Composition of the genus Musispirillum and method for treating cancer therewith

Abstract

The technology described herein is directed to compositions comprising the genus Musispirillum (e.g., M. schaedleri). Also described herein are methods of treating cancer using a Musispirillum composition; a diet rich in sulfur amino acids (SAA) or a supplement containing SAA; and / or an XCL1 polypeptide (or an XCR1 agonist). Also described herein are methods of stratifying cancer treatment related to the detection of the levels of M. schaedleri, XCL1 polypeptide, NKT, and / or CD103+ cDC1, and the corresponding modification or stratification of treatment. TIFF2025524662000062.tif117128

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority under 35 U.S.C.§119(e) to U.S. Provisional Patent Application No. 63 / 389,382, filed on July 15, 2022, the content of which is hereby incorporated by reference in its entirety.

[0002] Government Support This invention was made with government support under CA202704 and CA154426 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing This application contains a sequence listing that was submitted via Patent Center in XML format and is hereby incorporated by reference in its entirety. The XML copy created on July 6, 2023, is named 002806 - 099880WOPT_SL.xml and is 61,077 bytes in size.

[0004] Technical Field The technology described herein relates to compositions containing Mucispirillum and related methods of treating cancer therewith.

Background Art

[0005] Background Colorectal cancer (CRC) is the second leading cause of cancer - related death worldwide, and its global incidence rate is on the rise. Immunotherapy, specifically immune checkpoint inhibitors (ICIs), has been a revolutionary advance in the treatment of many cancers. However, the majority of CRCs are not ICI - responsive due to proficient DNA mismatch repair and tolerogenic immune regulators in the colon. Beyond intrinsic factors of tumor cells such as DNA mismatch repair (MMR) status, the gut microbiota also affects responsiveness to immunotherapy treatments and is an environmental factor in CRC development. The gut microbiota is associated with the efficacy of ICI treatment, and various bacterial species have been identified as mediators of responsiveness. See, for example, Andrews et al., Nat Med 27, 1432 - 1441 (2021) (Non - Patent Document 1); Frankel et al., Neoplasia 19, 848 - 855 (2017) (Non - Patent Document 2); Gopalakrishnan et al., Science 359, 97 - 103 (2018) (Non - Patent Document 3); Lee et al., Nat Microbiol 6, 277 - 288 (2021) (Non - Patent Document 4); Matson et al., Science 359, 104 - 108 (2018) (Non - Patent Document 5); Routy et al., Science 359, 91 - 97 (2018) (Non - Patent Document 6), each of which is incorporated herein by reference in its entirety. There is a need for additional treatment options for immunologically "cold" cancers such as CRC that are non - responsive to ICIs.

Prior Art Documents

Non - Patent Documents

[0006]

Non - Patent Document 1

Non - Patent Document 2

Non - Patent Document 3

[0007] Summary The techniques described herein are directed to compositions comprising members of the genus Mucispirillum (e.g., M. schaedleri). The present disclosure describes how a diet rich in sulfur-containing amino acids can increase the gastrointestinal tract levels of M. schaedleri, which in turn increases XCL1 secretion by NKT cells. An increase in XCL1 secretion by NKT cells, for example, increases the number and / or activation of CD103+ conventional dendritic cells (cDC1) in tumor draining lymph nodes, and thus recruits and activates CD8+ T cells with anti-tumor immune activity (see, e.g., FIG. 13). Accordingly, methods of treating cancer using a Mucispirillum composition as described herein; a diet rich in sulfur-containing amino acids (SAA) or a supplement containing SAA; and / or an XCL1 polypeptide (or XCR1 agonist) are described herein. Also described herein are cancer treatment stratification methods related to the detection of levels of M. schaedleri, XCL1 polypeptide, NKT, and / or CD103+ cDC1, and corresponding treatment modifications or stratifications.

[0008] Accordingly, in one aspect, a composition comprising Mucispirillum schaedleri (M. schaedleri) bacteria formulated for delivery to the intestine is described herein.

[0009] In some aspects of any of the situations, M. schaedleri bacteria are either alive or inactivated.

[0010] In some aspects of any of the situations, M. schaedleri bacteria are in a dried viable form.

[0011] In some aspects of any of the situations, M. schaedleri bacteria are encapsulated.

[0012] In some aspects of any of the situations, M. schaedleri bacteria are contained in enteric capsules.

[0013] In some aspects of any of the situations, M. schaedleri bacteria are maintained in an anaerobic state in the formulation.

[0014] In some aspects of any of the situations, M. schaedleri bacteria are in a mixture with prebiotics.

[0015] In some aspects of any of the situations, M. schaedleri bacteria are in a mixture with sulfur-containing amino acids.

[0016] In some aspects of any of the situations, the sulfur-containing amino acids are methionine, cysteine, or derivatives thereof.

[0017] In some aspects of any of the situations, M. schaedleri bacteria are formulated in a food composition.

[0018] In some aspects of any of the situations, the food composition is supplemented with sulfur-containing amino acids and / or prebiotics.

[0019] In some aspects of any of the situations, the composition further contains 1 to 20 additional bacteria.

[0020] In some aspects of any of the aspects, the composition comprises 20 or fewer bacteria.

[0021] In one aspect, a composition is described herein that comprises live M. schaedleri bacteria, dead M. schaedleri bacteria, conditioned M. schaedleri culture medium, or an organic solvent extract or fraction thereof that promotes XCL1 secretion by NKT cells, and the composition is formulated for delivery to the intestine.

[0022] In some aspects of any of the aspects, the M. schaedleri bacteria, medium, or solvent extract is in a dried form.

[0023] In some aspects of any of the aspects, the M. schaedleri bacteria, medium, or extract is encapsulated.

[0024] In some aspects of any of the aspects, the M. schaedleri bacteria are contained in enteric capsules.

[0025] In some aspects of any of the aspects, the M. schaedleri bacteria are maintained in an anaerobic state in the formulation.

[0026] In some aspects of any of the aspects, the M. schaedleri bacteria, medium, or extract is in a mixture with prebiotics and / or sulfur-containing amino acids or derivatives thereof.

[0027] In some aspects of any of the aspects, the conditioned M. schaedleri culture medium, or an organic solvent extract or fraction thereof, comprises at least one metabolite selected from Table 3 or Table 9 of U.S. Patent Application No. 63 / 389,382, or FIGS. 4H, 4I, or 16 herein.

[0028] In some embodiments of any of the aspects, the adjusted M. sheldleri culture medium, or an organic solvent extract or fraction thereof of the adjusted M. sheldleri culture medium, comprises at least one metabolite selected from the group consisting of succinic acid; propionic acid; nicotinic acid; aconitic acid (cis and / or trans); pentadecanoic acid; itaconic acid; 16-hydroxyhexadecanoic acid; C8H 15 NO3S; crotonic acid; myristic acid; 17-hydroxyheptadecanoic acid (C 17 H 34 O3); and 15-hydroxypentadecanoic acid (C 15 H 30 O3).

[0029] In some embodiments of any of the aspects, the adjusted M. sheldleri culture medium, or an organic solvent extract or fraction thereof of the adjusted M. sheldleri culture medium, comprises at least one metabolite selected from the group consisting of succinic acid; nicotinic acid; aconitic acid (cis and / or trans); pentadecanoic acid; itaconic acid; 16-hydroxyhexadecanoic acid; and crotonic acid.

[0030] In one aspect, a food composition comprising a composition as described herein is described herein.

[0031] In some embodiments of any of the aspects, the food composition further comprises 1 to 20 additional bacteria.

[0032] In one aspect, a method of treating cancer or promoting antitumor immune activity, the method comprising administering to a subject in need thereof a composition as described herein is described herein.

[0033] In some embodiments of any of the aspects, the cancer is colorectal cancer.

[0034] In some embodiments of any of the aspects, the method further comprises administering an immune checkpoint inhibitor.

[0035] Disclosed herein is a method for promoting responsiveness to an immune checkpoint inhibitor tumor therapy, the method comprising administering to a subject in need thereof a composition as described herein.

[0036] In some embodiments of any of the aspects, the method further comprises administering an immune checkpoint inhibitor.

[0037] In some embodiments of any of the aspects, the subject has colon cancer.

[0038] In some embodiments of any of the aspects, the subject's cancer has been determined to be resistant to immune checkpoint inhibitor therapy.

[0039] In some embodiments of any of the aspects, the composition promotes XCL1 secretion by NKT cells.

[0040] Disclosed herein is a method for increasing CD103+ conventional dendritic cells (cDC1), the method comprising administering to a subject in need thereof a composition as described herein.

[0041] In some embodiments of any of the aspects, the cDC1 is associated with the tumor.

[0042] In some embodiments of any of the aspects, the tumor is colon cancer.

[0043] In some embodiments of any of the aspects, the method further comprises administering a sulfur-containing amino acid.

[0044] In some embodiments of any of the aspects, the method further comprises administering an immune checkpoint inhibitor.

[0045] In one aspect, a method for increasing XCL1 secretion by NKT cells, the method comprising administering to a subject in need thereof a composition as described herein, is described herein.

[0046] In some embodiments of any of the aspects, the subject has cancer.

[0047] In some embodiments of any of the aspects, the subject has colon cancer.

[0048] In one aspect, a method for increasing infiltration of CD8+ T cells in colorectal tumors, the method comprising administering to a subject in need thereof a composition as described herein, is described herein.

[0049] In some embodiments of any of the aspects, cDC1 is associated with the tumor.

[0050] In some embodiments of any of the aspects, the tumor is colon cancer.

[0051] In some embodiments of any of the aspects, the method further comprises administering a sulfur-containing amino acid.

[0052] In some embodiments of any of the aspects, the method further comprises administering an immune checkpoint inhibitor.

[0053] In one aspect, a method for increasing infiltration of CD8+ T cells in colorectal tumors, the method comprising administering to a subject in need thereof a diet rich in sulfur-containing amino acids (SAA) or a supplement containing SAA, is described herein.

[0054] In some embodiments of any of the aspects, the diet rich in sulfur-containing amino acids contains more than 0.04 grams of SAA per kilogram of body weight per day.

[0055] In some aspects of any of the aspects, the method further comprises administering to a subject a composition as described herein.

[0056] In one aspect, a method for establishing or maintaining a tumor-suppressive gastrointestinal environment in a subject in need thereof, the method comprising administering a diet rich in sulfur-containing amino acids (SAA) or a supplement containing SAA is described herein.

[0057] In some aspects of any of the aspects, a diet rich in sulfur-containing amino acids or a supplement containing SAA contains more than 0.04 grams of SAA per kilogram of body weight per day.

[0058] In some aspects of any of the aspects, the method further comprises administering to a subject a composition as described herein.

[0059] In one aspect, a method for treating cancer, the method comprising administering an XCL1 polypeptide to a subject in need thereof is described herein.

[0060] In some aspects of any of the aspects, the cancer is colon cancer.

[0061] In some aspects of any of the aspects, the XCL1 polypeptide is administered to the gastrointestinal tract.

[0062] In one aspect, a method for treating cancer, the method comprising administering to a subject in need thereof a microorganism engineered to express an XCL1 polypeptide is described herein.

[0063] In one aspect, a method for treating cancer, the method comprising administering to a subject in need thereof an agonist of XCR1, which is an XCL1 receptor, is described herein.

[0064] In some aspects of any of the situations, the XCR1 agonist includes an amino acid sequence that is at least 95% identical to SEQ ID NOs: 9-11 and maintains its function.

[0065] In one aspect, a method of treating cancer in a subject in need thereof, the method comprising: detecting the level of M. sheddleri in a sample from the subject; administering a cancer immunotherapeutic agent when the level of M. sheddleri is equal to or greater than a predetermined threshold; and administering a cancer immunotherapeutic agent and a composition as described herein when the level of M. sheddleri is less than the predetermined threshold, is described herein.

[0066] In one aspect, a method of treating cancer in a subject in need thereof, the method comprising: obtaining a result from an assay that detects the level of M. sheddleri in a sample from the subject; administering a cancer immunotherapeutic agent when the level of M. sheddleri is equal to or greater than a predetermined threshold; and administering a cancer immunotherapeutic agent and a composition as described herein when the level of M. sheddleri is less than the predetermined threshold, is described herein.

[0067] In one aspect, a method of treating cancer in a subject in need thereof, the method comprising: detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 in a sample from the subject; administering a cancer immunotherapeutic agent when the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 is equal to or greater than a predetermined threshold; and administering a cancer immunotherapeutic agent and a composition as described herein when the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 is less than the predetermined threshold, is described herein.

[0068] In one aspect, a method of treating cancer in a subject in need thereof, the method comprising: obtaining a result from an assay that detects the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 in a sample derived from the subject; administering a cancer immunotherapeutic agent if the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 is equal to or greater than a predetermined threshold; and administering a cancer immunotherapeutic agent and a composition as described herein if the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 is less than a predetermined threshold, is described herein.

[0069] In one aspect, a method of stratifying a subject for cancer treatment, the method comprising: detecting the level of M. schaedleri in a sample derived from the subject; and classifying the subject as high risk if the level of M. schaedleri is less than a predetermined threshold; or classifying the subject as low risk if the level of M. schaedleri is equal to or greater than a predetermined threshold, is described herein.

[0070] In one aspect, a method of stratifying a subject for cancer treatment, the method comprising: obtaining a result from an assay that detects the level of M. schaedleri in a sample derived from the subject; and classifying the subject as high risk if the level of M. schaedleri is less than a predetermined threshold; or classifying the subject as low risk if the level of M. schaedleri is equal to or greater than a predetermined threshold, is described herein.

[0071] In one aspect, a method of stratifying a subject for cancer treatment, the method comprising: detecting the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 in a sample derived from the subject; and classifying the subject as high risk if the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 are below a predetermined threshold; or classifying the subject as low risk if the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 are above a predetermined threshold, is described herein.

[0072] In one aspect, a method of stratifying a subject for cancer treatment, the method comprising: obtaining results from an assay that detects the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 in a sample derived from the subject; and classifying the subject as high risk if the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 are below a predetermined threshold; or classifying the subject as low risk if the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 are above a predetermined threshold, is described herein.

[0073] In some embodiments of any of the aspects, the subject has colon cancer.

[0074] In some embodiments of any of the aspects, the method further comprises administering a composition as described herein.

[0075] In some embodiments of any of the aspects, the method further comprises administering a sulfur-containing amino acid.

[0076] In some embodiments of any of the aspects, the cancer immunotherapeutic agent is selected from the group consisting of immune checkpoint inhibitors; chemotherapy; dendritic cell vaccines; chimeric antigen receptor T cells (CAR-T); and NKT cell-based therapies.

[0077] In some aspects of any of the situations, the cancer immunotherapy agent includes an immune checkpoint inhibitor.

[0078] In some aspects of any of the situations, the method further includes the step of administering an immune checkpoint inhibitor.

[0079] In some aspects of any of the situations, the method further includes the step of administering a diet rich in sulfur-containing amino acids or a supplement containing SAA.

[0080] In some aspects of any of the situations, the method results in higher treatment efficacy compared to a method of treatment that does not initially involve the step of detecting the level of M. sheddleri; obtaining a result from an assay for detecting the level of M. sheddleri; detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1; or obtaining a result from an assay for detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1.

[0081] In some aspects of any of the situations, the method results in higher treatment efficacy compared to a method of treatment that does not initially involve stratifying the subject.

[0082] In some aspects of any of the situations, the method results in fewer treatment complications compared to a method of treatment that does not initially involve the step of detecting the level of M. sheddleri; obtaining a result from an assay for detecting the level of M. sheddleri; detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1; or obtaining a result from an assay for detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1.

[0083] In some aspects of any of the situations, the method results in fewer treatment complications compared to a method of treatment that does not initially involve stratifying the subject.

[0084] In one aspect, an enteric delivery formulation comprising at least one metabolite selected from Table 3 or Table 9 of U.S. Patent Application No. 63 / 389,382, or FIGS. 4H, 4I, or 16 herein is described herein.

[0085] In one aspect, an enteric delivery formulation comprising at least one metabolite selected from the group consisting of succinic acid; propionic acid; nicotinic acid; aconitic acid (cis and / or trans); pentadecanoic acid; itaconic acid; 16-hydroxyhexadecanoic acid; C8H 15 NO3S; crotonic acid; myristic acid; 17-hydroxyheptadecanoic acid (C 17 H 34 O3); and 15-hydroxypentadecanoic acid (C 15 H 30 O3) is described herein.

