underway.

[0007] However, considering the cost and low cure rate of immunotherapy, there is still a need for biomarkers that can more accurately predict the treatment response of immunotherapy and companion diagnostics using these biomarkers to provide personalized, precise treatment to patients.

[0008] Accordingly, the inventors of the present invention aimed to select a biomarker that can predict immunotherapy response or prognosis by identifying the gut microbiota composition of patients with high and low treatment response to immunotherapy.

[0009]

[0010] The present inventors confirmed the microbial composition after fecal microbiota transplantation (FMT) in patients receiving immunotherapy, and confirmed that the Lactobacillus salivarius and Bacteroides plebeius strains were significantly reduced in patients whose clinical characteristics improved after FMT, and completed the present invention based on this.

[0011]

[0012] Accordingly, the purpose of the present invention is to provide a composition for predicting the response to immunotherapy or a composition for predicting the prognosis of a cancer patient after immunotherapy, which comprises as an active ingredient an agent for detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius.

[0013] Another object of the present invention is to provide a kit for predicting immunotherapy responsiveness or a kit for predicting prognosis after immunotherapy treatment of a cancer patient, comprising the composition and instructions.

[0014] Another object of the present invention is to provide a method for providing information for predicting immunotherapy responsiveness or a method for predicting prognosis of a cancer patient after immunotherapy treatment, the method comprising a step of detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius in a biological sample isolated from a subject.

[0015] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating autoimmune diseases, which comprises as an active ingredient one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius.

[0016]

[0017] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0018]

[0019] To achieve the above purpose, the present invention provides a composition for predicting immunotherapy response, comprising as an active ingredient an agent for detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius.

[0020] In addition, the present invention provides a composition for predicting the prognosis of a cancer patient after immunotherapy treatment, comprising as an active ingredient an agent for detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius.

[0021] As one embodiment of the present invention, the immuno-oncology agent may be at least one selected from the group consisting of an immune checkpoint inhibitor, an immune cell therapy agent, and an immune virus therapy agent, but is not limited thereto.

[0022] In another embodiment of the present invention, the immune checkpoint inhibitor may be at least one selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA4 inhibitor, a LAG3 inhibitor, a TIM3 inhibitor, a 4-1BB inhibitor, a B7-H4 inhibitor, a VISTA inhibitor, a KIR inhibitor, and a BTLA inhibitor, but is not limited thereto.

[0023] As another embodiment of the present invention, the preparation may be at least one selected from the group consisting of a primer pair, a probe, an antisense oligonucleotide, an antibody, and an aptamer that specifically binds to 16S rRNA of the microorganism, but is not limited thereto.

[0024] As another embodiment of the present invention, the primer pair may include, but is not limited to, the base sequences of SEQ ID NOs: 1 and 2; or the base sequences of SEQ ID NOs: 3 and 4.

[0025] In another embodiment of the present invention, the cancer may be at least one selected from the group consisting of colorectal cancer, rectal cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, glioblastoma, lung cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, breast cancer, uterine cancer, stomach cancer, bone cancer, lymphoma, blood cancer, squamous cell carcinoma, lung adenocarcinoma, peritoneal cancer, skin cancer, skin melanoma, ocular melanoma, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, glioblastoma, endometrial cancer, salivary gland cancer, vulvar cancer, and head and neck cancer, but is not limited thereto.

[0026] In addition, the present invention provides a kit for predicting immunotherapy responsiveness, comprising a composition for predicting immunotherapy responsiveness and an instruction manual.

[0027] In addition, the present invention provides a kit for predicting the prognosis of a cancer patient after immunotherapy treatment, comprising a composition and instructions for predicting the prognosis of a cancer patient after immunotherapy treatment.

[0028] In addition, the present invention provides a method for providing information for predicting immunotherapy responsiveness, comprising a step of detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius in a biological sample isolated from a subject.

[0029] In addition, the present invention provides a method for providing information for predicting the prognosis of a cancer patient after immunotherapy treatment, comprising a step of detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius in a biological sample isolated from a subject.

[0030] As one embodiment of the present invention, the biological sample may be at least one selected from the group consisting of blood, whole blood, plasma, stool, urine, tissue, cells, organs, bone marrow, fine needle aspiration specimen, fine needle lavage fluid, core needle biopsy specimen, and saliva, but is not limited thereto.

[0031] As another embodiment of the present invention, the method for providing information for predicting immunotherapy responsiveness may further include, but is not limited to, a step of predicting that immunotherapy responsiveness will be low when the number of Lactobacillus salivarius or Bacteroides plebeius is higher than that of the control group.

[0032] As another embodiment of the present invention, the method for providing information for predicting the prognosis of a cancer patient after immunotherapy treatment may further include, but is not limited to, a step of predicting that the prognosis of the cancer patient after immunotherapy treatment will be poor when the number of Lactobacillus salivarius or Bacteroides plebeius is higher than that of the control group.

[0033] In addition, the present invention provides a pharmaceutical composition for preventing or treating an autoimmune disease, which comprises as an active ingredient one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius or a culture thereof.

[0034] As one embodiment of the present invention, the autoimmune disease may be at least one selected from the group consisting of multiple sclerosis, inflammatory bowel disease, graft-versus-host disease, asthma, atopy, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus, but is not limited thereto.

[0035] In another embodiment of the present invention, the composition may suppress immune activity, but is not limited thereto.

[0036] In another embodiment of the present invention, the composition may inhibit proliferation of T cells or reduce secretion of interferon gamma (IFN-γ) of T cells, but is not limited thereto.

[0037] In addition, the present invention provides a use for predicting immunotherapy response of a composition comprising, as an active ingredient, an agent for detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius.

[0038] In addition, the present invention provides a use for preparing a formulation for predicting immunotherapy response of an anticancer drug, wherein the formulation detects one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius.

[0039] In addition, the present invention provides a method for preventing or treating an autoimmune disease, comprising a step of administering to a subject in need thereof a composition comprising, as an active ingredient, one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius or a culture thereof.

[0040] In addition, the present invention provides a composition comprising, as an active ingredient, one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius or a culture thereof, for use in preventing or treating autoimmune diseases.

[0041] In addition, the present invention provides a use for producing a preparation for preventing or treating an autoimmune disease using one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius or a culture thereof.

[0042]

[0043] In the present invention, Lactobacillus salivarius and Bacteroides plebeius strains were selected as strains that suppress the effect of immunotherapy in a cohort of patients receiving fecal microbiota transplantation and receiving immunotherapy. Therefore, the present invention has the effect of predicting immunotherapy responsiveness or prognosis after immunotherapy by detecting the strains, and ensuring the effectiveness of existing fecal microbiota transplantation. In addition, the selected strains are strains existing in the human intestine and have excellent safety due to low treatment side effects, and are expected to be useful for the treatment of autoimmune diseases and the like through suppression of immune activity.