[0086] In one aspect, an enteric delivery formulation comprising at least one metabolite selected from the group consisting of succinic acid; nicotinic acid; aconitic acid (cis and / or trans); pentadecanoic acid; itaconic acid; 16-hydroxyhexadecanoic acid; and crotonic acid is described herein.

[0087] In some embodiments of any of the aspects, the enteric delivery formulation is formulated for delivery to the intestine. BRIEF DESCRIPTION OF THE DRAWINGS

[0088]

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Mode for Carrying Out the Invention

[0089] Detailed Description Aspects of the technology described herein are directed to compositions comprising members of the genus Musispirillum (e.g., M. schaedleri). The present disclosure describes how a diet rich in sulfur-containing amino acids can increase the gastrointestinal levels of M. schaedleri, which in turn increases XCL1 secretion by NKT cells. An increase in XCL1 secretion by NKT cells, for example, increases the number and / or activation of CD103+ conventional dendritic cells (cDC1) in tumor-draining regional lymph nodes, and thus recruits and activates CD8+ T cells with anti-tumor immune activity (see, e.g., FIG. 13). Accordingly, methods of treating cancer using a Musispirillum composition as described herein; a diet rich in sulfur-containing amino acids (SAA) or a supplement containing SAA; and / or an XCL1 polypeptide (or XCR1 agonist) are described herein. Also described herein are cancer treatment stratification methods related to the detection of levels of M. schaedleri, XCL1 polypeptide, NKT, and / or CD103+ cDC1, and corresponding treatment modification or stratification.

[0090] Composition In several aspects, compositions comprising bacteria of the genus Musispirillum are described herein. In one aspect, compositions comprising bacteria of the genus Musispirillum formulated for delivery to the intestine are described herein. In some embodiments of any of the aspects, the composition is formulated for delivery to the intestine via oral administration. In some embodiments of any of the aspects, the composition comprises an enteric coating or the like to survive gastric acidity and enable delivery into the small or large intestine. In one aspect, compositions comprising Musispirillum schaedleri (M. schaedleri) bacteria formulated for delivery to the intestine are described herein. In some embodiments, M. schaedleri is formulated for delivery to the small intestine, duodenum, jejunum, ileum, cecum, ileocecal region, appendix, ascending colon, transverse colon, descending colon, sigmoid colon, rectum, or anus.

[0091] The genus Musispirillum is a genus of the phylum Deferribacteres. It is represented by a single species, Musispirillum schaedleri. The genus Musispirillum is a spiral-shaped bacterium found in the mucus layer of the gastrointestinal tract of several rodents and is considered a symbiont. This species has been found in cockroaches, mice, guinea pigs, dogs, pigs, goats, termites, and humans. The genus Musispirillum is anaerobic and does not form spores. The genus Musispirillum is motile, flagellated, and capable of moving through mucus.

[0092] In some aspects of any of the scenarios, the M. schaedleri bacterium is the M. schaedleri strain ASF457 bacterium. In some aspects of any of the scenarios, the M. schaedleri bacterium comprises a 16S sequence comprising SEQ ID NO: 4, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 4. In some aspects of any of the scenarios, the M. schaedleri bacterium comprises a 16S sequence comprising SEQ ID NO: 4, or a nucleic acid sequence that is at least 95%, or more identical to SEQ ID NO: 4. In some aspects of any of the scenarios, the M. schaedleri bacterium comprises a 16S sequence comprising SEQ ID NO: 4, or a nucleic acid sequence that is at least 97%, or more identical to SEQ ID NO: 4.

[0093] SEQ ID NO: 4, "Lior" Musispirillum schaedleri 16S ribosomal RNA (see, e.g., Example 1) TIFF2025524662000002.tif166161

[0094] In some aspects of any of the scenarios, the M. schaedleri bacterium comprises a 16S sequence comprising one of SEQ ID NO: 4, 40 - 49, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NO: 4, 40 - 49 (see, e.g., FIG. 15). In some aspects of any of the scenarios, the M. schaedleri bacterium comprises a 16S sequence comprising one of SEQ ID NO: 4, 40 - 49, or a nucleic acid sequence that is at least 95%, or more identical to one of SEQ ID NO: 4, 40 - 49. In some aspects of any of the scenarios, the M. schaedleri bacterium comprises a 16S sequence comprising one of SEQ ID NO: 4, 40 - 49, or a nucleic acid sequence that is at least 97%, or more identical to one of SEQ ID NO: 4, 40 - 49.

[0095] SEQ ID NO: 40, Musispirillum schaedleri strain HRI I17 16S ribosomal RNA, partial sequence, NCBI Reference Sequence: NR_042896.1, 1471 nucleotides (nt) TIFF2025524662000003.tif95161

[0096] SEQ ID NO: 41, GENBANK accession number AF059186.1 Bacterium of the genus Flexistipes group UNSW2.6liv 16S ribosomal RNA gene, partial sequence TIFF2025524662000004.tif153161

[0097] SEQ ID NO: 42, GENBANK accession number AF059187.1 Bacterium of the genus Flexistipes group HRI1cae 16S ribosomal RNA gene, partial sequence TIFF2025524662000005.tif153161

[0098] SEQ ID NO: 43, GENBANK accession number AF059188.1, partial sequence of the 16S ribosomal RNA gene of the bacterium HRI3liv of the genus Flexistipes TIFF2025524662000006.tif153161

[0099] SEQ ID NO: 44, GENBANK accession number AF059189.1, partial sequence of the 16S ribosomal RNA gene of the bacterium UNSWMCS1 of the genus Flexistipes TIFF2025524662000007.tif160161

[0100] SEQ ID NO: 45, GENBANK accession number AF059190.1, partial sequence of the 16S ribosomal RNA gene of the bacterium UNSWRSp12 of the genus Flexistipes TIFF2025524662000008.tif154161

[0101] SEQ ID NO: 46, GENBANK accession number AY387668.1, partial sequence of the 16S ribosomal RNA gene of the strain ABHU I23 of Musispirillum schadei TIFF2025524662000009.tif153161

[0102] SEQ ID NO: 47, GENBANK accession number AY387669.1, partial sequence of the 16S ribosomal RNA gene of the strain HRI I12 of Musispirillum schadei TIFF2025524662000010.tif153161

[0103] SEQ ID NO: 48, GENBANK accession number AY387670.1, partial sequence of the 16S ribosomal RNA gene of the strain HRI I17 of Musispirillum schadei TIFF2025524662000011.tif153161

[0104] SEQ ID NO: 49, GENBANK ACCESSION NO. AY387671.1, Mucidospirillum schaedleri strain UNSW I23, 16S ribosomal RNA gene, partial sequence TIFF2025524662000012.tif153161

[0105] In some embodiments of any aspect, the composition comprises about 10 1 ~10 12 M. schaedleri cells / mL, for example, about 10 1 cells / mL, about 10 2 cells / mL, about 10 3 cells / mL, about 10 4 cells / mL, about 10 5 cells / mL, about 10 6 cells / mL, about 10 7 cells / mL, about 10 8 cells / mL, about 10 9 cells / mL, about 10 10 cells / mL, about 10 11 cells / mL, about 10 12 cells / mL, or more. In some embodiments of any aspect, the composition comprises about 10 1 ~10 12 colony forming units (CFU; e.g., as a measure of viable bacterial cells) of M. schaedleri, for example, about 10 1 CFU / mL, about 10 2 CFU / mL, about 10 3 CFU / mL, about 10 4 CFU / mL, about 10 5 CFU / mL, about 10 6 CFU / mL, about 10 7 CFU / mL, about 10 8 CFU / mL, about 10 9 CFU / mL, about 10 10 CFU / mL, about 10 11 CFU / mL, about 10 12 CFU / mL, or more.

[0106] In some embodiments of any of the aspects, the composition comprises about 10 1 to 10 12 M. schaedleri cells / g, for example, about 10 1 cells / g, about 10 2 cells / g, about 10 3 cells / g, about 10 4 cells / g, about 10 5 cells / g, about 10 6 cells / g, about 10 7 cells / g, about 10 8 cells / g, about 10 9 cells / g, about 10 10 cells / g, about 10 11 cells / g, about 10 12 cells / g, or more. In some embodiments of any of the aspects, the composition comprises about 10 1 to 10 12 colony forming units (CFU; e.g., as a measure of viable bacterial cells) of M. schaedleri, for example, about 10 1 CFU / g, about 10 2 CFU / g, about 10 3 CFU / g, about 10 4 CFU / g, about 10 5 CFU / g, about 10 6 CFU / g, about 10 7 CFU / g, about 10 8 CFU / g, about 10 9 CFU / g, about 10 10 CFU / g, about 10 11 CFU / g, about 10 12 CFU / g, or more.

[0107] In some embodiments of any of the aspects, the composition comprises a centrifuged Musispirillum schaedleri culture (e.g., O.D. 600nm about 0.8), and the bacterial pellet is resuspended in a pharmaceutically acceptable carrier. In some embodiments of any of the aspects, the composition comprises 5 mL of a centrifuged Musispirillum schaedleri culture (e.g., O.D. 600nmcontaining about 0.8), and the bacterial pellet is resuspended in 1 mL of a pharmaceutically acceptable carrier, and each unit dose of the composition contains 100 uL of the solution.

[0108] In some embodiments of any of the scenarios, the M. shadleri bacteria are alive. In some embodiments of any of the scenarios, the M. shadleri bacteria are inactivated. Non-limiting examples of bacterial inactivation methods include ethanol (e.g., 40% ethanol), ultraviolet irradiation, heating, or autoclaving. As a non-limiting example, the bacterial inactivation method includes heating the bacteria at a temperature of at least 100°C (e.g., at least 105°C, at least 110°C, at least 115°C, at least 120°C, or higher) for at least 10 minutes (e.g., at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 60 minutes, or higher). As another non-limiting example, the bacterial inactivation method includes heating the bacteria at a temperature of at least 70°C (e.g., at least 75°C, at least 80°C, at least 90°C, at least 95°C, or higher) for at least 40 minutes (e.g., at least 45 minutes, at least 50 minutes, at least 60 minutes, or higher). As a non-limiting example, the bacterial inactivation method includes autoclaving the bacteria at a temperature of at least 120°C (e.g., at least 121°C, at least 125°C, at least 130°C, or higher) for at least 30 minutes (e.g., at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 60 minutes, or higher) using saturated steam under a pressure of at least 15 psi (e.g., at least 20 psi, at least 25 psi, at least 30 psi).

[0109] In some aspects of any scenario, M. shadleri bacteria are in a viable dried form. In some aspects of any scenario, M. shadleri bacteria are viable bacteria that have been spray-dried or freeze-dried. In some aspects of any scenario, M. shadleri bacteria are non-viable. In some aspects of any scenario, the composition comprises fresh M. shadleri bacteria, viable M. shadleri bacteria, freeze-dried M. shadleri bacteria, spray-dried M. shadleri bacteria, non-viable M. shadleri bacteria, or any combination thereof.

[0110] In one aspect, a composition comprising a conditioned M. shadleri culture medium formulated for delivery to the intestine is described herein. In some aspects of any scenario, the conditioned culture medium can be prepared by incubating M. shadleri bacteria in the culture medium for a predetermined amount of anaerobic incubation time, such as about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, or more. In some aspects of any scenario, the culture medium is modified brain-heart infusion (mBHI) medium. In some aspects of any scenario, the mBHI medium comprises 37 g of BHI, 5 g of yeast extract, 2 mg of vitamin K, 5 mg of hemin, 0.5 g of L-cysteine, and 150 ml of fetal bovine serum per liter of water. The medium can be adjusted to a pH of 7.2 and filtered through 0.2 μm.

[0111] In some aspects of any of the scenarios, the conditioned culture medium is prepared by removing M. chalybea bacteria from the conditioned culture medium after a predetermined amount of anaerobic incubation time has elapsed; for example, the M. chalybea bacteria can be removed by centrifugation and removal of the supernatant from the bacterial pellet. In some aspects of any of the scenarios, the conditioned culture medium is prepared by inactivating M. chalybea from the conditioned culture medium after a predetermined amount of anaerobic incubation time has elapsed; for example, the M. chalybea bacteria can be inactivated by ethanol (e.g., 40% ethanol), ultraviolet irradiation, heating, or autoclaving.

[0112] As a non-limiting example, the bacterial inactivation method includes heating the bacteria at a temperature of at least 100°C (e.g., at least 105°C, at least 110°C, at least 115°C, at least 120°C, or higher) for at least 10 minutes (e.g., at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 60 minutes, or higher). As another non-limiting example, the bacterial inactivation method includes heating the bacteria at a temperature of at least 70°C (e.g., at least 75°C, at least 80°C, at least 90°C, at least 95°C, or higher) for at least 40 minutes (e.g., at least 45 minutes, at least 50 minutes, at least 60 minutes, or higher). As a non-limiting example, the bacterial inactivation method includes autoclaving the bacteria at a temperature of at least 120°C (e.g., at least 121°C, at least 125°C, at least 130°C, or higher) for at least 30 minutes (e.g., at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 60 minutes, or higher) by using saturated steam under a pressure of at least 15 psi (e.g., at least 20 psi, at least 25 psi, at least 30 psi).

[0113] In one aspect, a composition is described herein that comprises an organic solvent extract of a conditioned M. schreibersii culture medium formulated for delivery to the gut. Non-limiting examples of organic solvents used for extraction include methanol, chloroform, ethyl acetate, ethanol, acetone, or any combination thereof. In some embodiments of any of the aspects, the organic solvent extract of the conditioned M. schreibersii culture medium is prepared by methanol:chloroform extraction (see, e.g., the materials and methods in Example 1). In some embodiments of any of the aspects, the organic solvent extract of the conditioned M. schreibersii culture medium comprises organic compounds (e.g., organic solvent soluble, non-polar) secreted by M. schreibersii or otherwise produced. In some embodiments of any of the aspects, the conditioned M. schreibersii culture medium or the organic solvent extract of the conditioned M. schreibersii culture medium is heat treated, e.g., incubated at a temperature of at least 100 °C for at least 10 minutes.

[0114] In some embodiments of any of the aspects, the conditioned M. schreibersii culture medium, or the organic solvent extract or fraction thereof of the conditioned M. schreibersii culture medium, comprises at least one metabolite selected from Table 3 or Table 9 of U.S. Provisional Application No. 63 / 389,382, or FIG. 4H, FIG. 4I, or FIG. 16 herein.

[0115] In some embodiments of any of the aspects, the conditioned M. schreibersii culture medium, or the organic solvent extract or fraction thereof of the conditioned M. schreibersii culture medium, comprises succinic acid; propionic acid; nicotinic acid; aconitic acid (cis and / or trans); pentadecanoic acid; itaconic acid; 16-hydroxyhexadecanoic acid; C8H 15 NO3S; crotonic acid; myristic acid; 17-hydroxyheptadecanoic acid (C 17 H 34 O3); and 15-hydroxypentadecanoic acid (C 15 H 30comprises at least one metabolite selected from the group consisting of O3) (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or 12 metabolites).

[0116] In some embodiments of any of the aspects, the adjusted M. schizoneura culture medium, or an organic solvent extract or a fraction thereof of the adjusted M. schizoneura culture medium, comprises succinic acid; propionic acid; nicotinic acid; aconitic acid (cis and / or trans); pentadecanoic acid; itaconic acid; 16-hydroxyhexadecanoic acid; C8H 15 NO3S; crotonic acid; myristic acid; 17-hydroxyheptadecanoic acid (C 17 H 34 O3); and 15-hydroxypentadecanoic acid (C 15 H 30 O3) and comprises at most 12 metabolites selected from the group consisting of (e.g., at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, or at most 12 metabolites).

[0117] In some embodiments of any of the aspects, the adjusted M. schizoneura culture medium, or an organic solvent extract or a fraction thereof of the adjusted M. schizoneura culture medium, comprises at most 7 metabolites selected from the group consisting of succinic acid; nicotinic acid; aconitic acid (cis and / or trans); pentadecanoic acid; itaconic acid; 16-hydroxyhexadecanoic acid; and crotonic acid (e.g., at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, or at most 7 metabolites).

[0118] In some aspects of any of the situations, the adjusted M. sheldleri culture medium, or the organic solvent extract or fraction thereof of the adjusted M. sheldleri culture medium, contains succinic acid; propionic acid; nicotinic acid; aconitic acid (cis and / or trans); pentadecanoic acid; itaconic acid; 16-hydroxyhexadecanoic acid; crotonic acid; myristic acid; 17-hydroxyheptadecanoic acid (C 17 H 34 O3); 15-hydroxypentadecanoic acid (C 15 H 30 O3), or any combination thereof (see, for example, Formulas 4-15 in Table 3, respectively).

[0119] (Table 3) Exemplary metabolites TIFF2025524662000013.tif198161TIFF2025524662000014.tif185161TIFF2025524662000015.tif71161

[0120] In some aspects of any of the situations, the adjusted M. sheldleri culture medium, or the organic solvent extract or fraction thereof of the adjusted M. sheldleri culture medium, contains propionic acid, cis-aconitic acid, and / or trans-aconitic acid, or any combination thereof (see, for example, Pool 1 in FIG. 18). In some aspects of any of the situations, the adjusted M. sheldleri culture medium, or the organic solvent extract or fraction thereof of the adjusted M. sheldleri culture medium, contains crotonic acid, succinic acid, and / or itaconic acid, or any combination thereof (see, for example, Pool 2 in FIG. 18). In some aspects of any of the situations, the adjusted M. sheldleri culture medium, or the organic solvent extract or fraction thereof of the adjusted M. sheldleri culture medium, contains nicotinic acid (see, for example, Pool 3 in FIG. 18). In some aspects of any of the situations, the adjusted M. sheldleri culture medium, or the organic solvent extract or fraction thereof of the adjusted M. sheldleri culture medium, contains myristic acid, pentadecanoic acid, 15-hydroxypentadecanoic acid, 16-hydroxyhexadecanoic acid, and / or 17-hydroxyheptadecanoic acid, or any combination thereof (see, for example, Pool 4 in FIG. 18).

[0121] In some aspects of any of the scenarios, a composition comprising M. sheddleri bacteria, a culture medium, and / or a solvent extract promotes XCL1 secretion by NKT cells. X-C motif chemokine ligand 1 (XCL1) is also known as lymphotactin, lymphotoxin, or small inducible cytokine subfamily C, member 1. XCL1 is a chemokine that functions in inflammation and immune responses and induces leukocyte migration and activation. XCL1 contributes to chemotaxis in CD8+ T cells. NK cells release XCL1 along with IFN-γ and some other chemokines when encountering certain bacteria, and CD8+ cells work together to cross-present antigens and convey the activation of CD8+ T cells. In some aspects of any of the scenarios, XCL1 secretion can further activate cDC1, for example, in tumor-infiltrating regional lymph nodes, which can ultimately lead to an enhanced CD8+ T cell anti-tumor response. In some aspects of any of the scenarios, a composition comprising M. sheddleri bacteria, a culture medium, and / or a solvent extract further comprises an XCL1 polypeptide (e.g., SEQ ID NOs: 5 - 6).

[0122] In some aspects of any of the scenarios, XCL1 comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 5 and maintains the function of SEQ ID NO: 5 (e.g., binding to XCR1 and / or activation of XCR1). In some aspects of any of the scenarios, the mature human XCL1 peptide comprises residues 22 - 114 or residues 22 - 93 of SEQ ID NO: 5.

[0123] SEQ ID NO: 5, Lymphotactin (XCL1) precursor, Homo sapiens, NCBI Reference Sequence: NP_002986.1, 114 amino acids (aa) TIFF2025524662000016.tif10161

[0124] In some aspects of any of the scenarios, XCL1 comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 6, or maintains its function (e.g., binding to XCR1 and / or activation of XCR1). In some aspects of any of the scenarios, the mature mouse XCL1 peptide comprises residues 22 - 114 or residues 22 - 93 of SEQ ID NO: 6.

[0125] SEQ ID NO: 6, Lymphotactin (XCL1) precursor, Mus musculus, GenBank: AAA56752.1, 114 aa TIFF2025524662000017.tif10158

[0126] In some aspects of any of the scenarios, a composition comprising Mycobacterium chelonae bacteria, a culture medium, and / or a solvent extract increases the secretion of XCL1 by NKT cells by at least 100% (see, e.g., FIGS. 4F-4G, FIGS. 17-18). In some aspects of any of the scenarios, a composition comprising Mycobacterium chelonae bacteria, a culture medium, and / or a solvent extract increases the secretion of XCL1 by NKT cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400%, or more, compared to NKT cells not exposed to the composition.

[0127] In some aspects of any of the scenarios, Mycobacterium chelonae bacteria, a culture medium, and / or a solvent extract are in a dried form, e.g., spray-dried or freeze-dried. In some aspects of any of the scenarios, Mycobacterium chelonae bacteria, a culture medium, and / or a solvent extract are encapsulated. In some aspects of any of the scenarios, Mycobacterium chelonae bacteria, a culture medium, and / or a solvent extract are contained in enteric capsules. In some aspects of any of the scenarios, the composition comprises an enteric coating or the like to survive gastric acidity and enable delivery into the small intestine or large intestine. In some aspects of any of the scenarios, the composition is formulated for delivery in capsules, enteric capsules, tablets, caplets, pills, compressed pills, lozenges, troches, powders, granules, nutraceuticals, medical foods, sachets, liquids, suspensions, oil suspensions, gels, gel tabs, semi-solids, or any combination thereof.

[0128] In some aspects of any of the scenarios, the M. schaedleri bacteria, the culture medium, and / or the solvent extract are maintained in an anaerobic state in the formulation. As used herein, the term "anaerobic state" refers to levels of oxygen (e.g., dissolved oxygen or gaseous oxygen) below those normally found in the human intestinal lumen. By way of non-limiting example, an anaerobic formulation of the M. schaedleri bacteria, the culture medium, and / or the solvent extract can be prepared by sweeping oxygen from the formulation using an inert gas such as nitrogen. In some aspects of any of the scenarios, an anaerobic formulation comprising the M. schaedleri bacteria, the culture medium, and / or the solvent extract does not contain detectable dissolved oxygen or gaseous oxygen, or substantially does not contain dissolved oxygen or gaseous oxygen. In some aspects of any of the scenarios, an anaerobic formulation comprising the M. schaedleri bacteria, the culture medium, and / or the solvent extract contains at most 0.01%, at most 0.1%, or at most 1% dissolved oxygen or gaseous oxygen.

[0129] In some aspects of any of the scenarios, M. chadreieri bacteria, medium, and / or solvent extract are in a mixture with prebiotics. Non-limiting examples of prebiotics include amino acids (e.g., arginine, glutamic acid, and ornithine), short-chain fatty acids (SCFAs; e.g., acetic acid, propionic acid, butyric acid), biotin, fructooligosaccharides, galactooligosaccharides, hemicelluloses (e.g., arabinoxylan, xylan, xyloglucan, and glucomannan), inulin, chitin, lactulose, mannan oligosaccharides, oligofructose-enriched inulin, gums (e.g., guar gum, gum arabic, and carrageenan), oligofructose, oligodextrins, tagatose, resistant maltodextrins (e.g., resistant starch), trans-galactooligosaccharides, pectins (e.g., xylogalactouronan, citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fibers (e.g., soy fiber, beet fiber, pea fiber, corn bran, and oat fiber), xylooligosaccharides, polyamines (such as, but not limited to, spermidine and putrescine).

[0130] In some aspects of any of the scenarios, M. chadreieri bacteria, medium, and / or solvent extract are in a mixture with sulfur-containing amino acids (SAA). In some aspects of any of the scenarios, the sulfur-containing amino acids are methionine, cysteine, or derivatives thereof. In some aspects of any of the scenarios, the sulfur-containing amino acids are methionine, cysteine, homocysteine, taurine, or derivatives thereof. In some aspects of any of the scenarios, the composition comprises any one combination of methionine, cysteine, homocysteine, and / or taurine (see, for example, Table 9). In some aspects of any of the scenarios, the composition comprises methionine. In some aspects of any of the scenarios, the composition comprises cysteine. In some aspects of any of the scenarios, the composition comprises homocysteine. In some aspects of any of the scenarios, the composition comprises taurine.

[0131] Methionine derivatives or cysteine derivatives necessarily contain sulfur, but can be different from the structures of methionine and cysteine and can include homocysteine or taurine. In some embodiments, the methionine derivative or cysteine derivative is ribose-cysteine, ribose-methionine, N-acetylcysteine, or acetylcysteine.

[0132] As used herein, the term "methionine derivative" refers to an amino acid derivative resulting from a reaction at the amino or carboxy group of methionine or from replacement of any hydrogen of methionine by a heteroatom; the definition usually excludes peptides containing methionine residues. Non-limiting examples of methionine derivatives include ribose-methionine; (2S)-2-[[[4-[[(2R)-2-amino-3-mercaptopropyl]amino]-2-phenylphenyl]-oxomethyl]amino]-4-(methylthio)butanoic acid; 2-(1,3-benzothiazol-2-ylamino)-4-(methylthio)butanoic acid; 2-[(6-bromo-4-quinazolinyl)amino]-4-(methylthio)butanoic acid; methyl 2-[[(4-ethylphenyl)-oxomethyl]amino]-4-(methylthio)butanoate; 2-amino-4-(methylsulfanyl)-N-(2-naphthyl)butanamide; N-acetylmethionine; D-methionine; L-methionine; L-methionine methylsulfonium iodide; l-methionine, trimethylsilyl ester; methionine S-oxide; methionine sulfone; methionine sulfoximine; methionine hydroxamate; N-(1-deoxy-1-fructosyl)methionine; N-[(2S)-2-hydroxypropanoyl]methionine; N-formyl-DL-methionine; N-oleoylmethionine; or peptidyl-methionine. In some embodiments, the methionine derivative is ribose-methionine.

[0133] As used herein, the term "cysteine derivative" refers to an amino acid derivative resulting from the reaction of the amino, carboxy, or thiol group of cysteine, or the replacement of any hydrogen of cysteine by a heteroatom; the definition usually excludes peptides containing cysteine residues. Non-limiting examples of cysteine derivatives include ribose-cysteine; N-acetylcysteine; acetylcysteine; (2R;2'S)-isobutane; 2-amino-3-(hydroxysulfonylthio)propionic acid; 2-amino-3-{[(1E)-3-(prop-2-en-1-sulfinyl)prop-1-en-1-yl]disulfanyl}propanoic acid; 2-ammonio-3-disulfanylpropanoate; N-acetyl-S-(1Z)-propenyl-cysteine-sulfoxide; S-(5-acetamido-2-hydroxyphenyl)cysteine; S-2-chloroethylcysteine; S-propylcysteine; D-cysteine derivative; L-cysteine derivative; allothiostatin; allylcysteine; cysteic acid; cysteinyl-amino acid; cystine (cysteine dimer); gamma-glutamylcysteinylglutamate; glyxazone B; hawkinsin; L-cysteine-glycine; peptidyl-cysteine; prenylcysteine; S-(3-oxo-3-carboxy-n-propyl)cysteine; S-(allylthio)-L-cysteine; S-acetamidomethylcysteine; S-cystenosuccinic acid; or trans-S-(1-propenyl)-L-cysteine. In some embodiments, the cysteine derivative is ribose-cysteine; N-acetylcysteine; or acetylcysteine.

[0134] (Table 9) Exemplary SAA in the composition ("X" indicates being included in the composition) TIFF2025524662000018.tif83160

[0135] In some aspects of any of the situations, the composition comprises at least 2.0 g of SAA (e.g., methionine, cysteine, homocysteine, and / or taurine, or derivatives thereof). In some aspects of any of the situations, the composition comprises at least 2.4 g of SAA. In some aspects of any of the situations, the composition comprises at least 5 g, at least 10 g, at least 20 g, at least 30 g, at least 40 g, at least 50 g, at least 60 g, at least 70 g, at least 80 g, at least 90 g, at least 100 g, at least 150 g, at least 200 g, at least 300 g, or at least 400 g of SAA. In some aspects of any of the situations, the composition comprises at most 5 g, at most 10 g, at most 20 g, at most 30 g, at most 40 g, at most 50 g, at most 60 g, at most 70 g, at most 80 g, at most 90 g, at most 100 g, at most 150 g, at most 200 g, at most 300 g, or at most 400 g of SAA. In some aspects of any of the situations, the composition comprises 2.0 g to 400 g of SAA. In some aspects of any of the situations, the composition comprises 2.0 g to 400 g of SAA, 2.0 g to 300 g of SAA, 2.0 g to 200 g of SAA, 2.0 g to 100 g of SAA, 10.0 g to 400 g of SAA, 10 g to 100 g of SAA, 50 g to 400 g of SAA, 100 g to 400 g of SAA, 200 g to 400 g of SAA, or 300 g to 400 g of SAA.

[0136] In some aspects of any of the situations, the composition further comprises 1 to 20 additional bacteria. In some aspects of any of the situations, the composition comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 additional bacteria. In some aspects of any of the situations, the composition comprises 20 or fewer bacteria. In some aspects of any of the situations, the composition comprises at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, or at most 20 bacteria, including M. schadeleyi bacteria.

[0137] In some aspects of any of the scenarios, additional bacteria in compositions other than M. schaedleri bacteria are derived from sources such as environmental isolates, commercially available isolates, at least a portion of a human microbiome sample, at least a portion of a non-human mammalian (e.g., mouse) microbiome sample, isolates from a human microbiome sample, isolates from a non-human mammalian microbiome sample, etc. The benefits of specific microbiome species depend on the specific indication but are beneficial (e.g., to the human gastrointestinal system), and non-limiting examples of genera of bacteria that can be included in the composition include Akkermansia; Alistipes; Bacillus; Bacteroides; Bifidobacterium; Blautia; Clostridium; Collinsella; Eggerthella; Enterococcus; Eubacterium; Faecalibacterium; Fusobacterium; Gemmiger; Lactobacillus; Parabacteroides; Paraprevotella; Phascolarctobacterium; Peptococcus; Peptostreptococcus; Prevotella; Roseburia; Ruminococcus; Ruthenibacterium; Streptococcus; and Subdoligranulum.

[0138] In some aspects of any situation, the composition is substantially free of pathogens. As used herein, the term "substantially" refers to a complete or nearly complete range or degree. For example, a composition that is "substantially" free of pathogens means that the composition is completely or nearly completely free of any pathogens, e.g., any viable pathogens. The exact allowable deviation from absolute completeness may, in some cases, depend on the particular circumstances. However, generally speaking, the near-completion would be such that it would have the same overall result as if absolute and total completion had been achieved. In some aspects of any situation, the pathogens in the therapeutic composition are either absent or reduced to levels acceptable for human administration, as determined, for example, by the FDA.

[0139] As used herein, the term "pathogen" refers to any infectious microorganism that causes disease in an organism. In one aspect, pathogens include bacteria, fungi, archaea (e.g., methanogens, halophiles, thermophiles, and psychrophiles), protists (e.g., Plasmodium, Entamoeba histolytica, Trypanosoma brucei, Giardia lamblia), viruses, prions (e.g., PrPres and PrPSc), microphytes (e.g., Shewanella algae, Shewanella putrefaciens, and Shewanella xiamenensis), and / or microanimals / parasites (e.g., plankton, planaria, helminths, Schistosoma mansoni, and Trypanosoma). In some aspects, pathogenic viruses include RNA viruses such as flaviviruses, picornaviruses, rhabdoviruses, filoviruses, retroviruses (including lentiviruses), or DNA viruses such as adenoviruses, poxviruses, herpesviruses, cytomegaloviruses, hepadnaviruses, or others, but are not limited thereto.