[0044]

[0045] Figure 1 is a diagram schematically showing the research design of a clinical trial according to one embodiment of the present invention.

[0046] FIG. 2 is a diagram schematically illustrating a combined treatment process of FMT and anti-PD-(L)1 inhibitor according to one embodiment of the present invention.

[0047] FIG. 3 is a diagram showing the results of confirming the microbial composition of recipients R#1, R#2, and R#7 after FMT according to one embodiment of the present invention through beta diversity analysis.

[0048] Figures 4a and 4b are drawings showing the results of confirming clinical efficacy for 13 FMT recipients according to one embodiment of the present invention.

[0049] FIGS. 4c and 4d are drawings showing the results of checking the alpha-fetoprotein (AFP) level after performing the first FMT using the donor stool of D#1 (FIG. 4c) or the second FMT using the donor stool of D#5 (FIG. 4d) in FMT recipient R#7 according to one embodiment of the present invention.

[0050] Figure 4e is a drawing showing the results of confirming systemic immune changes in peripheral blood in FMT recipient R#7 according to one embodiment of the present invention.

[0051] FIG. 4f is a diagram showing the results of confirming immune activation in the tumor microenvironment in FMT recipient R#7 according to one embodiment of the present invention.

[0052] FIG. 4g is a diagram showing the test results showing that immune-related gastritis occurred due to the immune enhancement effect after the second FMT in FMT recipient R#7 according to one embodiment of the present invention.

[0053] FIG. 5a is a diagram showing the results of confirming changes in microbial abundance at the genus level in FMT recipient R#7 according to one embodiment of the present invention.

[0054] FIG. 5b is a Venn diagram showing the number of microorganisms in FMT recipient R#7 according to one embodiment of the present invention and the decrease in the number of microorganisms after the second FMT compared to the first FMT.

[0055] FIG. 5c is a diagram showing the results of confirming the abundance of Bacteroides plebeius strains after the first and second FMTs in FMT recipient R#7 according to one embodiment of the present invention.

[0056] FIG. 5d is a diagram showing the results of confirming the responsiveness to immunotherapy in FMT recipients R#6 and R#7 according to one embodiment of the present invention.

[0057] FIG. 5e is a diagram showing the results of comparing the abundance of B. plebeius in FMT recipients R#6 and R#7 according to one embodiment of the present invention.

[0058] FIG. 5f is a drawing showing the results of confirming the abundance of Lactobacillus salivarius strains after the first FMT and the second FMT in FMT recipients R#6 and R#7 according to one embodiment of the present invention.

[0059] Figure 6a is a drawing showing the results of confirming the T cell proliferation inhibitory effect of B. plebeius and L. salivarius strains according to one embodiment of the present invention.

[0060] Figure 6b is a drawing showing the results of confirming the interferon gamma secretion inhibition effect of T cells of B. plebeius and L. salivarius strains according to one embodiment of the present invention.

[0061] FIG. 7 is a diagram showing the results of confirming the survival rate according to the presence or absence of B. plebeius and L. salivarius strains within an independent biomarker cohort according to one embodiment of the present invention.

[0062]

[0063] In one experimental example of the present invention, as a result of checking the microbial composition after fecal microbiota transplantation (FMT), it was confirmed that recipients R#1, R#2, and R#7 showed a microbial composition similar to that of the donor (see Experimental Example 1), and as a result of checking various clinical responses to immunotherapy administration in recipient R#7, which showed a partial response among them, it was found that there was no effect of enhancing the responsiveness to immunotherapy after the first FMT using the stool of donor D#1, and that the responsiveness to immunotherapy was improved after the second FMT using the stool of donor D#5 (see Experimental Example 2).

[0064] In another experimental example of the present invention, the causative agent responsible for the therapeutic effect of FMT was identified. As a result, the Bacteroides plebeius strain was found to be more prevalent in the first FMT donors than in the second FMT donors in recipient R#7, and was found to be significantly more prevalent in the first FMT than after the second FMT in patient R#7. Therefore, it was found that the B. plebeius strain is a strain that suppresses the therapeutic effect of immunotherapy. In addition, as a result of analyzing many strains from patient R#6, whose immunotherapy response did not improve compared to patient R#7 after the second FMT, the Lactobacillus salivarius strain was selected. This was the strain originally present in patient R#6, and it persisted or increased after FMT. Since it was the strain present in patient R#7's first FMT, which did not show an improvement in immunotherapy response, it was found to be a strain that suppresses the immunotherapy effect (see Experimental Example 3).

[0065] In another experimental example of the present invention, the immunosuppressive activity of B. plebeius and L. salivarius strains was confirmed, and when the B. plebeius strain and L. salivarius strain cultures were treated, it was confirmed that the proliferation of both CD4 T cells / CD8 T cells was inhibited, and when the B. plebeius strain and L. salivarius strain cultures were treated, it was confirmed that the IFN-γ concentration was significantly reduced compared to the control group, and it was confirmed that the B. plebeius strain and L. salivarius strain could suppress the immune activity of the host (see Experimental Example 4).

[0066] In another experimental example of the present invention, the survival rate was confirmed according to the presence or absence of B. plebeius and L. salivarius strains, and it was confirmed that the overall survival (OS) period was significantly longer in the patient group without B. plebeius strains and L. salivarius (see Experimental Example 5).

[0067]

[0068] Hereinafter, the present invention will be described in detail.

[0069]

[0070] The present invention provides a composition for predicting immunotherapy response, comprising as an active ingredient an agent for detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius.

[0071] In addition, the present invention provides a composition for predicting the prognosis of a cancer patient after immunotherapy treatment, comprising as an active ingredient an agent for detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius.

[0072] In the present invention, the specific strain of the “Lactobacillus salivarius” microorganism is not limited as long as it is a bacterium known in the art or determined (classified) as Lactobacillus salivarius species by a genome analysis means known in the art.

[0073] In the present invention, the specific strain of the “Bacteroides plebeius” microorganism is not limited as long as it is a bacterium known in the art or determined (classified) as a Bacteroides plebeius species by a genome analysis means known in the art.

[0074] In the present invention, the preparation may be at least one selected from the group consisting of a primer pair, a probe, an antisense oligonucleotide, an antibody, and an aptamer that specifically binds to the 16S rRNA of the microorganism, but is not limited thereto.

[0075] In the present invention, a "primer" refers to a short sequence of bases having a short free 3' hydroxyl group at the end, which can form base pairs with a complementary template and serves as a starting point for copying the template strand. In the present invention, a sense primer and an antisense primer can be provided as a pair (set). The primer can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. By performing PCR amplification, the responsiveness to immunotherapy for cancer can be determined (predicted) based on whether a desired product is produced or / and whether the amount of the product increases. PCR conditions and the lengths of the sense and antisense primers can be modified based on those known in the art.