[0140] Non-limiting examples of pathogenic bacteria include spirochetes (e.g., Borrelia), actinomycetes (e.g., Actinomyces), Mycoplasma, Rickettsia, Gram-negative aerobic bacilli, Gram-negative aerobic cocci, Gram-negative facultative anaerobic bacilli (e.g., Erwinia and Yersinia), Gram-negative cocci, Gram-negative coccobacilli, Gram-positive cocci (e.g., Staphylococcus and Streptococcus), endospore-forming bacilli, and endospore-forming cocci. Further non-limiting examples of bacterial pathogens include certain species of Bacillus, Brucella, Burkholderia, Francisella, Yersinia, Streptococcus, Haemophilus, Nisseria, Listeria, Clostridium, Klebsiella, Legionella, Escherichia (e.g., E. coli), Mycobacterium, Staphylococcus, Campylobacter, Vibrio, and Salmonella, as well as drug-resistant and multi-drug resistant strains and highly virulent strains of these pathogenic bacteria. Non-limiting examples of bacterial pathogens carried by known foods include certain species of Salmonella, Clostridium, Campylobacter, Staphylococcus, Salmonella, Escherichia (e.g., E. coli), and Listeria.In some aspects, non-limiting examples of bacterial pathogens include Bacillus anthracis, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Yersinia pestis, group A and B streptococci, MRSA, Streptococcus pneumonia, Haemophilus influenza, Nisseria meningitides, Listeria monocytegenes, Clostridium difficile, Klebsiella spp., highly virulent pathogenic strains of Escherichia coli, Mycobacterium tuberculosis, Staphylococcus aureus, species of Campylobacter, species of Salmonella, and Clostridium perfringens, as well as drug-resistant and multi-drug resistant strains and highly virulent strains of these pathogenic bacteria. In some aspects, non-limiting examples of bacterial pathogens carried by known foods include species of non-typhoidal Salmonella, Clostridium perfringens, species of Campylobacter, Staphylococcus aureus, non-typhoidal Salmonella, species of Campylobacter, Escherichia coli (STEC) O157, and Listeria monocytogenes.

[0141] In some aspects of any of the scenarios, the composition is substantially free of human pathogens (e.g., as described above or as known in the art). In some aspects of any of the scenarios, the composition is substantially free of non-human mammalian pathogens. In some aspects of any of the scenarios, the composition is substantially free of non-human mammalian pathogens that can infect humans and / or cause disease in humans.

[0142] In some aspects of any of the scenarios, M. shedleri bacteria, a culture medium, and / or a solvent extract are formulated into a food composition. In some aspects of any of the scenarios, the food composition includes yogurt or a yogurt beverage. In some aspects of any of the scenarios, M. shedleri bacteria, a culture medium, and / or a solvent extract are formulated into a medical food. In some aspects of any of the scenarios, M. shedleri bacteria, a culture medium, and / or a solvent extract are formulated into a supplement. In one scenario, a food composition comprising M. shedleri bacteria, a culture medium, and / or a solvent extract as described herein is described herein. In one scenario, a medical food comprising M. shedleri bacteria, a culture medium, and / or a solvent extract as described herein is described herein. In one scenario, a supplement comprising M. shedleri bacteria, a culture medium, and / or a solvent extract as described herein is described herein. In some aspects of any of the scenarios, the food composition, medical food, or supplement is fortified with sulfur-containing amino acids and / or prebiotics. In some aspects of any of the scenarios, the food composition, medical food, or supplement further comprises 1 to 20 additional bacteria.

[0143] In some aspects, the technology described herein relates to a pharmaceutical composition comprising M. shedleri bacteria, a culture medium, and / or a solvent extract as described herein and optionally a pharmaceutically acceptable carrier. In some aspects, the active ingredient of the pharmaceutical composition comprises M. shedleri bacteria, a culture medium, and / or a solvent extract as described herein. In some aspects, the active ingredient of the pharmaceutical composition consists essentially of M. shedleri bacteria, a culture medium, and / or a solvent extract as described herein. In some aspects, the active ingredient of the pharmaceutical composition consists of M. shedleri bacteria, a culture medium, and / or a solvent extract as described herein.

[0144] In some embodiments, the technology described herein relates to pharmaceutical compositions of M. sheddleri bacteria, media, and / or solvent extracts as described herein, further comprising sulfur-containing amino acids (SAA). In some embodiments, the active ingredient of the pharmaceutical composition comprises SAA as described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists essentially of SAA as described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists of SAA as described herein.

[0145] Pharmaceutically acceptable carriers and diluents include physiological saline, aqueous buffer solutions, solvents, and / or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials that can act as pharmaceutically acceptable carriers include the following: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) celluloses and their derivatives such as sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic physiological saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL, and LDL; (24) C2-C12 Alcohol; and other non-toxic compatible substances employed in (25) pharmaceutical formulations. Humectants, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, fragrances, preservatives, and antioxidants can also be present in the formulation. Terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent, such as the M. shedleri bacterium, medium, and / or solvent extract, and / or SAA, as described herein.

[0146] Conventional dosage forms generally provide rapid or immediate release of the active ingredient from the formulation. Depending on the pharmacology and pharmacokinetics of the active ingredient, the use of conventional dosage forms can result in wide fluctuations in the concentration of the active ingredient in the patient's blood and other tissues. These fluctuations can affect a number of parameters such as dosing frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, etc. Advantageously, controlled release formulations can be used to control the onset of action, duration of action, levels within the therapeutic window (e.g., gastrointestinal levels), and peak levels (e.g., gastrointestinal levels) of the active ingredient. In particular, controlled release or sustained release dosage forms or formulations can be used to minimize potential adverse effects and safety concerns that can arise from both under-dosing of the active ingredient (i.e., below the minimum therapeutic level) and over-exceeding the toxic level of the active ingredient, while ensuring that the maximum effectiveness of the active ingredient is achieved. In some embodiments, the M. shedleri composition can be administered in a sustained release formulation.

[0147] Controlled-release pharmaceuticals have the common goal of improving therapy beyond what can be achieved by their uncontrolled-release counterparts. Ideally, the use of optimally designed controlled-release preparations in medical treatment is characterized by the minimum amount of active ingredient employed to cure or control a condition in the minimum amount of time. The advantages of controlled-release formulations include the following: 1) extension of the activity of the active ingredient; 2) reduction in dosing frequency; 3) increased patient compliance with taking the medicine; 4) use of a smaller total amount of pharmaceutical composition; 5) reduction of local or systemic side effects; 6) minimization of accumulation of the active ingredient; 7) reduction of fluctuations in levels (e.g., gastrointestinal levels); 8) improvement in the effectiveness of treatment; 9) enhancement of active ingredient activity or reduction of loss of activity; and 10) improvement in the rate of control of a disease or condition. See, for example, Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

[0148] Most controlled-release formulations are designed to first release an amount of the active ingredient that produces the desired therapeutic effect rapidly and then to gradually and continuously release other amounts of the active ingredient to maintain this level of therapeutic or prophylactic effect over a long period of time. To maintain this constant level of the active ingredient in the body, the active ingredient must be released from the dosage form at a rate that replaces the amount of active ingredient that is metabolized, excreted from the body, and / or inactivated in another way. The controlled release of the active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.

[0149] A variety of known controlled release or sustained release dosage forms, formulations, and devices can be adapted for use with the compositions described herein. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1, each of which is incorporated herein by reference. These dosage forms can be used, for example, with hydroxypropylmethylcellulose, other polymeric matrices, gels, permeable membranes, osmotic systems (such as OROS (登録商標) (Alza Corporation, Mountain View, Calif. USA), etc.) or combinations thereof to provide sustained or controlled release of one or more active ingredients to provide a desired release profile at various rates.

[0150] Treatment methods The compositions described herein can be administered to a subject in need thereof, for example, to treat cancer, to promote anti-tumor immune activity, to promote responsiveness to immune checkpoint inhibitor tumor therapy, to increase CD103+ conventional dendritic cells (cDC1), to increase XCL1 secretion by NKT cells, to increase infiltration of CD8+ T cells in colorectal tumors, to establish or maintain a tumor-suppressive gastrointestinal environment, and / or for cancer treatment stratification. Such methods can include administration of one or more of a Musispirillum composition as described herein, a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA), and / or an XCL1 polypeptide (or an XCR1 agonist). In some embodiments of any of the aspects, the method is a Musispirillum composition as described herein; a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA); an XCL1 polypeptide (or an XCR1 agonist); a Musispirillum composition as described herein and a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA); a Musispirillum composition as described herein and an XCL1 polypeptide (or an XCR1 agonist); a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA) and an XCL1 polypeptide (or an XCR1 agonist); or administration of a Musispirillum composition as described herein, a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA), and an XCL1 polypeptide (or an XCR1 agonist).

[0151] In one aspect of any of the embodiments, a method of treating cancer, promoting anti-tumor immune activity, promoting responsiveness to immune checkpoint inhibitor tumor therapy, increasing CD103+ conventional dendritic cells (cDC1), increasing XCL1 secretion by NKT cells, increasing infiltration of CD8+ T cells in colorectal tumors, and / or establishing or maintaining a tumor-suppressive gastrointestinal environment in a subject in need thereof, the method comprising administering a Musispirillum composition as described herein, a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA), and / or an XCL1 polypeptide (or an XCR1 agonist) is described herein.

[0152] In some embodiments, the method of treatment can include the step of prescribing to the subject a treatment as disclosed herein instead of administering the treatment. As used herein, the term "prescribing" refers to, for example, advising and / or authorizing the use of a treatment for a subject in writing. In one aspect of any of the embodiments, a method of treating cancer, promoting anti-tumor immune activity, promoting responsiveness to immune checkpoint inhibitor tumor therapy, increasing CD103+ conventional dendritic cells (cDC1), increasing XCL1 secretion by NKT cells, and / or establishing or maintaining a tumor-suppressive gastrointestinal environment in a subject in need thereof, the method comprising prescribing a Musispirillum composition as described herein, a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA), and / or an XCL1 polypeptide (or an XCR1 agonist) is described herein.

[0153] In some embodiments, a method of treatment can include first diagnosing a subject or patient who can benefit from treatment with a composition described herein. In some embodiments, such diagnosis involves detecting or measuring in a sample from the subject or patient, for example, low levels of Musispirillum spp. (e.g., Musispirillum schaedleri), low levels of sulfur-containing amino acids (SAA), low levels of XCL1 (RNA or protein), low levels of CD103+ conventional dendritic cells (cDC1), low levels of XCL1-expressing NKT cells, low levels of anti-tumor immune activity, or low levels of responsiveness to immune checkpoint inhibitor cancer therapy, each of which is an example of an abnormal level of each analyte. In some embodiments, the method further includes administering to the patient a Musispirillum spp. composition, a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA), and / or an XCL1 polypeptide (or XCL1 agonist) as described herein.

[0154] In some aspects of any of the situations, a low level of Musispirillum (e.g., Musispirillum schaedleri) is less than that measured in normal controls, such as that measured in a gastrointestinal tract, fecal, or mucosal sample. In some aspects of any of the situations, the level of Musispirillum (e.g., Musispirillum schaedleri) is measured using the 16S rRNA abundance determined by 16S sequencing, or the level is determined using bacterial culture (e.g., CFU / mL). As a non-limiting example, the relative 16S rRNA abundance of Musispirillum in the feces of normal controls (e.g., healthy mouse controls) is 1E-5 to 0.05 (e.g., 1.33E-05 to 0.0441517) or 0.01 to 0.05 (e.g., 0.01117764 to 0.0441517). In some aspects of any of the situations, a low level of Musispirillum in the feces is a relative 16S rRNA abundance of Musispirillum in the feces that is less than 0.01, less than 0.009, less than 0.008, less than 0.007, less than 0.006, less than 0.005, less than 0.004, less than 0.003, less than 0.002, less than 1E-3, less than 1E-4, less than 1E-5, or less than 1E-6. In some aspects of any of the situations, a low level of Musispirillum is a relative 16S rRNA abundance of Musispirillum in the feces that is 0 to 0.01. In some aspects of any of the situations, a low level of Musispirillum is a relative 16S rRNA abundance of Musispirillum in the feces that is 0 to 1E-5.

[0155] In some aspects of any situation, the level of the genus Musispirillum (e.g., Musispirillum schaedleri) is measured in an intestinal sample, such as a mucosal sample or a luminal sample, which may have a high prevalence of the genus Musispirillum in human mucosal biopsy samples (e.g., an average prevalence of 42%) but a low prevalence in fecal specimens (e.g., a prevalence of 3%). In some aspects, the intestinal sample is selected from the group consisting of jejunal lumen, jejunal mucosa, terminal ileum lumen, terminal ileum mucosa, cecal lumen, cecal mucosa, ascending colon mucosa, transverse colon mucosa, descending colon lumen, and descending colon mucosa. In some aspects, the intestinal sample is from the cecal lumen.

[0156] As a non-limiting example, the relative 16S rRNA abundance of Musispirillum schaedleri in intestinal samples of normal controls (e.g., healthy human controls) can range from 0.02 to 1.0 depending on the sample site: e.g., 0.2 (small intestinal lumen), 0.4 (small intestinal mucosa), 0.1 (terminal ileum lumen), 0.1 (terminal ileum mucosa), 1 (cecal lumen), 0.02 (cecal mucosa), 0.05 (ascending colon mucosa), 0.08 (transverse colon mucosa), 0.08 (descending colon lumen), 0.2 (descending colon mucosa), or 0.02 (stool). In some aspects of any situation, a low level of the genus Musispirillum is a relative 16S rRNA abundance of the genus Musispirillum in the intestinal sample that is less than 1.0, less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.1, less than 0.09, less than 0.08, less than 0.07, less than 0.06, less than 0.05, less than 0.04, less than 0.03, less than 0.02, less than 0.01. In some aspects of any situation, a low level of the genus Musispirillum is a relative 16S rRNA abundance of the genus Musispirillum in the intestinal sample that is from 0 to 0.01.

[0157] As another non-limiting example, the relative abundance of 16S rRNA of Musispirillum schaedleri in intestinal samples of normal controls (e.g., healthy human controls) is 0.06% (e.g., 0.057%), or 0.001% - 1.4% (e.g., 0.001% - 1.319%), or 0.001% - 9.6% (e.g., 0.001% - 9.524%). In some aspects of any of the aspects, a low level of Musispirillum schaedleri is a relative abundance of 16S rRNA of Musispirillum schaedleri in an intestinal sample that is less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, less than 0.01%, less than 0.009%, less than 0.008%, less than 0.007%, less than 0.006%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, or less than 0.001%. In some aspects of any of the aspects, a low level of Musispirillum schaedleri is a relative abundance of 16S rRNA of Musispirillum schaedleri in an intestinal sample that is 0% - 0.001%. In some aspects of any of the aspects, a low level of Musispirillum schaedleri is a relative abundance of 16S rRNA of Musispirillum schaedleri in an intestinal sample that is 0% - 0.06%. See, for example, Herp et al., Cell Host & Microbe 25(5): 681-694 (2019); Zmora et al., "Personalized gut mucosal colonization resistance to empiric probiotics is associated with unique host and microbiome features," Cell 174 (2018): 1388-1405, each of which is incorporated herein by reference in its entirety.

[0158] In some embodiments, low levels of sulfur-containing amino acids (SAA) are less than those measured in normal controls, such as those measured in plasma, the gastrointestinal tract, or serum. As a non-limiting example, the level of methionine in the plasma of normal controls (e.g., normal human controls) is 14 - 48 μmol / L (1.40 - 4.80 μmol / dL) of methionine. In some embodiments of any of the aspects, low levels of methionine are less than 14 μmol / L, less than 13 μmol / L, less than 12 μmol / L, less than 11 μmol / L, less than 10 μmol / L, less than 5 μmol / L in a plasma sample. In some embodiments of any of the aspects, low levels of methionine are 0 - 14 μmol / L of methionine in a plasma sample.

[0159] As a non-limiting example, the level of cysteine can be measured using cysteine derivatives such as cystine. Cystine is the oxidized disulfide form of cysteine (Cys) and is the predominant form of cysteine in the blood due to its higher relative stability. Cystine is derived from dietary proteins and is formed endogenously from cysteine. In some embodiments of any of the aspects, the level of cystine in the plasma of normal controls (e.g., normal human controls) is 0.8 - 27.5 μmol / L of cystine. In some embodiments of any of the aspects, low levels of cysteine (e.g., cystine) are less than 0.8 μmol / L, less than 0.7 μmol / L, less than 0.6 μmol / L, less than 0.5 μmol / L, less than 0.4 μmol / L, less than 0.3 μmol / L, less than 0.2 μmol / L, less than 0.1 μmol / L in a plasma sample. In some embodiments of any of the aspects, low levels of cysteine (e.g., cystine) are 0 - 0.8 μmol / L of cystine in a plasma sample.