[0076] In the present invention, "16S rRNA" is an rRNA that constitutes the 30S subunit of prokaryotic ribosomes, and since it has a conserved region common to all species and a hypervariable region that can classify specific species, microorganisms can be identified through base sequence analysis. In particular, since diversity is almost non-existent between the same species, diversity appears between different species, so prokaryotes can be usefully identified by comparing the sequences of 16S rRNA. In addition, since 16S rDNA is a gene that encodes 16S rRNA, microorganisms can also be identified using 16S rDNA.

[0077] In the present invention, the 16s rRNA of Lactobacillus salivarius may be composed of or include the sequence of NCBI Reference Sequence: NR_028725.2 (https: / www.ncbi.nlm.nih.gov / nuccore / NR_028725.2), but is not limited thereto.

[0078] In the present invention, the 16s rRNA of the Bacteroides plebius may be composed of or include the sequence of GenBank: AB200217.1 (https: / www.ncbi.nlm.nih.gov / nuccore / AB200217), but is not limited thereto.

[0079] In the present invention, the primer pair refers to a primer pair that specifically binds to a 16s rRNA base sequence that specifically exists in Lactobacillus salivarius and Bacteroides plebius.

[0080] In the present invention, the primer pair for Lactobacillus salivarius may be composed of or include 5'-AGCGCAGACGGGTCGTTAAG-3' (forward, SEQ ID NO: 1) and 5'-TCACATCAGACTTAAAAGACCGC-3' (reverse, SEQ ID NO: 2), but is not limited thereto. The primer pair for Bacteroides plebius may be composed of or include 5'-AGCGCAGACGGGTCGTTAAG-3' (forward, SEQ ID NO: 3) and 5'-GTGTGCTGCCTTCGCAATCG-3' (reverse, SEQ ID NO: 4), but is not limited thereto.

[0081] As used herein, the term "probe" refers to a typically labeled oligonucleotide having the sequence of a target nucleic acid and forming a duplex structure by complementary base pairing. The probe will comprise a "hybridization region" corresponding to a region of the target sequence, preferably at least 30 nucleotides, and in some cases at least 50 nucleotides. "Corresponding" means identical to or complementary to the designated nucleic acid. The probe may preferably not contain a sequence complementary to the sequence(s) used to prepare the PCR. Typically, the 3' end of the probe will be "blocked" to inhibit incorporation of the probe into the primer extension product. "Blocking" can be achieved by using a non-complementary base or by adding a chemical moiety, such as biotin, a phosphate group, or a fluorescent agent, to the 3' hydroxyl of the base nucleotide, which can serve a dual purpose by acting as a label for subsequent detection or capture of the labeled nucleic acid, depending on the selected moiety. Blocking can be achieved by removing the 3'-OH or using nucleotides lacking a 3'-OH, such as dideoxynucleotides.

[0082] The term "antisense oligonucleotide" as used herein is defined as an oligonucleotide capable of modulating the expression of a target gene by hybridizing to a target nucleic acid, particularly a contiguous sequence on the target nucleic acid. This includes modified or substituted oligomers comprising non-naturally occurring monomers or portions thereof that function similarly to the present invention. The incorporation of substituted oligomers is based on factors including enhanced cellular uptake or increased nuclease resistance, and may be selected as known in the art. The entire oligonucleotide or a portion thereof may contain substituted oligomers. Furthermore, the antisense oligonucleotides of the present invention may include oligomeric mimetics, such as peptide nucleic acids (PNAs) and locked nucleic acids (LNAs), modified by methods known in the art to increase their affinity for their target and provide tolerance to mismatches of the target sequence. In addition, the antisense oligonucleotide may be in the form of a modified oligonucleotide, including, but not limited to, a natural oligonucleotide, a phosphorothioate-type oligodeoxyribonucleotide, a phosphorodithioate-type oligodeoxyribonucleotide, a methylphosphonate-type oligodeoxyribonucleotide, a phosphoramidate-type oligodeoxyribonucleotide, an H-phosphonate-type oligodeoxyribonucleotide, a triester-type oligodeoxyribonucleotide, an alpha-anomeric oligodeoxyribonucleotide, a peptide nucleic acid, other artificial nucleic acids, and nucleic acid-modified compounds.

[0083] The length of the antisense oligonucleotide is not particularly limited, but may preferably be 6 to 100 bases, more preferably 8 to 60 bases, and even more preferably 10 to 40 bases. The antisense oligonucleotide may be synthesized in vivo according to a conventional method used in the art, or may be synthesized in vitro and administered into a body. A non-limiting example of a method for synthesizing antisense RNA in vivo includes a method in which antisense RNA is transcribed using a vector in which the origin of a multi-cloning site (MCS) is in the opposite direction. A non-limiting example of a method for synthesizing antisense RNA in vitro includes a method using RNA polymerase I.

[0084] In the present invention, "microorganism-specific antibody" or "antibody against microorganism" refers to a specific protein molecule directed against an antigenic site (or a marker protein, etc., that distinguishes the microorganism from other organisms) of the target microorganism (bacteria). For the purposes of the present invention, the antibody refers to an antibody that specifically binds to an antigenic site (or antigenic substance) of the microorganism (bacteria), and includes polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. For the purposes of the present invention, it may be preferable that the antibody be a monoclonal antibody, which is a group of antibodies in which the amino acid sequences of the heavy and light chains of the antibodies are substantially identical.

[0085] The production of antibodies as described above can be readily accomplished using techniques well known in the art. Polyclonal antibodies can be produced by methods well known in the art, including injecting an antigen into an animal and collecting blood from the animal to obtain serum containing the antibody. Such polyclonal antibodies can be produced from any animal species, including goats, rabbits, sheep, monkeys, horses, pigs, cattle, and dogs.

[0086] In the present invention, an "aptamer" refers to a single-stranded DNA (ssDNA) or RNA that has high specificity and affinity for a specific substance. Aptamers have very high affinity for a specific substance and are stable, can be synthesized by a relatively simple method, can be modified in various ways to increase binding affinity, and can target cells, proteins, and even small organic substances. Therefore, their specificity and stability are much higher than those of antibodies that have already been developed. For example, an aptamer may be a small nucleic acid, 15-50 bases in length, that folds into a defined secondary or tertiary structure, such as a stem-loop structure, as is known in the art. Aptamers can bind to target molecules with very high specificity. Furthermore, aptamers may be composed of multiple ribonucleotide units, deoxyribonucleotide units, or a mixture of two types of nucleotide residues. Aptamers may additionally include one or more modified base, sugar, or phosphate backbone units.