[0160] In some aspects of any of the situations, low levels of XCL1 (RNA or protein) are secreted XCL1 polypeptides of 10 pg / mL to less than 30 pg / mL, as measured, for example, in the digestive tract or serum (see, for example, FIGS. 4D, 4F-4G, 17-18). In some aspects of any of the situations, low levels of CD103+ conventional dendritic cells (cDC1) are MHCII+CD11c+ cells that are less than 10-15% CD103+CD11b-, or 0.5×10 4 to 1×10 4 less than cells of CD103+CD11b- (see, for example, FIGS. 3G-3I, 27C-27G). In some aspects of any of the situations, low levels of NKT cells (e.g., XCL1-expressing NKT cells) are CD3+ cells that are less than 0.25% CD1d tetramer-binding NKT cells, or 1×10 4 less than NKT cells, as measured, for example, in the digestive tract, tumor draining lymph nodes, or tumors (see, for example, FIG. 33D).

[0161] As used herein, the term "anti-tumor immune activity" refers to an immune-mediated attack against tumor cells or tissues. For example, anti-tumor immune activity, such as mediated by the compositions and methods described herein, can include a shift in the tumor microenvironment from an immunosuppressive immune profile to an immune profile that permits and / or promotes an immune-mediated attack against tumor cells or tissues. An immunosuppressive immune profile can be characterized by the presence and / or activation of regulatory T cells (Tregs), regulatory B cells (Bregs), exhausted T cells, increased expression of immune checkpoint proteins, and / or decreased activation of immune cells (e.g., cDC1, NKT, CD8+ T cells, etc.). A shift in the tumor microenvironment that permits and / or promotes an immune-mediated attack against tumor cells or tissues can include an infiltration or increased activation of activated immune cells (e.g., antigen-presenting cells such as cDC1; NKT; CD8+ T cells, etc.), a decrease in the local concentration or activation of Tregs or Bregs, and a decrease in the expression of immune checkpoint proteins. In some embodiments of any of the aspects, low levels of anti-tumor immune activity are low levels of CD8+ T cell anti-tumor immunity, e.g., less than 0.1 CD3+CD8+ cells per CD3+ cell per field of view, less than 20% IFN-γ+ CD3+CD8+ cells (see, e.g., FIG. 3D), or less than 30% granzyme B+ CD3+CD8+ cells (see, e.g., FIGS. 3C-3E).

[0162] In some aspects of any situation, responsiveness to low-level immune checkpoint inhibitor (ICI) tumor therapy means that, despite administration of an ICI (e.g., known to target checkpoint molecules expressed on tumors), tumor growth (e.g., tumor volume, tumor mass) is no slower by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more compared to non-treatment with the ICI. Alternatively, or in addition, "low responsiveness" means that tumor infiltrating lymphocytes (TILs) do not increase after CPI administration and / or activated CD8+ TILs (e.g., IFNγ + and / or GZMB + ) do not increase after CPI administration.

[0163] In some aspects of any situation, the frequency of CD8+ T cells can be measured in the tumor or tumor draining regional lymph nodes. In some aspects of any situation, low anti-tumor immune activity, or low responsiveness to immune checkpoint inhibitor tumor therapy, may be associated with no change or an increase in a tumor measurement (e.g., tumor weight or tumor volume) or a tumor- or cancer-related symptom or complication.

[0164] In some aspects, the subject has previously been determined to have an abnormal level of an analyte described herein as compared to a reference. In some aspects, the reference level can be the level in a sample from a sample / subject and a subject with a similar cell type, sample type, sample processing treatment, and / or similar age, gender, and other demographic parameters. In some aspects, the test sample and the control reference sample are of the same type, i.e., obtained from the same biological source and containing the same composition, e.g., the same number and type of cells.

[0165] As used herein, the term “sample” or “test sample” means a sample taken or isolated from an organism, e.g., a blood or plasma sample from a subject. In some aspects of any of the aspects, the techniques described herein include some examples of biological samples. In some aspects of any of the aspects, the biological sample is a cell, or a tissue, or peripheral blood, or a body fluid. Exemplary biological samples include, but are not limited to, biopsies, tumor samples, biofluid samples; blood; serum; plasma; urine; semen; mucus; tissue biopsies; organ biopsies; synovial fluid; bile fluid; cerebrospinal fluid; mucosal secretions; exudates; sweat; saliva; and / or tissue samples. The term also includes mixtures of the above-described samples. The term “test sample” also includes a biological sample that has not been treated or has been pre-treated (or pre-processed). In some aspects of any of the aspects, the test sample can contain cells from a subject.

[0166] In some aspects of any of the scenarios, the step of determining whether a subject has an abnormal level of an analyte described herein can include: i) obtaining or having obtained a sample from the subject, and ii) performing or having performed an assay on the sample obtained from the subject to determine / measure the level of the analyte in the subject. In some aspects of any of the scenarios, the step of determining whether a subject has an abnormal level of an analyte described herein can include performing or having performed an assay on the sample obtained from the subject to determine / measure the level of the analyte in the subject. In some aspects of any of the scenarios, the step of determining whether a subject has an abnormal level of an analyte described herein can include ordering or requesting an assay on the sample obtained from the subject to determine / measure the level of the analyte in the subject. In some aspects of any of the scenarios, the step of determining whether a subject has an abnormal level of an analyte described herein can include receiving the results of an assay on the sample obtained from the subject to determine / measure the level of the analyte in the subject. In some aspects of any of the scenarios, the step of determining whether a subject has an abnormal level of an analyte described herein can include receiving a report, result, or other means of identifying the subject as a subject with a reduced level of the analyte.

[0167] In one aspect of any of the embodiments, a method of treating cancer, promoting anti-tumor immune activity, promoting responsiveness to immune checkpoint inhibitor tumor therapy, increasing CD103+ conventional dendritic cells (cDC1), increasing XCL1 secretion by NKT cells, and / or establishing or maintaining a tumor-suppressive gastrointestinal environment in a subject in need thereof, comprising: a) determining whether the subject has an abnormal level of an analyte described herein; and b) if the level of the analyte is abnormal (e.g., decreased) compared to a reference, instructing or directing the subject to administer a Musispirillum composition as described herein, a diet rich in sulfur-containing amino acids (SAA) (or a supplement containing SAA), and / or an XCL1 polypeptide herein. In some embodiments of any of the aspects, the step of instructing or directing the subject to administer a particular treatment can include providing a report of the assay results. In some embodiments of any of the aspects, the step of instructing or directing the subject to administer a particular treatment can include providing a report of the assay results and / or a treatment recommendation taking into account the assay results.

[0168] Method of the genus Musispirillum In a plurality of aspects, methods are described herein for using a Musispirillum composition described herein for methods including, but not limited to, methods of treating cancer, promoting anti-tumor immune activity, promoting responsiveness to immune checkpoint inhibitor tumor therapy, increasing CD103+ conventional dendritic cells (cDC1), increasing XCL1 secretion by NKT cells, and / or increasing infiltration of CD8+ T cells in colorectal tumors.

[0169] In one aspect, a method of treating cancer or promoting anti-tumor immune activity, the method comprising administering to a subject in need thereof a Musispirillum composition as described herein, is described herein. In one aspect, a method of treating colorectal cancer, the method comprising administering to a subject in need thereof a Musispirillum composition as described herein, is described herein. In one aspect, a method of promoting anti-tumor immune activity, the method comprising administering to a subject in need thereof a Musispirillum composition as described herein, is described herein. In one aspect, a method of increasing infiltration of CD8+ T cells in colorectal tumors, the method comprising administering to a subject in need thereof a Musispirillum composition as described herein, is described herein.

[0170] In one aspect, a method of treating cancer, promoting anti-tumor immune activity, promoting responsiveness to immune checkpoint inhibitor tumor therapy, increasing CD103+ conventional dendritic cells (cDC1), increasing XCL1 secretion by NKT cells, and / or increasing infiltration of CD8+ T cells in colorectal tumors, the method comprising formulating for a subject in need thereof a Musispirillum composition as described herein, is described herein.

[0171] In one aspect, there is provided a method for promoting responsiveness to immune checkpoint inhibitor tumor therapy, the method comprising administering to a subject in need thereof a composition of the genus Musispirillum as described herein. In some embodiments of any of the aspects, the method further comprises administering an immune checkpoint inhibitor. In one aspect, there is provided a method for promoting responsiveness to immune checkpoint inhibitor tumor therapy, the method comprising administering to a subject in need thereof an immune checkpoint inhibitor (ICI) and a composition of the genus Musispirillum as described herein. In some embodiments of any of the aspects, the ICI is administered before the administration of the composition of the genus Musispirillum. In some embodiments of any of the aspects, the ICI is administered after the administration of the composition of the genus Musispirillum. In some embodiments of any of the aspects, the ICI is administered at the same time as the administration of the composition of the genus Musispirillum. In some embodiments of any of the aspects, the subject has cancer. In some embodiments of any of the aspects, the subject has colon cancer. In some embodiments of any of the aspects, the subject has cancer of the mucosal epithelial tissue. In some embodiments of any of the aspects, the subject's cancer is determined to be resistant to immune checkpoint inhibitor therapy.

[0172] In one aspect, there is provided a method for increasing XCL1 secretion by NKT cells, the method comprising administering to a subject in need thereof a composition of the genus Musispirillum as described herein. In some embodiments of any of the aspects, the subject has cancer. In some embodiments of any of the aspects, XCL1 secretion recruits cDCs and / or activates cDC1s. In some embodiments of any of the aspects, the subject has colon cancer. In some embodiments of any of the aspects, the subject has cancer of the mucosal epithelial tissue.

[0173] In one aspect, a method of increasing CD103+ conventional dendritic cells (cDC1) comprising administering to a subject in need thereof a Musispirillum composition as described herein. In some embodiments of any of the aspects, the administration increases CD103+CD11b - conventional dendritic cells (cDC1). In some embodiments of any of the aspects, the administration increases the number of cDCs. In some embodiments of any of the aspects, the administration increases the number of cDCs in tumor-draining lymph nodes (TDLNs). In some embodiments of any of the aspects, the administration increases the activation of cDCs (e.g., increased expression of the XCL1 receptor XCR1). In some embodiments of any of the aspects, the administration increases the activation of cDCs in tumor-draining lymph nodes (TDLNs) (e.g., increased expression of the XCL1 receptor XCR1). In some embodiments of any of the aspects, the cDCs are mobilized to and / or activated by XCL1 in tumor-draining lymph nodes (TDLNs). In some embodiments of any of the aspects, the cDC1 is associated with a tumor. In some embodiments of any of the aspects, the cDC1 is associated with tumor-draining lymph nodes (TDLNs). In some embodiments of any of the aspects, the tumor is a colon cancer. In some embodiments of any of the aspects, the tumor is a tumor of the mucosal epithelial tissue. In some embodiments of any of the aspects, the tumor is an adenoma, which is a non-cancerous tumor that begins in glandular cells of the epithelial tissue. In some embodiments of any of the aspects, the tumor is a carcinoma, which is a cancer that begins in the tissue lining or covering the skin or internal organs.

[0174] In some aspects of any of the situations, the cancer is colorectal cancer. In some aspects of any of the situations, the cancer is colorectal cancer (CRC). In some aspects of any of the situations, the cancer is a cancer of the mucosal epithelial tissue or a cancer of the mucosa. As used herein, the term "mucosa" refers to a membrane rich in mucosa or mucus glands that lines the body's passages and cavities (e.g., the digestive tract or the respiratory tract) that are directly or indirectly connected to the outside. In some aspects of any of the situations, the cancer is a cancer of the gastrointestinal tract, including but not limited to oral cancer, esophageal cancer, gastric cancer, small intestine cancer, colorectal cancer, or anal cancer. In some aspects of any of the situations, the cancer is a cancer of the respiratory tract, including but not limited to lung cancer, laryngeal cancer, or bronchial adenoma.

[0175] In some aspects of any of the scenarios, the method further comprises administering an immune checkpoint inhibitor. In some aspects of any of the scenarios, the immune checkpoint inhibitor comprises an immune checkpoint inhibitor antibody. In some aspects of any of the scenarios, the checkpoint inhibitor immunotherapy is directed against a checkpoint molecule selected from the group consisting of programmed cell death 1 (PD-1), programmed death-ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), adenosine A2A receptor (A2AR), CD276, CD39, CD73, B7 family immune checkpoint molecules, V-set domain-containing T cell activation inhibitor 1 (B7H4), B and T lymphocyte attenuator (BTLA), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene-3 (LAG-3), nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2 (NOX2), T cell immunoglobulin domain and mucin domain 3 (TIM-3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), V-domain Ig suppressor of T cell activation (VISTA), and sialic acid-binding immunoglobulin-type lectin 7 (SIGLEC7).

[0176] Non-limiting examples of immune checkpoint inhibitors (ICIs) include pembrolizumab (Keytruda®), nivolumab (Opdivo®), cemiplimab (Libtayo®), spartalizumab, camrelizumab (AiRuiKa (商標) )), sintilimab (TYVYT®), tislelizumab, toripalimab (Tuoyi (商標)) Dostarlimab (JEMPERLI), INCMGA00012, AMP-224, AMP-514 (MEDI0608), atezolizumab (Tecentriq®), avelumab (Bavencio®), enobafolimab (KN035), cosibelimab (CK-301), AUNP12, CA-170, BMS-986189, BMS-936559 (MDX-1105), durvalumab (IMFINZI®), tremelimumab, and ipilimumab (Yervoy®) are included. See, for example, U.S. Patents US5811097, US5855887, US6051227, US668,2736, US6984720, US7595048, US7605238, US7943743, US8008449, US8217149, US8354509, US8383796, US8728474, US8735553, US8779105, US8779108, US8907053, US8900587, US8952136, US9067999, US9073994, US9683048, US9987500, US10160736, US10316089, US10441655, US10590199, US11225522, U.S. Patent Application Publication US2014341917; Storz et al., MAbs. 2016 Jan; 8(1): 10-26, the contents of each of which are hereby incorporated by reference in their entirety.

[0177] In some aspects of any of the scenarios, administration of a Musispirillum composition promotes XCL1 secretion by NKT cells. In some aspects of any of the scenarios, the Musispirillum composition increases the secretion of XCL1 by NKT cells by at least 100% (see, for example, FIGS. 4F - 4G, FIGS. 17 - 18). In some aspects of any of the scenarios, the Musispirillum composition increases the secretion of XCL1 by NKT cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400%, or more, relative to NKT cells not exposed to the composition. In some aspects of any of the scenarios, XCL1 secretion is measured using an XCL1 - specific ELISA, such as the R&D SYSTEMS MOUSE XCL1 / LYMPHOTACTIN DUOSET ELISA KIT, the ONESTEP MOUSE XCL1 ELISA KIT (ABCAM), the ONESTEP HUMAN XCL1 ELISA (ABCAM). In some aspects of any of the scenarios, NKT cells are measured and / or isolated using flow cytometry, for example, by using CD1d tetramer - binding CD3+ cells (for NKT cell gating strategies, see, for example, FIGS. 8F, 30E). In some aspects of any of the scenarios, NKT cells can be expanded from cell lines (e.g., GW1 NKT cells).

[0178] In some aspects of any of the scenarios, administration of the Musispirillum composition increases the number and / or activation of cDC1 by at least 50% in, for example, tumor-draining regional lymph nodes (see, e.g., FIGS. 3G-3I, FIGS. 27C-27F). In some aspects of any of the scenarios, administration of the Musispirillum composition increases the number and / or activation of cDC1 by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400%, or more, compared to a subject not administered the Musispirillum composition. In some aspects of any of the scenarios, the number of cDC1 is measured by quantifying the number of CD103+CD11b− cells from the population of MHCII+CDC11c+ dendritic cells using flow cytometry (see, e.g., FIGS. 8E, 30D for gating strategies for dendritic cells expressing CD103 / CD11b). In some aspects of any of the scenarios, cDC1 activation can be measured by quantifying the number of cDC1 cells that have migrated from the intestine (e.g., lamina propria) into the tumor-draining regional lymph nodes. In some aspects of any of the scenarios, activation of cDC1 by XCL1 can be measured by quantifying the RNA or protein expression of XCR1. Additional non-limiting examples of activation markers of cDC1 include Clec9a and Irf8.