[0087] In the present invention, "immunotherapy" refers to a drug that enhances the body's own immune system and thus its ability to fight cancer. Because immunotherapy agents treat cancer by strengthening the patient's own immune system, they have fewer side effects, improve the quality of life of cancer patients, and significantly extend survival. Immunotherapy agents exhibit anticancer effects by enhancing the specificity, memory, and adaptability of the immune system. Examples of immunotherapy agents include, but are not limited to, agents for immune checkpoint blockade (ICB), immune cell therapy, immunoviral therapy, therapeutic antibodies, and immune checkpoint enhancers. In the present invention, the agent for blocking the immune checkpoint, i.e., the immune checkpoint inhibitor, has a mechanism in which, unlike existing immunotherapeutic agents (cytokine therapeutic agents, anticancer vaccines, etc.), it binds to the binding site of cancer cells and T cells to block immune evasion signals, thereby preventing the formation of immunological synapses, and thus allowing T cells that are not immune evasion-resistant to destroy cancer cells.

[0088] In the present invention, the immune checkpoint inhibitor may be at least one selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA4 inhibitor, a LAG3 inhibitor, a TIM3 inhibitor, a 4-1BB inhibitor, a B7-H4 inhibitor, a VISTA inhibitor, a KIR inhibitor, and a BTLA inhibitor, and may include, but is not limited to, nivolumab, atezolizumab, pembrolizumab, duvalumab, avelumab, ipilimumab, or tremelimumab.

[0089] In the present invention, “detection” includes both measuring and confirming the presence (expression) of a target substance, or measuring and confirming a change in the presence level (expression level) of the target substance. In the same context, detecting the microorganism in the present invention means measuring the presence of the microorganism (i.e., measuring the presence or absence) or measuring the qualitative and quantitative change level of the microorganism. The measurement can be performed without limitation, including both qualitative methods (analysis) and quantitative methods. The types of qualitative and quantitative methods for measuring the presence or absence of microorganisms are well known in the art, and the experimental methods described herein are included therein.

[0090] In the present invention, the cancer may be at least one selected from the group consisting of colon cancer, rectal cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, glioblastoma, lung cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, breast cancer, uterine cancer, stomach cancer, bone cancer, lymphoma, blood cancer, squamous cell carcinoma, lung adenocarcinoma, peritoneal cancer, skin cancer, skin melanoma, ocular melanoma, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, glioblastoma, endometrial cancer, salivary gland cancer, vulvar cancer, and head and neck cancer, but is not limited thereto.

[0091]

[0092] In addition, the present invention provides a kit for predicting immunotherapy responsiveness, comprising a composition for predicting immunotherapy responsiveness and an instruction manual.

[0093] In addition, the present invention provides a kit for predicting the prognosis of a cancer patient after immunotherapy treatment, comprising a composition and instructions for predicting the prognosis of a cancer patient after immunotherapy treatment.

[0094] In the present invention, the term "kit" refers to a tool that includes a preparation that detects the presence level of the microorganism, thereby enabling prediction of immunotherapy responsiveness or prediction of the prognosis of a cancer patient after immunotherapy treatment. In addition to the preparation, the kit of the present invention may include other components, compositions, solutions, devices, etc. that are typically required for the measurement or detection methods thereof. In this case, the substance that measures the presence level of the microorganism may be applied one or more times without limitation, and there is no restriction on the order in which each substance is applied, and the application of each substance may be performed simultaneously or microscopically.

[0095] In the present invention, the kit may include a container; instructions; and a preparation for detecting the presence level of the microorganism. The container may serve to package the preparation, and may also serve to store and fix the preparation. The material of the container may take the form of, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, etc., and these may be formed partially or wholly from plastic, glass, paper, foil, wax, etc. The container may be initially equipped with a completely or partially detachable stopper, which may be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also be equipped with a stopper for allowing access to the contents by means of a syringe needle. The kit may include an outer package, which may include instructions for the use of the components.

[0096] In addition, the present invention provides a use for predicting immunotherapy response of a composition comprising, as an active ingredient, an agent for detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius.

[0097] In addition, the present invention provides a use for preparing a formulation for predicting immunotherapy response of an anticancer drug, wherein the formulation detects one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius.

[0098]

[0099] In addition, the present invention provides a method for providing information for predicting immunotherapy responsiveness, comprising a step of detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius in a biological sample isolated from a subject.

[0100] In addition, the present invention provides a method for predicting immunotherapy responsiveness, comprising a step of detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius in a biological sample isolated from a subject.

[0101] In addition, the present invention provides a method for providing information for predicting the prognosis of a cancer patient after immunotherapy treatment, comprising a step of detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius in a biological sample isolated from a subject.

[0102] In addition, the present invention provides a method for predicting the prognosis of a cancer patient after immunotherapy treatment, comprising the step of detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius in a biological sample isolated from a subject.

[0103] In the present invention, the intestinal microorganisms may be measured by measuring the genome or gene amount specific to each microorganism using a primer set or probe that specifically binds to the 16S rRNA gene of the microorganism, but is not limited thereto. At this time, the amount of the microbial-specific genome or gene may be measured using one or more methods selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, quantitative or semi-quantitative RT-PCR, quantitative or semi-quantitative real-time RT-PCR, digital PCR, droplet digital PCR, in situ hybridization, fluorescence in situ hybridization (FISH), RNase protection assay (RPA), northern blotting, southern blotting, RNA sequencing, DNA chip, and RNA chip, but is not limited thereto.

[0104] In the present invention, the “subject” may include both a cancer patient who wishes to receive immunotherapy or a cancer patient who has received immunotherapy.

[0105] In the present invention, the biological sample may be at least one selected from the group consisting of blood, whole blood, plasma, feces, urine, tissue, cells, organs, bone marrow, fine needle aspiration specimen, fine needle lavage fluid, core needle biopsy specimen, and saliva, and according to one embodiment or experimental example of the present invention, it may be feces, but is not limited thereto.

[0106] In the present invention, the method for providing information for predicting immunotherapy responsiveness or the method for predicting immunotherapy responsiveness may further include, but is not limited to, a step of predicting that immunotherapy responsiveness will be low when the number of Lactobacillus salivarius or Bacteroides plebeius is higher than that of the control group. In this case, the control group may be a cancer patient with high immunotherapy responsiveness, and may be a patient who shows a complete response (CR) or a partial response (PR) after immunotherapy treatment, but is not limited thereto.

[0107] In the present invention, the method for providing information for predicting immunotherapy responsiveness or the method for predicting immunotherapy responsiveness may further include, but is not limited to, a step of predicting that immunotherapy responsiveness is high when the number of Lactobacillus salivarius or Bacteroides plebeius is lower or not detected compared to the control group. In this case, the control group may be a cancer patient with low immunotherapy responsiveness, and may be a patient who exhibits progressive disease (PD) or stable disease (SD) after immunotherapy treatment, but is not limited thereto.

[0108] In the present invention, the method for providing information for predicting the prognosis of a cancer patient after immunotherapy treatment or the method for predicting the prognosis of a cancer patient after immunotherapy treatment may further include, but is not limited to, a step of predicting that the prognosis of the cancer patient after immunotherapy treatment will be poor if the number of Lactobacillus salivarius or Bacteroides plebeius is higher than that of the control group. In this case, the control group is a cancer patient with a good prognosis after immunotherapy treatment, and may be a patient who shows a complete response (CR) or a partial response (PR) after immunotherapy treatment, but is not limited thereto.

[0109] In the present invention, the method for providing information for predicting the prognosis of a cancer patient after immunotherapy treatment or the method for predicting the prognosis of a cancer patient after immunotherapy treatment may further include, but is not limited to, a step of predicting that the cancer patient will have a good prognosis after immunotherapy treatment if the number of Lactobacillus salivarius or Bacteroides plebeius is lower or not detected compared to a control group. In this case, the control group is a cancer patient with a poor prognosis after immunotherapy treatment, and may be a patient who shows progressive disease (PD) or stable disease (SD) after immunotherapy treatment, but is not limited thereto.

[0110] In the present invention, “prognosis prediction” means predicting the future tumor response or course of cancer after immunotherapy treatment of a cancer patient, and means predicting the probability of cancer metastasis, recurrence, or metastatic recurrence, etc., through the microbial level.

[0111] In addition, the present invention provides a method for providing information for determining / analyzing whether a subject is highly susceptible to predicting immunotherapy responsiveness or predicting prognosis after immunotherapy, wherein the number of at least one selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius is measured in biological samples isolated from subjects suspected of having low immunotherapy responsiveness or subjects suspected of having poor prognosis after immunotherapy and a control group, and if the difference in the level increases (by a factor of two or more), it is considered to indicate that the subject is low in immunotherapy responsiveness or subjects with poor prognosis after immunotherapy.

[0112] In addition, the present invention provides a method for improving immunotherapy responsiveness or increasing immune activity, comprising the steps of: detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius in a biological sample isolated from a subject; and, if the number of Lactobacillus salivarius or Bacteroides plebeius is higher than that of a control group, receiving fecal microbiota transplantation from the control group.

[0113] In the present invention, there is no limitation on the number of times the fecal microbiota transplantation is performed, and may be, for example, 1 to 4 times, 1 to 3 times, or 1 to 2 times. The step of receiving the fecal microbiota transplantation from the control group may be a step of reducing the number of Lactobacillus salivarius or Bacteroides plebius.

[0114] In the present invention, the control group in the method for improving the responsiveness to immunotherapy or the method for increasing immune activity is a cancer patient with a high responsiveness to immunotherapy or a cancer patient with a good prognosis after immunotherapy, and may be a patient who shows a complete response (CR) or partial response (PR) after immunotherapy, but is not limited thereto.

[0115]

[0116] In addition, the present invention provides a pharmaceutical composition for preventing or treating an autoimmune disease, which comprises as an active ingredient one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius or a culture thereof.

[0117] In the present invention, the Lactobacillus salivarius and Bacteroides plebeius are microorganisms existing in the human intestine and may be isolated from a human fecal sample, but are not limited thereto, and the origin thereof is not limited as long as it is a bacterium known in the art or determined (classified) as Lactobacillus salivarius species and Bacteroides plebeius species by a genome analysis means known in the art.

[0118] In the present invention, the autoimmune disease is a disease caused by the immune system inside the human body attacking normal cells inside the body rather than external antigens, and may be at least one selected from the group consisting of multiple sclerosis, inflammatory bowel disease, graft-versus-host disease, asthma, atopy, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus, but is not limited thereto.

[0119] In the present invention, the composition may suppress immune activity, but is not limited thereto.

[0120] In the present invention, the composition may suppress proliferation of T cells or reduce secretion of interferon gamma (IFN-γ) of T cells, but is not limited thereto.

[0121] In addition, the present invention provides a method for preventing or treating an autoimmune disease, comprising a step of administering to a subject in need thereof a composition comprising, as an active ingredient, one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius or a culture thereof.

[0122] In addition, the present invention provides a composition comprising, as an active ingredient, one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius or a culture thereof, for use in preventing or treating autoimmune diseases.

[0123] In addition, the present invention provides a use for producing a preparation for preventing or treating an autoimmune disease using one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius or a culture thereof.

[0124] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The excipients may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

[0125] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0126] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0127] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0128] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.;

[0129] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0130] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity increasing agent, and the like, as needed.

[0131] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0132] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0133] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, and dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0134] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0135] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0136] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0137] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0138] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0139] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0140] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, route of administration, age, sex, weight of the patient, and severity of the disease.

[0141] In the present invention, “subject” means a subject requiring treatment for a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0142] In the present invention, "administration" means providing a given composition of the present invention to a subject by any suitable method. Therefore, in the present invention, "administration" includes not only injection or ingestion into a subject, but also application.

[0143] In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, and “treatment” means any action that improves or beneficially changes the target disease and its accompanying metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention.

[0144]

[0145] Hereinafter, preferred examples and experimental examples are presented to aid in understanding the present invention. However, the following examples and experimental examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples and experimental examples.

[0146]

[0147] [Example]

[0148] Example 1. Clinical trial design for a prospective study

[0149] The present invention was conducted as a prospective, single-arm, single-center clinical trial (NCT04264975) using a combination of fecal microbiota transplantation (FMT) and an anti-PD-(L)1 inhibitor as an immunotherapy agent for patients with advanced solid tumors refractory to anti-PD-(L)1 inhibitors. The study design of this clinical trial is schematically depicted in Figure 1.

[0150]

[0151] Example 2. Selection of donors and recipients for fecal microbiota transplantation (FMT).

[0152] Donors for fecal microbiota transplantation were selected based on the following criteria:

[0153] - Age ≥ 19 years old

[0154] -Having a histologically confirmed solid tumor (in the case of hepatocellular carcinoma, a clinically confirmed diagnosis according to the American Association for the Study of Liver Diseases (AASLD) is also acceptable)

[0155] - Sustained complete or partial response for at least 6 months according to RECIST v1.1 with anti-PD-(L)1 monotherapy for unresectable or metastatic solid tumors

[0156] -No concurrent infectious diseases

[0157] - No history or risk behavior for infectious diseases such as human immunodeficiency virus or other viral infections, and no travel to a country with endemic diarrhea or a country with a high risk of traveler's diarrhea within the past 6 months.

[0158] - No history of chronic gastrointestinal disease, including inflammatory bowel disease

[0159] -No recent ingestion of any allergen to which the recipient is known to be allergic

[0160] - Only donors who pass serological and stool screening tests are considered eligible to donate stool samples for FMT transplantation.