[0179] In some aspects of any of the scenarios, administration of the Musispirillum composition increases the number and / or activation of CD8+ T cells by at least 50%. In some aspects of any of the scenarios, administration of the Musispirillum composition increases the number and / or activation of CD8+ T cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400%, or more, compared to a subject not receiving the Musispirillum composition. In some aspects of any of the scenarios, administration of the Musispirillum composition increases the infiltration of CD8+ T cells in colorectal tumors. In some aspects of any of the scenarios, the number of CD8+ T cells is quantified using flow cytometry in tumor-draining regional lymph nodes or in tumors. In some aspects of any of the scenarios, activation of CD8+ T cells is quantified by measuring an increase in the expression of effectors such as IFNγ or GZMB, or by measuring a decrease in the expression of immune checkpoint receptors such as PD-1, LAG-3, TIM-3, or CTLA-4 (for gating strategies for the expression of CD8+ T cells, and their immune checkpoint receptors, and their IFNγ and GZMB expression, see, for example, FIGS. 8B, 30B).

[0180] In some embodiments of any of the aspects, the method further comprises administering a sulfur - containing amino acid (SAA). In some embodiments of any of the aspects, the sulfur - containing amino acid (SAA) is administered at or above the recommended daily intake. The recommended daily intake of methionine is 10.4 mg per kilogram of body weight, or 4.5 mg per pound. A person weighing 70 kg (about 154 pounds) should consume at least 728 mg of methionine per day. The recommended daily intake of cysteine is 4.1 mg per kilogram of body weight, or 1.9 mg per pound. A person weighing 70 kg (about 154 pounds) should consume at least 287 mg of cysteine per day. In some embodiments of any of the aspects, the administration of SAA includes administering foods rich in natural SAA, foods supplemented with SAA, supplements containing SAA, or pharmaceutical compositions containing SAA.

[0181] In some aspects of any of the scenarios, the method further includes a step of administering a food rich in sulfur-containing amino acids, such as a food rich in sulfur-containing amino acids naturally. Non-limiting examples of foods rich in sulfur-containing amino acids (e.g., methionine and / or cysteine) include the following: turkey (e.g., ground turkey; e.g., 931 mg of methionine per 100 g of ground turkey, 128% of the recommended daily intake (RDI) of methionine) or chicken (e.g., 40 - 195% of the RDI of methionine; e.g., skinless chicken breast; e.g., 336 mg of cysteine per 100 g of skinless chicken breast; 117% of the RDI of cysteine) and other poultry; red meat, such as beef (e.g., sirloin steak; e.g., 931 mg of methionine per 100 g of sirloin steak; 124% of the RDI of methionine, 345 mg of cysteine per 100 g of sirloin steak; 120% of the RDI of cysteine), lamb, veal, or buffalo meat; fish or seafood, such as tuna (e.g., 885 mg of methionine per 100 g of tuna; 122% of the RDI of methionine, e.g., 321 mg of cysteine per 100 g of tuna; 112% of the RDI of cysteine), halibut, salmon, cod, tilapia, mahi-mahi, or clams; pork, such as pork chop (e.g., 850 mg of methionine per 100 g of pork chop; 117% of the RDI of methionine, 350 mg of cysteine per 100 g of pork chop; 122% of the RDI of cysteine), pork rib, lean ham, pork sausage, ground pork, salami, roast ham, sausage link, or bacon; soy products, such as soft tofu (e.g., 211 mg of methionine per 100 g of soft tofu; 29% of the RDI of methionine), boiled soybeans, cooked soybeans sprouts, or soy milk; dairy products, such as milk (e.g., 88 mg of methionine per 100 g of milk; 12% of the RDI of methionine), yogurt (e.g., 52 mg of cysteine per 100 g of yogurt; 18% of the RDI of cysteine), or buttermilk; cheese products, such as ricotta (e.g., 284 mg of methionine per 100 g of ricotta;(39% of the RDI for methionine), Swiss cheese (e.g., 290 mg of cysteine per 100 g of Swiss cheese; 133% of 101 of the RDI for cysteine), Parmesan, Gruyère, Gouda, or Fontina; nuts or seeds, e.g., Brazil nuts (e.g., 1124 mg of methionine per 100 g of Brazil nuts; 154% of the RDI for methionine), sunflower seeds (e.g., 383 mg of cysteine per 100 g of sunflower seeds; 133% of the RDI for cysteine), hemp seeds, squash seeds, pumpkin seeds, chia seeds, sesame seeds, flaxseeds, cashew nuts, pistachios, or peanuts; beans, e.g., large white beans (e.g., 146 mg of methionine per 100 g of large white beans; 20% of the RDI for methionine), lentils (e.g., 118 mg of cysteine per 100 g of lentils; 41% of the RDI for cysteine), navy beans, kidney beans, black beans, great northern beans, fava beans, split peas; grains, e.g., quinoa (e.g., 96 mg of methionine per 100 g of quinoa; 13% of the RDI for methionine), oatmeal (e.g., 97 mg of cysteine per 100 g of oatmeal; 34% of the RDI for cysteine), teff, wild rice, kamut, rice, or whole grain pasta; or chicken eggs (e.g., 292 mg of cysteine per 100 g of eggs; 102% of the RDI for cysteine).;

[0182] In some embodiments of any of the aspects, the method further comprises administering a foodstuff supplemented with at least one sulfur - containing amino acid. In some embodiments of any of the aspects, the foodstuff is supplemented with at least 2.0 g, at least 5 g, at least 10 g, at least 20 g, at least 30 g, at least 40 g, at least 50 g, at least 60 g, at least 70 g, at least 80 g, at least 90 g, at least 100 g, at least 150 g, at least 200 g, at least 300 g, or at least 400 g of SAA. In some embodiments of any of the aspects, the foodstuff is supplemented with 2.0 g - 400 g of SAA, 2.0 g - 300 g of SAA, 2.0 g - 200 g of SAA, 2.0 g - 100 g of SAA, 10.0 g - 400 g of SAA, 10 g - 100 g of SAA, 50 g - 400 g of SAA, 100 g - 400 g of SAA, 200 g - 400 g of SAA, or 300 g - 400 g of SAA.

[0183] In some aspects of any of the situations, the method further comprises administering a supplement comprising at least one sulfur-containing amino acid. In some aspects of any of the situations, the supplement comprises at least 2.0 g, at least 5 g, at least 10 g, at least 20 g, at least 30 g, at least 40 g, at least 50 g, at least 60 g, at least 70 g, at least 80 g, at least 90 g, at least 100 g, at least 150 g, at least 200 g, at least 300 g, or at least 400 g of SAA. In some aspects of any of the situations, the supplement comprises 2.0 g to 400 g of SAA, 2.0 g to 300 g of SAA, 2.0 g to 200 g of SAA, 2.0 g to 100 g of SAA, 10.0 g to 400 g of SAA, 10 g to 100 g of SAA, 50 g to 400 g of SAA, 100 g to 400 g of SAA, 200 g to 400 g of SAA, or 300 g to 400 g of SAA. In some aspects of any of the situations, the sulfur-containing amino acid is methionine, cysteine, or a derivative thereof. In some aspects of any of the situations, the sulfur-containing amino acid is methionine, cysteine, homocysteine, taurine, or a derivative thereof. In some aspects of any of the situations, the composition comprises any one combination of methionine, cysteine, homocysteine, and / or taurine (see, for example, Table 9).

[0184] In some embodiments of any of the situations, the method further comprises administering at least 2.0 g of SAA (e.g., methionine, cysteine, homocysteine, and / or taurine, or derivatives thereof). In some embodiments of any of the situations, the method further comprises administering at least 2.4 g of SAA. In some embodiments of any of the situations, the method further comprises administering at least 5 g, at least 10 g, at least 20 g, at least 30 g, at least 40 g, at least 50 g, at least 60 g, at least 70 g, at least 80 g, at least 90 g, at least 100 g, at least 150 g, at least 200 g, at least 300 g, or at least 400 g of SAA. In some embodiments of any of the situations, the method further comprises administering at most 5 g, at most 10 g, at most 20 g, at most 30 g, at most 40 g, at most 50 g, at most 60 g, at most 70 g, at most 80 g, at most 90 g, at most 100 g, at most 150 g, at most 200 g, at most 300 g, or at most 400 g of SAA. In some embodiments of any of the situations, the method further comprises administering 2.0 g to 400 g of SAA. In some embodiments of any of the situations, the method further comprises administering 2.0 g to 400 g of SAA, 2.0 g to 300 g of SAA, 2.0 g to 200 g of SAA, 2.0 g to 100 g of SAA, 10.0 g to 400 g of SAA, 10 g to 100 g of SAA, 50 g to 400 g of SAA, 100 g to 400 g of SAA, 200 g to 400 g of SAA, or 300 g to 400 g of SAA.

[0185] In some aspects of any of the scenarios, the method involves administering SAA once a day, twice a day, three times a day (e.g., with meals), four times a day, five times a day, or more than five times a day, over a period of one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, or six months, or longer (e.g., in foods rich in natural SAA, foods supplemented with SAA, supplements containing SAA, or pharmaceutical compositions containing SAA). In some aspects of any of the scenarios, the administration of SAA corresponds to the duration of cancer treatment. In some aspects of any of the scenarios, the administration of SAA continues after the cessation of cancer treatment.

[0186] In some aspects of any of the scenarios, the method further involves administering at least a second composition as described herein, in addition to the Musispirillum composition. In some aspects of any of the scenarios, the Musispirillum composition is co-administered with a diet rich in sulfur - containing amino acids (SAA) (or a supplement containing SAA), and / or an XCL1 polypeptide (or an XCR1 agonist). In some aspects of any of the scenarios, the Musispirillum composition is co - administered with a diet rich in sulfur - containing amino acids (SAA) (or a supplement containing SAA). In some aspects of any of the scenarios, the Musispirillum composition is co - administered with an XCL1 polypeptide (or an XCR1 agonist). In some aspects of any of the scenarios, the Musispirillum composition is co - administered with a diet rich in sulfur - containing amino acids (SAA) (or a supplement containing SAA), and an XCL1 polypeptide (or an XCR1 agonist). For exemplary treatment combinations, see, for example, Table 10. In some aspects of any of the scenarios, the treatment combinations can be administered sequentially or simultaneously.

[0187] (Table 10) Exemplary treatment combinations ("X" indicates inclusion in the treatment method) TIFF2025524662000019.tif87160

[0188] Method of SAA diet or supplementation In one aspect, a method of establishing or maintaining a tumor-suppressive gastrointestinal environment in a subject in need thereof, the method comprising administering a diet rich in sulfur amino acids (SAA) (or a supplement containing SAA), is described herein. In one aspect, a method of establishing a tumor-suppressive gastrointestinal environment in a subject in need thereof, the method comprising administering a diet rich in sulfur amino acids (SAA) (or a supplement containing SAA), is described herein. In one aspect, a method of maintaining a tumor-suppressive gastrointestinal environment in a subject in need thereof, the method comprising administering a diet rich in sulfur amino acids (SAA) (or a supplement containing SAA), is described herein. In one aspect, a method of increasing infiltration of CD8+ T cells in colorectal tumors, the method comprising administering a diet rich in sulfur amino acids (SAA) (or a supplement containing SAA) to a subject in need thereof, is described herein. As used herein, the term "tumor-suppressive gastrointestinal environment" refers to a gastrointestinal environment associated with tumor suppression (e.g., host gastrointestinal cells, host immune cells, and / or associated gastrointestinal microbiota); as described herein, an increase in the gastrointestinal levels of sulfur amino acids (SAA), Muris sp., XCL1, XCL1-expressing NKT, and / or CD103+ conventional dendritic cells (cDC1) may be associated with tumor suppression (see, e.g., FIG. 13). In some embodiments of any of the aspects, the method establishes or maintains a colon cancer tumor-suppressive gastrointestinal environment in a subject in need thereof.

[0189] As used herein, the term "diet rich in sulfur-containing amino acids" refers to a diet containing more than the recommended daily intake (e.g., for a person weighing 70 kg, at least 728 mg of methionine per day and at least 287 mg of cysteine per day). In some embodiments of any of the aspects, a diet rich in sulfur-containing amino acids includes foods that are naturally rich in SAA as described herein or foods supplemented with SAA. Thus, a diet rich in sulfur-containing amino acids includes not only diets containing an increased amount of SAA as described herein, but also diets in which one or more SAAs are provided as supplements. In this aspect, SAA can be provided as a supplement to a normal diet or to increase the diet rich in or abundant in SAA.

[0190] In some aspects of any of the scenarios, the method includes the step of administering a foodstuff supplemented with at least one sulfur-containing amino acid. In some aspects of any of the scenarios, the foodstuff is supplemented with at least 2.0 g, at least 5 g, at least 10 g, at least 20 g, at least 30 g, at least 40 g, at least 50 g, at least 60 g, at least 70 g, at least 80 g, at least 90 g, at least 100 g, at least 150 g, at least 200 g, at least 300 g, or at least 400 g of SAA. In some aspects of any of the scenarios, the foodstuff is supplemented with 2.0 g to 400 g of SAA, 2.0 g to 300 g of SAA, 2.0 g to 200 g of SAA, 2.0 g to 100 g of SAA, 10.0 g to 400 g of SAA, 10 g to 100 g of SAA, 10 g to 20 g of SAA, 20 g to 30 g of SAA, 30 g to 40 g of SAA, 40 g to 50 g of SAA, 50 g to 400 g of SAA, 100 g to 400 g of SAA, 200 g to 400 g of SAA, or 300 g to 400 g of SAA. In some aspects of any of the scenarios, the foodstuff is supplemented with about 15 g of L-methionine and / or about 8 g of L-cysteine. In some aspects of any of the scenarios, the foodstuff is supplemented with about 15 g of L-methionine and / or about 8 g of L-cystine (see, for example, Table 2).

[0191] In some aspects of any of the situations, the method includes the step of administering a supplement containing at least one sulfur-containing amino acid. In some aspects of any of the situations, the supplement contains at least 2.0 g, at least 5 g, at least 10 g, at least 20 g, at least 30 g, at least 40 g, at least 50 g, at least 60 g, at least 70 g, at least 80 g, at least 90 g, at least 100 g, at least 150 g, at least 200 g, at least 300 g, or at least 400 g of SAA. In some aspects of any of the situations, the supplement contains 2.0 g - 400 g of SAA, 2.0 g - 300 g of SAA, 2.0 g - 200 g of SAA, 2.×g - 100 g of SAA, 10.0 g - 400 g of SAA, 10 g - 100 g of SAA, 10 g - 20 g of SAA, 20 g - 30 g of SAA, 30 g - 40 g of SAA, 40 g - 50 g of SAA, 50 g - 400 g of SAA, 100 g - 400 g of SAA, 200 g - 400 g of SAA, or 300 g - 400 g of SAA. In some aspects of any of the situations, the supplement contains about 15 g of L-methionine and / or about 8 g of L-cysteine. In some aspects of any of the situations, the supplement contains about 15 g of L-methionine and / or about 8 g of L-cystine (see, for example, Table 2).

[0192] In some aspects of any of the scenarios, a diet high in sulfur - containing amino acids (or a supplement containing SAA) contains elevated levels of methionine, cysteine, or their derivatives compared to a diet low in sulfur - containing amino acids or compared to a normal or typical diet, as described herein. In some aspects of any of the scenarios, a diet high in sulfur - containing amino acids (or a supplement containing SAA) contains elevated levels of methionine, cysteine, homocysteine, taurine, or their derivatives compared to a diet low in sulfur - containing amino acids. In some aspects of any of the scenarios, a diet high in sulfur - containing amino acids (or a supplement containing SAA) contains an elevated level of any one combination of methionine, cysteine, homocysteine, and / or taurine (see, e.g., Table 9).

[0193] In some aspects of any of the scenarios, a diet low in sulfur - containing amino acids contains from 0.01 grams to 0.04 grams of SAA per kilogram of (subject's) body weight per day. In some aspects of any of the scenarios, a diet high in sulfur - containing amino acids contains more than 0.04 grams of SAA per kilogram of (subject's) body weight per day. In some aspects of any of the scenarios, a diet high in sulfur - containing amino acids contains at least 6 grams of SAA per kilogram of body weight per day.

[0194] In some aspects of any of the situations, a diet rich in sulfur - containing amino acids contains at least 0.04 g, at least 0.05 g, at least 0.06 g, at least 0.07 g, at least 0.08 g, at least 0.09 g, at least 0.1 g, at least 0.2 g, at least 0.3 g, at least 0.4 g, at least 0.5 g, at least 0.6 g, at least 0.7 g, at least 0.8 g, at least 0.9 g, at least 1 g, at least 2 g, at least 3 g, at least 4 g, at least 5 g, at least 6 g, or more SAA per kilogram of (the subject's) body weight per day. In some aspects of any of the situations, a diet rich in sulfur - containing amino acids contains at most 0.05 g, at most 0.06 g, at most 0.07 g, at most 0.08 g, at most 0.09 g, at most 0.1 g, at most 0.2 g, at most 0.3 g, at most 0.4 g, at most 0.5 g, at most 0.6 g, at most 0.7 g, at most 0.8 g, at most 0.9 g, at most 1 g, at most 2 g, at most 3 g, at most 4 g, at most 5 g, at most 6 g SAA per kilogram of (the subject's) body weight per day. In some aspects of any of the situations, a diet rich in sulfur - containing amino acids contains 0.04 g - 0.1 g, 0.1 g - 1.0 g, 1.0 g - 6.0 g, 0.04 g - 1.0 g, 0.04 g - 6.0 g, or 0.1 g - 6.0 g SAA per kilogram of (the subject's) body weight per day.

[0195] Assuming an average body weight of 60 kg, in some aspects of any of the scenarios, a diet high in sulfur amino acids (SAA) contains more than 2.4 grams of SAA per day. In some aspects of any of the scenarios, a diet high in SAA contains at least 5 g, at least 10 g, at least 20 g, at least 30 g, at least 40 g, at least 50 g, at least 60 g, at least 70 g, at least 80 g, at least 90 g, at least 100 g, at least 150 g, at least 200 g, at least 300 g, or at least 400 g of SAA per day. In some aspects of any of the scenarios, a diet high in SAA contains at most 5 g, at most 10 g, at most 20 g, at most 30 g, at most 40 g, at most 50 g, at most 60 g, at most 70 g, at most 80 g, at most 90 g, at most 100 g, at most 150 g, at most 200 g, at most 300 g, or at most 400 g of SAA per day. In some aspects of any of the scenarios, a diet high in SAA contains from 2.0 g to 400 g of SAA per day. In some aspects of any of the scenarios, a diet high in SAA contains from 2.0 g to 400 g of SAA, from 2.0 g to 300 g of SAA, from 2.0 g to 200 g of SAA, from 2.0 g to 100 g of SAA, from 10.0 g to 400 g of SAA, from 10 g to 100 g of SAA, from 50 g to 400 g of SAA, from 100 g to 400 g of SAA, from 200 g to 400 g of SAA, or from 300 g to 400 g of SAA per day. In some aspects of any of the scenarios, the method further comprises administering at least a second treatment as described herein, in addition to a diet high in sulfur amino acids (SAA) (see, for example, Table 10).

[0196] In some aspects of any of the scenarios, administration of a diet rich in sulfur - containing amino acids (or a supplement containing SAA) promotes XCL1 secretion by NKT cells. In some aspects of any of the scenarios, a diet rich in sulfur - containing amino acids increases the secretion of XCL1 by NKT cells by at least 100%. In some aspects of any of the scenarios, a diet rich in sulfur - containing amino acids increases the secretion of XCL1 by NKT cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400%, or more, compared to the secretion of XCL1 by NKT cells in a subject not receiving a diet rich in sulfur - containing amino acids.

[0197] In some aspects of any of the scenarios, administration of a sulfur amino acid-rich diet (or a supplement containing SAA) increases the number and / or activation of cDC1 by at least 25% in, for example, tumor-inflow region lymph nodes (see, e.g., FIGS. 3G-3I, 27C-27F). In some aspects of any of the scenarios, administration of a sulfur amino acid-rich diet increases the number and / or activation of cDC1 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400%, or more, compared to a subject not receiving a sulfur amino acid-rich diet. In some aspects of any of the scenarios, the cDC1 number is measured by quantifying the number of CD103+CD11b− cells from the population of MHCII+CDC11c+ dendritic cells using flow cytometry (see, e.g., FIGS. 8E, 30D for gating strategies for dendritic cells expressing CD103 / CD11b). In some aspects of any of the scenarios, cDC1 activation can be measured by quantifying the number of cDC1 cells that have migrated from the intestine (e.g., lamina propria) into the tumor-inflow region lymph nodes. In some aspects of any of the scenarios, cDC1 activation by XCL1 can be measured by quantifying the RNA or protein expression of XCR1. Additional non-limiting examples of activation markers for cDC1 include Clec9a and Irf8.

[0198] In some aspects of any of the scenarios, administration of a diet rich in sulfur-containing amino acids (or a supplement containing SAA) increases the number and / or activation of CD8+ T cells by at least 50% (see, e.g., FIGS. 3B-3E). In some aspects of any of the scenarios, administration of a diet rich in sulfur-containing amino acids increases the number and / or activation of CD8+ T cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400%, or more, compared to a subject not receiving a diet rich in sulfur-containing amino acids. In some aspects of any of the scenarios, the number of CD8+ T cells is quantified by flow cytometry in tumor-inflow region lymph nodes or in tumors. In some aspects of any of the scenarios, activation of CD8+ T cells is quantified by measuring an increase in the expression of effectors such as IFNγ or GZMB, or by measuring a decrease in the expression of immune checkpoint receptors such as PD-1, LAG-3, TIM-3, or CTLA-4 (for gating strategies for the expression of CD8+ T cells and their immune checkpoint receptors and their IFNγ and GZMB expression, see, e.g., FIGS. 8B, 30B).

[0199] Method of XCL1 polypeptide or XCR1 agonist In one aspect, a method of treating cancer, the method comprising administering to a subject in need thereof an XCL1 polypeptide (see, e.g., SEQ ID NOs: 5-6), is described herein. In one aspect, a method of treating cancer, the method comprising administering to a subject in need thereof an XCR1 agonist, is described herein. In one aspect, a method of treating cancer, the method comprising administering to a subject in need thereof an XCL1 polypeptide and an XCR1 agonist, is described herein. In one aspect, a method of treating cancer, the method comprising administering to a subject in need thereof a microorganism engineered to express an XCL1 polypeptide, is described herein. In some embodiments of any of the aspects, the cancer is colon cancer.

[0200] X-C motif chemokine receptor 1 (XCR1) is a receptor for XCL1 and XCL2 (lymphotactin-1 and lymphotactin-2, respectively). XCR1 can also be referred to as CCXCR1 or G protein-coupled receptor (GPR5). XCR1 is a chemokine receptor belonging to the G protein-coupled receptor superfamily. Family members are characterized by the presence of seven transmembrane domains and a number of conserved amino acids. XCR1 can be expressed on dendritic cells such as cDC1 cells. Cross-presenting dendritic cells (DCs) in the spleen develop in the small intestine, the T cell area of Peyer's patches, and the T cell area and sinus of the mesenteric lymph nodes and become XCR1+ DCs. XCR1+ DCs are specialized in the cross-presentation of orally applied antigens.

[0201] In some aspects of any of the situations, XCR1 comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 7 or SEQ ID NO: 8, or that maintains its function (e.g., binding to XCL1 and / or intracellular signaling associated with XCR1).

[0202] SEQ ID NO: 7, Chemokine XC Receptor 1, Homo sapiens, NCBI Reference Sequence: NP_001019815.1, 333 aa TIFF2025524662000020.tif36160

[0203] SEQ ID NO: 8, Chemokine XC Receptor 1, Mus musculus, NCBI Reference Sequence: NP_035928.2, 338 aa TIFF2025524662000021.tif36160

[0204] In some aspects of any of the situations, the XCR1 agonist is a functional variant of XCL1, such as human XCL1 or murine XCL1. In some aspects of any of the situations, the XCR1 agonist is a functional variant of XCL1 that further comprises at least one additional disulfide bridge (e.g., at least two residues are mutated to cysteine). In some aspects of any of the situations, the XCR1 agonist comprises at least one of the following mutations: V21C, A59C, V59C, T10C mutation, and / or the addition of the "AC" dipeptide at residue 32, and is a functional variant of XCL1 (e.g., SEQ ID NO: 5, SEQ ID NO: 6, residues 22-114 of SEQ ID NO: 5, residues 22-93 of SEQ ID NO: 5, residues 22-114 of SEQ ID NO: 6).

[0205] In some aspects of any of the situations, the XCR1 agonist comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NO: 5, SEQ ID NO: 6, residues 22-114 of SEQ ID NO: 5, residues 22-93 of SEQ ID NO: 5, residues 22-114 of SEQ ID NO: 6, or residues 22-93 of SEQ ID NO: 6 and maintains its function (e.g., binding to XCR1 and / or activation of XCR1).

[0206] In some aspects of any of the scenarios, the XCR1 agonist is mXCL1-V21C / A59C, a highly active form of mXCL1 that includes the V21C and A59C mutations (see, e.g., SEQ ID NO: 9). In some aspects of any of the scenarios, the XCR1 agonist is CC1 Ltn or CC3 Ltn, which includes at least one additional disulfide bond in hXCL to limit XCL1 to a chemokine-like structure having XCR1 agonist activity (see, e.g., SEQ ID NOs: 10-11). In some aspects of any of the scenarios, the XCR1 agonist is selected from the group consisting of mXCL1-V21C / A59C, CC1 Ltn, and CC3 Ltn; see, e.g., Matsuo et al., Front Immunol. 2018, 9: 2775; Tuinstra et al., Biochemistry 2007, 46(10): 2564-73, the contents of each of which are hereby incorporated by reference in their entirety. In some aspects of any of the scenarios, the XCR1 agonist is selected from the group consisting of SEQ ID NOs: 9-11.

[0207] In some aspects of any of the scenarios, the XCR1 agonist comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 9-11 and that maintains its function (e.g., binding to XCR1 and / or activation of XCR1).

[0208] SEQ ID NO: 9, mXCL1-V21C / A59C, 93 aa, the bold underlined text indicates the V21C and A59C mutations compared to residues 22-114 of mXCL1 (see, e.g., SEQ ID NO: 6). TIFF2025524662000022.tif10159

[0209] SEQ ID NO: 10, CC1 Ltn, 95 aa. The bold underlined text indicates the T10C mutation and the addition of the "AC" dipeptide at residue 32 compared to residues 22 - 114 of hXCL1 (see, for example, SEQ ID NO: 5). TIFF2025524662000023.tif10161

[0210] SEQ ID NO: 11, CC3 Ltn, 93 aa. The bold underlined text indicates the V21C and V59C mutations compared to residues 22 - 114 of hXCL1 (see, for example, SEQ ID NO: 5). TIFF2025524,662,000,024.tif10160

[0211] In some aspects of any of the aspects, the XCL1 polypeptide is administered to the gastrointestinal tract. In some aspects of any of the aspects, the XCR1 agonist is administered to the gastrointestinal tract. In some aspects of any of the aspects, the XCL1 polypeptide is administered using bacteria engineered to express the XCL1 polypeptide and / or the XCR1 agonist (e.g., commensal gastrointestinal bacteria; e.g., Lactobacillus). In some aspects of any of the aspects, the method further comprises administering at least a second composition as described herein in addition to the XCL1 polypeptide or the XCR1 agonist (see, for example, Table 10).

[0212] In some aspects of any of the scenarios, administration of an XCL1 polypeptide or an XCR1 agonist increases the number and / or activation of cDC1 by at least 50% in, for example, tumor-draining regional lymph nodes. In some aspects of any of the scenarios, administration of an XCL1 polypeptide or an XCR1 agonist increases the number and / or activation of cDC1 by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400%, or more, compared to a subject not administered an XCL1 polypeptide or an XCR1 agonist. In some aspects of any of the scenarios, the number of cDC1 is measured by quantifying the number of CD103+CD11b− cells from a population of MHCII+CDC11c+ dendritic cells using flow cytometry (see, for example, FIGS. 8E and 30D for gating strategies for dendritic cells expressing CD103 / CD11b). In some aspects of any of the scenarios, cDC1 activation can be measured by quantifying the number of cDC1 cells that have migrated from the intestine (e.g., lamina propria) into tumor-draining regional lymph nodes. In some aspects of any of the scenarios, cDC1 activation by XCL1 can be measured by quantifying the RNA or protein expression of XCR1. Additional non-limiting examples of activation markers for cDC1 include Clec9a and Irf8.

[0213] In some aspects of any of the situations, administration of an XCL1 polypeptide or an XCR1 agonist increases the number and / or activation of CD8+ T cells by at least 50%. In some aspects of any of the situations, administration of an XCL1 polypeptide or an XCR1 agonist increases the number and / or activation of CD8+ T cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400%, or more, compared to a subject not administered the XCL1 polypeptide or the XCR1 agonist. In some aspects of any of the situations, the number of CD8+ T cells is quantified by flow cytometry in tumor draining lymph nodes or in tumors. In some aspects of any of the situations, the activation of CD8+ T cells is quantified by measuring an increase in the expression of effectors such as IFNγ or GZMB, or by measuring a decrease in the expression of immune checkpoint receptors such as PD-1, LAG-3, TIM-3, or CTLA-4 (see, for example, FIGS. 8B and 30B for gating strategies for the expression of CD8+ T cells and their immune checkpoint receptors and their expression of IFNγ and GZMB).

[0214] Treatment stratification method In multiple situations, methods of treatment stratification related to the detection of M. schaedleri are described herein. In one situation, a method of treating cancer in a subject in need thereof, the method comprising: detecting the level of M. schaedleri in a sample from the subject; administering a cancer immunotherapeutic agent if the level of M. schaedleri is above a predetermined threshold; and administering a cancer immunotherapeutic agent and a Musispirillum composition as described herein if the level of M. schaedleri is below a predetermined threshold.

[0215] In one aspect, a method of treating cancer in a subject in need thereof, the method comprising: obtaining a result from an assay that detects the level of M. sheidleri in a sample from the subject; administering a cancer immunotherapeutic agent if the level of M. sheidleri is equal to or greater than a predetermined threshold; and administering a cancer immunotherapeutic agent and a Musispirillum composition as described herein if the level of M. sheidleri is less than the predetermined threshold, is described herein.

[0216] In one aspect, a method of treating cancer in a subject in need thereof, the method comprising: detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 in a sample from the subject; administering a cancer immunotherapeutic agent if the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 is equal to or greater than a predetermined threshold; and administering a cancer immunotherapeutic agent and a Musispirillum composition as described herein if the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 is less than the predetermined threshold, is described herein.

[0217] In one aspect, a method of treating cancer in a subject in need thereof, the method comprising: obtaining a result from an assay that detects the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 in a sample from the subject; administering a cancer immunotherapeutic agent if the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 is equal to or greater than a predetermined threshold; and administering a cancer immunotherapeutic agent and a Musispirillum composition as described herein if the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 is less than the predetermined threshold, is described herein.

[0218] In one aspect, a method for stratifying a subject for cancer treatment, the method comprising: detecting the level of M. schaedleri in a sample derived from the subject; and classifying the subject as at high risk (e.g., for symptoms or complications of colorectal cancer) if the level of M. schaedleri is less than a predetermined threshold; or classifying the subject as at low risk (e.g., for symptoms or complications of colorectal cancer) if the level of M. schaedleri is greater than or equal to the predetermined threshold, is described herein.

[0219] In one aspect, a method for stratifying a subject for cancer treatment, the method comprising: obtaining a result from an assay that detects the level of M. schaedleri in a sample derived from the subject; and classifying the subject as at high risk (e.g., for symptoms or complications of colorectal cancer) if the level of M. schaedleri is less than a predetermined threshold; or classifying the subject as at low risk (e.g., for symptoms or complications of colorectal cancer) if the level of M. schaedleri is greater than or equal to the predetermined threshold, is described herein.