[0161]

[0162] Additionally, recipients of fecal microbiota transplantation were selected based on the following criteria:

[0163] - Age ≥ 19 years old

[0164] -ECOG (Eastern Cooperative Oncology Group) performance status: 0-2

[0165] -Having a histologically confirmed solid tumor (in the case of hepatocellular carcinoma, a clinically confirmed diagnosis is also acceptable according to AASLD)

[0166] - Disease progression confirmed during anti-PD-(L)1-based treatment (monotherapy or combination therapy) for unresectable or metastatic solid tumors

[0167] -Has at least one measurable lesion according to RECIST v1.1

[0168] - No history of other active malignant tumors within 3 years prior to study entry (except for curatively treated non-melanoma skin cancer, superficial bladder cancer, or intraepithelial carcinoma of the prostate, cervix, breast, or stomach)

[0169] - No history of active autoimmune disease requiring systemic treatment

[0170] - No concurrent conditions requiring immunosuppression or active infection

[0171] -There are no contraindications to colonoscopy.

[0172]

[0173] Example 3. Combination therapy with FMT and anti-PD-(L)1 inhibitor

[0174] A combination treatment of FMT and anti-PD-(L)1 inhibitor was performed on recipients who satisfied the conditions described in Example 2 above, and the process is schematically shown in Figure 2.

[0175] Specifically, a clinical trial of FMT + anti-PD-(L)1 inhibitor combination therapy was conducted on patients (recipients) with unresectable or metastatic solid tumors whose cancer had progressed during anti-PD-(L)1 inhibitor treatment. The stool donors for FMT were patients with unresectable or metastatic solid tumors who had maintained complete remission (CR) or partial remission (PR) for at least 6 months on anti-PD-(L)1 monotherapy. To reduce the recipients' native gut microbiome, oral antibiotics (Augmentin®amoxicillin clavulanate; 625 mg three times daily) were administered for 5 days (from D-5 to D-1) before FMT, and the last dose of antibiotics was completed at least 12 hours before FMT. FMT was performed via colonoscopy, and bowel preparation before colonoscopy was performed according to the hospital protocol, and the thawed donor stool was administered to the recipient's intestine via colonoscopy. The day after FMT, recipients received standard doses of anti-PD-(L)1 inhibitors, which were continued until tumor progression or intolerable toxicity. Additional FMTs using stool from the same or a different donor were permitted at the investigator's discretion before tumor response assessment or if clinical response was unclear. Tumor response assessments were performed every 6–8 weeks using computed tomography according to RECIST v1.1.

[0176] All recipients in the study received intravenous (IV) infusions of 3 mg / kg nivolumab as an anti-PD-1 inhibitor every 2 weeks along with FMT. The median number of nivolumab administrations in the 13 recipients was 5 cycles (one administration was defined as a cycle), with a range of 1 to 27 cycles. Seven of the 13 recipients had received at least 2 FMTs from the same or different donors: 4 received 2 cycles, 2 received 3 cycles, and 1 received 4 cycles.

[0177] Amoxicillin / clavulanate, used as an oral antibiotic, was administered only before the first FMT. Anti-PD-(L)1 inhibitors were continued or reintroduced at standard doses and schedules until unacceptable toxicity or disease progression.

[0178] Tumor biopsies were performed within 1 week before FMT, within 2 weeks after FMT for the first FMT, and within 4 weeks after FMT for subsequent FMTs, and elective tumor biopsies were performed at the time of disease progression.

[0179]

[0180] Example 4. Clinical characteristics

[0181] The clinical characteristics of the recipients of the FMT of the present invention (N=13) are shown in Table 1 below.

[0182]

[0183] Additionally, the clinical characteristics of FMT donors (N=6) are shown in Table 2 below.

[0184]

[0185] In addition, adverse effects related to the combined treatment of FMT and anti-PD-(L)1 inhibitor were identified in the recipients of the above FMT, and the results are shown in Table 3 below.

[0186] Side effects include skin rash, skin pruritus, hypothyroidism, adrenal insufficiency, myalgia, fever, and gastritis.

[0187]

[0188] [Experimental Example]

[0189] Experimental Example 1. Determination of microbial composition after fecal microbiota transplantation (FMT)

[0190] After FMT, the microbiota composition of most recipients was confirmed through beta-diversity (Bray-Curtis distance) analysis, which revealed that the microbiota composition of the recipients underwent significant changes, and among them, recipients R#1, R#2, and R#7 showed a microbiota composition very similar to that of their respective donors, as shown in Figure 3.

[0191]

[0192] Experimental Example 2. Confirmation of clinical efficacy

[0193] As a result of confirming clinical efficacy for 13 recipients, as shown in Figures 4a and 4b, 5 of the recipients showed stable disease (SD), and 1 (R#7) achieved a partial response (PR) after FMT, recording a disease control rate of 46.2% (6 / 13) and an objective response rate of 7.7%.

[0194] Accordingly, we confirmed various clinical responses to nivolumab administration in FMT recipient R#7, who showed a partial response.

[0195]

[0196] 2-1. Confirmation of alpha-fetoprotein levels in recipient R#7

[0197] As shown in Fig. 4c, when the first FMT was performed from donor D#1 and nivolumab was administered to recipient R#7 (male, 47 years old, hepatocellular carcinoma) who showed progressive disease (PD) after 4 cycles of nivolumab administration, cancer progression continued, and an increase in α-fetoprotein (AFP) was confirmed one month after performing FMT.

[0198] In addition, as shown in Fig. 4d, when the second FMT was performed from the donor D#5 7 weeks after the first FMT in the recipient R#7 and nivolumab was administered, and then administered twice more at 2-week intervals, a partial response (PR) was observed, and it was confirmed that AFP was significantly reduced 3 months after the second FMT.

[0199] Accordingly, beneficiary R#7 showed no improvement in nivolumab treatment response after the first FMT, but showed improvement in nivolumab treatment response after the second FMT.

[0200]

[0201] 2-2. Confirmation of systemic immune activation in recipient R#7

[0202] Mass cytometry analysis was performed using CyTOF (Cytometry by time of flight).

[0203] Specifically, PBMCs from patients were obtained using the Maxpar Direct Immune Profiling Assay kit (Fluidigm), and then the Fc receptors of the immune cells were blocked using Human TruStain FcX (BioLegend). Subsequently, the cells were cultured with antibodies capable of detecting the markers and Cell ID Intercalator-Ir (Fluidigm) for cell differentiation. Then, changes in the immune cell system were identified through marker expression in each cell using a Helios mass cytometer (Fluidigm). Through this analysis, systemic immune changes in the peripheral blood of FMT recipient R#7 were evaluated.

[0204] As a result, as shown in Fig. 4e, compared to the baseline, CD8+ T cells and CD8+ effector T cells increased after the second FMT, while regulatory T cells (Treg) decreased. In Fig. 4e, EM stands for effector-memory, CM for central-memory, TM for terminal-memory, and TE for terminal-effector.

[0205]

[0206] 2-3. Confirmation of immune activation in the tumor microenvironment (TME) of recipient R#7

[0207] Multiplex immunohistochemistry (IHC) was used to confirm immune activation in the tumor microenvironment in FMT recipient R#7. Specifically, 4 μm-thick slices were cut from formalin-fixed, paraffin-embedded (FFPE) blocks, and the slides were heated in a drying oven at 60°C for at least 1 h. Subsequently, multiplex immunofluorescence staining was performed using the Leica Bond Rx™ Automated Stainer (Leica Biosystems). Multiplex staining was performed by performing the following processes for multiple antigens: antigen retrieval, blocking, antibody staining, tyramide signal amplification, and removal of bound antibodies.

[0208] As a result, as shown in Fig. 4f, a significant increase in tumor-infiltrating cytotoxic T cells and MHC-II+ M1 macrophages was observed 4 weeks after the second FMT, and the number of Treg cells was confirmed to remain consistently low.

[0209]

[0210] 2-4. Confirmation of immune-related gastritis in recipient R#7

[0211] The results of immune-related gastritis following immune activation after the second FMT in FMT recipient R#7 were confirmed by upper endoscopy, histological examination, and clinical examination, and are shown in Fig. 4g.

[0212]

[0213] Experimental Example 3. Identification of the causative agent responsible for the therapeutic effect of FMT.

[0214] 3-1. Confirmation of changes in abundance at the genus level in beneficiary R#7

[0215] We performed patient fecal metagenome analysis using 16s rRNA sequencing, and analyzed microbial abundance using QIIME2 and DADA2 tools. As a result of confirming the change in microbial abundance at the genus level in FMT recipient R#7, as shown in Fig. 5a, after the first FMT transplant from donor D#1, the genus Bacteroides increased and the genus Prevotella decreased. After the second FMT transplant from donor D#5, the genus Bacteroides decreased and the genus Prevotella increased.

[0216]

[0217] 3-2. Identification of microorganisms with decreased abundance after the second FMT in recipient R#7.

[0218] As a result of checking the abundance of microorganisms after FMT in recipient R#7, as shown in Fig. 5b and Table 4 below, 73 microorganisms showed a decrease in abundance after the second FMT compared to the first FMT, and among these, 21 microbial groups were originally present in R#7, but were found to have significantly decreased after the second FMT compared to the first FMT.

[0219]

[0220] In addition, as confirmed in Experimental Example 2 above, among the patients in the FMT cohort, recipient R#7 did not show an enhanced effect of immunotherapy responsiveness after the first FMT, but showed an improved responsiveness of immunotherapy after the second FMT. Accordingly, the strains that were significantly more abundant in first FMT donors than in second FMT donors and in patient R#7 after the first FMT than after the second FMT were analyzed, and as a result, the Bacteroides plebeius strain was selected, as shown in Fig. 5c.

[0221] Additionally, the B. plebeius strain was found to be the strain originally carried by patient R#7, and was found to be a strain that suppresses the effect of immunotherapy.

[0222]

[0223] 3-3. Comparison of responses in recipients who received FMT from donor D#5

[0224] Among the patients in the FMT cohort, recipient patient R#6 received a second FMT from the same donor (D#5) as patient R#7. As a result of examining the response to immunotherapy in patients R#6 and R#7, as shown in Figure 5d, patient R#7 maintained a sustained partial response (PR), whereas patient R#6 maintained a short-lived stable lesion (SD). Therefore, unlike patient R#7, patient R#6 did not show an improved response to immunotherapy.

[0225] In addition, when comparing the abundance of B. plebeius in patients R#6 and R#7, as shown in Fig. 5e, it was significantly reduced in patient R#7 after the second FMT, whereas in patient R#6, it was not abundant to begin with and did not increase after the first and second FMT.

[0226] In addition, using QIIME2 and ANCOM (Analysis of Composition of Microbiomes) analysis methods (Mandal, S., Van Treuren, W., White, RA, EggesbøM., Knight, R., and Peddada, SD (2015). Analysis of composition of microbiomes: a novel method for studying microbial composition. Microbial ecology in health and disease 26, 27663.), we analyzed more strains in patient R#6 than in patient R#7 after the second FMT, and as shown in Fig. 5f, the Lactobacillus salivarius strain was selected. The L. salivarius strain was the original strain of patient R#6, and it persisted or increased after FMT. In addition, it was a strain present in the first FMT of patient R#7, which did not show an effect of enhancing the response to immunotherapy, so it was found to be a strain that suppresses the effect of immunotherapy.

[0227]

[0228] Experimental Example 4. Confirmation of the immunosuppressive activity of B. plebeius and L. salivarius strains.

[0229] 4-1. Confirmation of T cell proliferation inhibition effect

[0230] To confirm the immunosuppressive activity of B. plebeius strains and L. salivarius strains, T cells and strain cultures were co-cultured, and the T cell proliferation inhibitory activity was confirmed using flow cytometry.

[0231] First, peripheral blood mononuclear cells (PBMCs) were separated from human blood using Ficoll, and then remaining red blood cells (RBCs) were removed using RBC lysis buffer. After that, CD4 T cells and CD8 T cells were separated using a CD4 / CD8 T cell separation kit (MACS kit) and LS column, and after staining with CFSE, each cell was seeded in a 96-well plate at a density of 2x10 5 Cells were divided into 200 μl and treated with strain cultures. 96-well plates were pre-incubated with anti-CD3 and anti-CD28 antibodies, which can induce T cell proliferation, 24 hours in advance. Strain cultures were harvested after culturing for at least 12 hours in RPMI medium, and 10% of the final T cell medium volume was used for treatment with T cells. After 72 hours of co-culturing, T cells from each well were harvested and the fluorescence of CFSE was detected using flow cytometry. When T cells proliferated significantly, the fluorescence intensity of each T cell decreased. In other words, a decrease in the intensity of CFSE or a shift in the peak to a lower value indicates that T cell proliferation has progressed. The degree of T cell proliferation by each strain was analyzed using this analysis method.

[0232] As a result, as shown in Fig. 6a, it was confirmed that when the cultures of B. plebeius strain and L. salivarius strain were treated, the proliferation of both CD4 T cells and CD8 T cells was inhibited.

[0233]

[0234] 4-2. Confirmation of the inhibitory effect of interferon gamma (IFN-γ) secretion by T cells

[0235] A co-culture solution was obtained by co-culturing T cells and the strain culture solution using the method of Experimental Example 4-1 above, and the concentration of interferon gamma (IFN-γ) secreted by the T cells was analyzed using ELISA. Since the co-culture solution is a state in which interferon gamma (IFN-γ) is secreted as T cells proliferate and are activated, a capture antibody capable of detecting interferon gamma (IFN-γ) in such a co-culture solution was coated on a 96-well plate, and the co-culture solution was treated and incubated at room temperature for 1 hour to induce binding of interferon gamma (IFN-γ) to the capture antibody. Afterwards, a washing process and secondary antibody staining, washing and HRP reagent staining were performed, and finally, the absorbance of each well was obtained using a microplate. The absorbance of each well was converted into an interferon-gamma concentration value using reference (interferon-gamma-treated wells with known concentrations) and the amount of interferon-gamma secretion induced between strains was compared and analyzed. As a result, as shown in Fig. 6b, it was confirmed that when the cultures of B. plebeius and L. salivarius strains were treated, the IFN-γ concentration was significantly reduced compared to the control group.

[0236]

[0237] Accordingly, through the above experimental examples 4-1 and 4-2, it was confirmed that the B. plebeius strain and the L. salivarius strain can suppress the immune activity of the host.

[0238]

[0239] Experimental Example 5. Survival Rate Verification According to the Presence or Absence of B. plebeius and L. salivarius Strains

[0240] Using an independent biomarker cohort, we analyzed survival rates according to the presence or absence of B. plebeius and L. salivarius strains. A total of 72 patients with available baseline stool samples and no prior antibiotic treatment within 4 weeks prior to immune checkpoint inhibitor (ICI) treatment were included in the analysis. Their clinical information is presented in Table 5.

[0241]

[0242] As a result of examining the survival rate according to the presence or absence of the two strains in an independent biomarker cohort, as shown in Figure 7, it was confirmed that the overall survival (OS) was significantly longer in the group of patients without the B. plebeius strain, and it was confirmed that there was a significant difference in the overall survival time depending on the presence or absence of L. salivarius.

[0243]

[0244] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0245]

[0246] The present invention can predict the response to immunotherapy or the prognosis after immunotherapy by detecting Lactobacillus salivarius and Bacteroides plebeius strains, and the strains are expected to be useful for the treatment of autoimmune diseases, etc., through suppression of immune activity, and thus have industrial applicability.

Claims

1. A composition for predicting immunotherapy response, comprising as an active ingredient an agent for detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius.

2. In paragraph 1, A composition for predicting immunotherapy response, characterized in that the immunotherapy agent is at least one selected from the group consisting of an immune checkpoint inhibitor, an immunocytotherapy agent, and an immunovirus therapy agent.

3. In paragraph 2, A composition for predicting immunotherapy response, characterized in that the immune checkpoint inhibitor is at least one selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA4 inhibitor, a LAG3 inhibitor, a TIM3 inhibitor, a 4-1BB inhibitor, a B7-H4 inhibitor, a VISTA inhibitor, a KIR inhibitor, and a BTLA inhibitor.

4. In paragraph 1, A composition for predicting immunotherapy response, characterized in that the preparation comprises at least one selected from the group consisting of a primer pair, a probe, an antisense oligonucleotide, an antibody, and an aptamer that specifically binds to 16S rRNA of the microorganism.

5. In paragraph 4, A composition for predicting immunotherapy responsiveness, characterized in that the primer pair comprises the base sequences of SEQ ID NOs: 1 and 2; or the base sequences of SEQ ID NOs: 3 and 4.

6. A composition for predicting the prognosis of a cancer patient after immunotherapy treatment, comprising as an active ingredient an agent for detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius.

7. In paragraph 6, A composition for predicting the prognosis of a cancer patient after immunotherapy treatment, characterized in that the cancer is at least one selected from the group consisting of colorectal cancer, rectal cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, glioblastoma, lung cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, breast cancer, uterine cancer, stomach cancer, bone cancer, lymphoma, blood cancer, squamous cell carcinoma, lung adenocarcinoma, peritoneal cancer, skin cancer, skin melanoma, ocular melanoma, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, glioblastoma, endometrial cancer, salivary gland cancer, vulvar cancer, and head and neck cancer.

8. A kit for predicting immunotherapy response, comprising a composition and instructions according to any one of claims 1 to 5.

9. A kit for predicting the prognosis of a cancer patient after immunotherapy treatment, comprising the composition and instructions of clause 6 or 7.

10. A method for providing information for predicting immunotherapy responsiveness, comprising a step of detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius in a biological sample isolated from a subject.

11. In paragraph 10, A method for providing information for predicting immunotherapy responsiveness, characterized in that the biological sample is at least one selected from the group consisting of blood, whole blood, plasma, stool, urine, tissue, cell, organ, bone marrow, fine needle aspiration specimen, fine needle lavage fluid, core needle biopsy specimen, and saliva.

12. In paragraph 10, A method for providing information for predicting immunotherapy responsiveness, characterized in that it further includes a step of predicting that immunotherapy responsiveness will be low when the number of Lactobacillus salivarius or Bacteroides plebeius is higher than that of the control group.

13. A method for providing information for predicting the prognosis of a cancer patient after immunotherapy treatment, comprising a step of detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius in a biological sample isolated from a subject.

14. In paragraph 13, A method for providing information for predicting the prognosis of a cancer patient after immunotherapy treatment, characterized in that it further includes a step of predicting that the prognosis of the cancer patient after immunotherapy treatment will be poor if the number of Lactobacillus salivarius or Bacteroides plebeius is higher than that of the control group.

15. A pharmaceutical composition for preventing or treating an autoimmune disease, comprising as an active ingredient one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius or a culture thereof.

16. In paragraph 15, A pharmaceutical composition, characterized in that the autoimmune disease is at least one selected from the group consisting of multiple sclerosis, inflammatory bowel disease, graft-versus-host disease, asthma, atopy, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

17. In paragraph 15, A pharmaceutical composition characterized in that the composition suppresses immune activity.

18. In paragraph 17, A pharmaceutical composition characterized in that the composition inhibits the proliferation of T cells or reduces the secretion of interferon gamma (IFN-γ) by T cells.

19. Use of a composition comprising as an active ingredient an agent for detecting one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius for predicting immunotherapy response to cancer treatment.

20. Use for the manufacture of a preparation for predicting the response to immunotherapy of cancer, wherein the preparation detects one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius.

21. A method for preventing or treating an autoimmune disease, comprising administering to a subject in need thereof a composition comprising, as an active ingredient, one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius or a culture thereof.

22. Use of a composition comprising one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius or a culture thereof as an active ingredient for preventing or treating autoimmune diseases.

23. Use for the manufacture of a preparation for preventing or treating autoimmune diseases comprising one or more intestinal microorganisms selected from the group consisting of Lactobacillus salivarius and Bacteroides plebeius or a culture thereof.

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