[0220] In one aspect, a method for stratifying a subject for cancer treatment, the method comprising: detecting the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 in a sample derived from the subject; and classifying the subject as at high risk (e.g., for symptoms or complications of colorectal cancer) if the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 are less than a predetermined threshold; or classifying the subject as at low risk (e.g., for symptoms or complications of colorectal cancer) if the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 are greater than or equal to the predetermined threshold, is described herein.

[0221] In one aspect, a method of stratifying a subject for cancer treatment, the method comprising obtaining a result from an assay that detects the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 in a sample from the subject; and classifying the subject as at high risk (e.g., for symptoms or complications of colorectal cancer) if the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 is below a predetermined threshold; or classifying the subject as at low risk (e.g., for symptoms or complications of colorectal cancer) if the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 is at or above a predetermined threshold, is described herein.

[0222] In some embodiments of any of the aspects, the subject has colorectal cancer. In some embodiments of any of the aspects, the stratification method further comprises administering a Musispirillum composition as described herein. In some embodiments of any of the aspects, the stratification method further comprises administering a sulfur-containing amino acid. In some embodiments of any of the aspects, the stratification method further comprises administering a diet rich in sulfur-containing amino acids. In some embodiments of any of the aspects, the stratification method further comprises administering a Musispirillum composition as described herein, a diet rich in sulfur-containing amino acids (SAA), and / or an XCL1 polypeptide (or XCR1 agonist), or any combination thereof (see, e.g., Table 10).

[0223] In some aspects of any of the scenarios, the cancer immunotherapy agent includes an immune checkpoint inhibitor. In some aspects of any of the scenarios, the stratification method further includes the step of administering an immune checkpoint inhibitor, and non-limiting examples thereof are provided herein. In some aspects of any of the scenarios, the cancer immunotherapy agent is selected from the group consisting of an immune checkpoint inhibitor; chemotherapy; dendritic cell vaccine; chimeric antigen receptor T cell (CAR-T); and NKT cell-based therapy. See, for example, Nelson, et al. Cancers vol. 13, 20 5174. 15 Oct. 2021, the content of which is incorporated herein by reference in its entirety.

[0224] In some aspects of any of the scenarios, the treatments described herein (e.g., a Musispirillum composition as described herein, a diet rich in sulfur-containing amino acids (SAA), and / or an XCL1 polypeptide (or XCR1 agonist)) are administered as monotherapy, e.g., no other treatment for cancer is administered to the subject. In some aspects of any of the scenarios, the methods described herein can further include, for example, the step of administering a second agent and / or treatment to the subject as part of a combination therapy.

[0225] Non-limiting examples of the second agent and / or treatment include radiation therapy, surgery, gemcitabine, cisplatin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as banzodopa, carbocone, meturedopa, and uredopa; ethyleneimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bratasin and bratasinone); camptothecin (including the synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs KW-2189 and CB1-TM1); eribulin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, noburemabicin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma 1I and calicheamicin omega I1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein engyin antibiotics chromophore), actinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, cardinophyllin, chromomycin, daunorubicin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, keramycin, rhodomycin, streptomycin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin and other antibiotics; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimethoprim; purine analogs such as fludarabine, 6-mercaptopurine, thiampurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, drostanolone propionate, epithioestanol, mepitiostane, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; dexamethasone; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocin; mitoguazone;Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Phenamet; Pirarubicin; Losoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Oreg.); Razoxane; Lysoxine; Sizofran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2',2''-Trichlorotriethylamine; Trichothecene (especially T-2 toxin, Verracurin A, Loridin A, and Anguizine); Urethane; VinDesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gasitocin; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, such as TAXOL (registered trademark) Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE (registered trademark) albumin-engineered nanoparticle formulation of Paclitaxel without Cremophor (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE (registered trademark) Docetaxel (Rhone-Poulenc Rorer, Antony, France); Chlorambucil; GEMZAR (registered trademark) Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs such as Cisplatin, Oxaliplatin, and Carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; NAVELBINE (registered trademark) Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11) (including treatment regimens of Irinotecan with 5-FU and Leucovorin); Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoids such as Retinoic acid; Capecitabine; Combretastatin; Leucovorin (LV);Oxaliplatin; lapatinib (Tykerb®); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing, can be included in a cancer therapy selected from the group consisting of oxaliplatin treatment regimens (FOLFOX).;

[0226] One of ordinary skill in the art can readily identify useful chemotherapeutic agents (see, e.g., Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abeloff's Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

[0227] In addition, the method of treatment can further include the use of radiation or radiotherapy. Further, the method of treatment can further include the use of surgical procedures.

[0228] In some aspects of any of the situations, the level of M. schaedleri is quantified using standard methods of detecting bacteria, including but not limited to quantitative 16S sequencing, serial dilution plate assays, direct counting by optical microscope chambers, etc. In some aspects of any of the situations, the level of the XCL1 polypeptide is quantified using standard methods of detecting polypeptides, including but not limited to ELISA (enzyme-linked immunosorbent assay), Western blot, immunoprecipitation, or immunofluorescence using a detection reagent such as an antibody or protein binder. In some aspects of any of the situations, the level of NKT and / or CD103+ cDC1 is quantified using standard methods of detecting immune cells, including but not limited to flow cytometry on blood or tissue samples, or laser capture microdissection, immunohistochemistry, or immunofluorescence on tissue samples.

[0229] In some aspects of any of the situations, the stratification method results in a higher treatment efficacy as compared to a method of treating that does not initially involve the step of detecting the level of M. schaedleri, the step of obtaining results from an assay for detecting the level of M. schaedleri, the step of detecting the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1, or the step of obtaining results from an assay for detecting the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1.

[0230] In some aspects of any of the situations, the stratification method results in treatment efficacy that is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400%, or more, compared to a method of treatment that does not initially involve detecting the level of M. shadeleli, obtaining results from an assay for detecting the level of M. shadeleli, detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1, or obtaining results from an assay for detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1.

[0231] In some aspects of any of the situations, the stratification method results in higher treatment efficacy compared to a method of treatment that does not initially involve stratifying the subject. In some aspects of any of the situations, the stratification method results in treatment efficacy that is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400%, or more, compared to a method of treatment that does not initially involve stratifying the subject.

[0232] In some aspects of any situation, the stratification method results in fewer treatment complications compared to a method of treatment that does not initially involve the step of detecting the level of M. schaedleri, the step of obtaining results from an assay for detecting the level of M. schaedleri, the step of detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1, or the step of obtaining results from an assay for detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1.

[0233] In some aspects of any situation, the stratification method results in treatment complications that are at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 400%, or more reduced compared to a method of treatment that does not initially involve the step of detecting the level of M. schaedleri, the step of obtaining results from an assay for detecting the level of M. schaedleri, the step of detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1, or the step of obtaining results from an assay for detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1.

[0234] In some aspects of any scenario, the stratification method results in fewer treatment complications compared to a method of treatment that does not initially involve stratifying the subject. In some aspects of any scenario, the stratification method results in at least a 5%, at least a 10%, at least a 15%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least an 80%, at least a 90%, at least a 100%, at least a 150%, at least a 200%, at least a 300%, or at least a 400%, or more reduction in treatment complications compared to a method of treatment that does not initially involve stratifying the subject.

[0235] Non-limiting examples of complications of cancer treatments (e.g., chemotherapy, radiation) that can be reduced using the methods of treatment or stratification as described herein include anemia; anorexia; bleeding and bruising (e.g., thrombocytopenia); constipation; delirium; diarrhea; edema; fatigue; problems with male and female reproductive ability; influenza-like symptoms; hair loss (e.g., alopecia); infections and neutropenia; lymphedema; problems with memory or concentration; mouth and throat problems; nausea and vomiting; nerve problems (e.g., peripheral neuropathy); immunotherapy and organ-related inflammation; pain; male sexual health problems; female sexual health problems; skin and nail changes; sleep problems; or urinary and bladder problems.

[0236] Administration In some aspects, the methods described herein relate to treating a subject having or diagnosed with cancer with a Musispirillum composition, a diet rich in sulfur-containing amino acids (SAA), and / or an XCL1 polypeptide (or XCR1 agonist) as described herein. A subject having cancer can be identified by a physician using current methods of diagnosing cancer.

[0237] Cancer symptoms and / or complications that characterize these conditions and assist in diagnosis are well known in the art and include fatigue or extreme tiredness that does not improve with rest; unexplained weight loss or gain of more than 10 pounds; eating problems such as not feeling full; difficulty swallowing, abdominal pain, or nausea and vomiting; swelling or lumps in any part of the body; thickening or lumps in the breast or other parts of the body; pain, especially new or unexplained; that does not go away or gets worse; bleeding or scaly lumps, new moles or changes in moles, non-healing sores, or skin changes such as yellowing of the skin or eyes (e.g., jaundice); persistent cough or hoarseness; abnormal bleeding or bruising of unknown cause; persistent constipation or diarrhea, or changes in the appearance of stools, such as changes in bowel habits; pain during urination, blood in the urine, or more or less frequent need to urinate, such as changes in the bladder; fever or night sweats; headache; vision or hearing problems; changes in the mouth, including but not limited to mouth sores, bleeding, pain, or numbness.

[0238] Colorectal cancer symptoms and / or complications that characterize these conditions and assist in diagnosis are well known in the art and include persistent changes in bowel habits, including diarrhea or constipation or changes in the firmness of stools; rectal bleeding or bloody stools; persistent abdominal discomfort, such as cramps, gas, or pain; a feeling that the bowel does not completely empty; weakness or fatigue; or unexplained weight loss, but are not limited to these.

[0239] For example, tests that can assist in the diagnosis of colorectal cancer include, but are not limited to, colonoscopy, sigmoidoscopy, biopsy of the colon or rectum, stool tests (e.g., Cologuard®), genetic tests (e.g., changes in the KRAS, NRAS, or BRAF genes; microsatellite instability (MSI); changes in any of the mismatch repair (MMR) genes (MLH1, MSH2, MSH6, and PMS2); changes in the EPCAM gene), CT-guided needle biopsy, ultrasound, MRI, and PET scan. Exposure to a family history of colorectal cancer, or risk factors for colorectal cancer (e.g., lack of regular physical activity; diet low in fruits and vegetables; diet low in fiber and high in fat, or high in processed meat; overweight and obesity; alcohol intake; or smoking) can also help determine whether a subject is at high risk of having colorectal cancer or assist in the diagnosis of colorectal cancer.

[0240] The compositions and methods described herein can be administered to a subject having cancer (e.g., colorectal cancer, also referred to herein as colon cancer) or diagnosed with cancer. In some embodiments, the methods described herein include administering to the subject a composition described herein, such as a Musispirillum composition as described herein, a diet rich in sulfur amino acids (SAA), and / or an effective amount of an XCL1 polypeptide (or XCR1 agonist) to alleviate the symptoms of cancer (e.g., colorectal cancer). As used herein, "alleviating the symptoms of cancer" means improving any condition or symptom associated with cancer (e.g., colorectal cancer). Such reduction, when compared to an equivalent untreated control, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%, or more, measured by any standard technique. Various means for administering the compositions described herein to a subject are known to those of skill in the art. Such methods can include, but are not limited to, oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, dermal, topical, injection, or intratumoral administration. Administration can be local or systemic.

[0241] As used herein, the term "effective amount" refers to the amount of a Musispirillum composition, a sulfur amino acid (SAA)-rich diet, and / or an XCL1 polypeptide (or XCR1 agonist) as described herein that is required to alleviate at least one or more symptoms of a disease or disorder, and relates to the amount of a pharmacological composition sufficient to provide the desired effect. Thus, the term "therapeutically effective amount" refers to the amount of a Musispirillum composition, a sulfur amino acid (SAA)-rich diet, and / or an XCL1 polypeptide (or XCR1 agonist) as described herein that is sufficient to provide a particular anti-cancer effect when administered to a typical subject. An effective amount as used herein also includes, in various contexts, an amount sufficient to delay the onset of symptoms of a disease, alter the course of a symptomatic disease (e.g., but not limited to, slow the progression of symptoms of a disease), or reverse the symptoms of a disease. Thus, it is generally not possible to specify an exact "effective amount". However, in any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0242] The effective amount, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage will vary depending upon the dosage form employed and the route of administration utilized. The dosage ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions and methods showing a large therapeutic index are preferred. The therapeutically effective dose can first be estimated from cell culture assays. Additionally, the dosage can be formulated in animal models to achieve a circulating plasma concentration range that includes, for example, the IC50 (i.e., the concentration of a Musispirillum genus composition, a sulfur amino acid (SAA)-rich diet, and / or an XCL1 polypeptide (or XCR1 agonist) as described herein that achieves inhibition of the maximum half of the symptoms) as measured in cell culture or in a suitable animal model. Levels in plasma or the gastrointestinal tract can be measured, for example, by high performance liquid chromatography. The effect of any particular dosage can be monitored by appropriate bioassays, for example, assays for levels of, inter alia, Musispirillum genus bacteria, XCL1 polypeptide, NKT, and / or CD103+ cDC1. The dosage is determined by the physician and can be adjusted as required to suit the observed effects of the treatment.

[0243] A pharmaceutical composition comprising a Musispirillum composition, a sulfur-containing amino acid (SAA), and / or an XCL1 polypeptide (or XCR1 agonist) as described herein can also be formulated, for example, as tablets (including, without limitation, scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as, without limitation, solutions or suspensions in syrups, elixirs, aqueous liquids, non-aqueous liquids, water-in-oil emulsions, or oil-in-water emulsions, etc., as individual dosage forms suitable for oral administration. Such compositions contain a predetermined amount of a pharmaceutically acceptable salt of the disclosed compounds and can be prepared by well-known pharmaceutical methods well known to those of skill in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).

[0244] In certain embodiments, an effective dose of a Musispirillum composition as described herein, a diet rich in sulfur - containing amino acids (SAA), and / or a composition comprising an XCL1 polypeptide (or XCR1 agonist) as described herein can be administered to a patient once. In certain embodiments, an effective dose of a Musispirillum composition as described herein, a diet rich in sulfur - containing amino acids (SAA), and / or a composition comprising an XCL1 polypeptide (or XCR1 agonist) as described herein can be repeatedly administered to a patient. For systemic administration, a therapeutically effective amount of a composition comprising a Musispirillum composition, sulfur - containing amino acids (SAA), and / or an XCL1 polypeptide (or XCR1 agonist) as described herein, e.g., 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or more, can be administered to the subject.

[0245] In some embodiments, after the initial treatment regimen, the treatment can be administered at a less frequent rate. For example, after bi - weekly treatment for 3 months, the treatment can be repeated monthly for 6 months or 1 year or more. Treatment according to the methods described herein can reduce the level of a marker or symptom of a condition, e.g., cancer (e.g., colorectal cancer), by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, or more.

[0246] The dosage of the composition as described herein is determined by the physician and can be adjusted as necessary to suit the observed effect of the treatment. Regarding the duration and frequency of the treatment, a skilled clinician typically monitors the subject to determine when the treatment is providing a therapeutic benefit and to decide whether to increase or decrease the dosage, increase or decrease the dosing frequency, discontinue the treatment, resume the treatment, or make other changes to the treatment regimen. The dosing schedule can vary from once a week to daily, depending on a number of clinical factors, such as the composition of the genus Musispirillum as described herein, a diet rich in sulfur-containing amino acids (SAA), and / or the subject's sensitivity to the XCL1 polypeptide (or XCR1 agonist). The desired dose or amount can be administered in a single dose or divided into sub-doses, e.g., 2 to 4 sub-doses, and administered over a period of time, e.g., at appropriate intervals throughout the day or other appropriate schedule. In some embodiments, the administration can be daily, one or more doses and / or treatments over a long-term period, e.g., a period of weeks or months. Examples of dosing and / or treatment schedules are administration once a day, twice a day, three times a day, or four or more times a day over a period of one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, or six months, or longer. The composition containing the genus Musispirillum as described herein, a diet rich in sulfur-containing amino acids (SAA), and / or the XCL1 polypeptide (or XCR1 agonist) can be administered over a period of time, such as a period of 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes.

[0247] The dosage ranges for administration of the compositions described herein, according to the methods described herein, depend, for example, on the mycospirillum genus compositions as described herein, a diet rich in sulfur-containing amino acids (SAA), and / or the form of the XCL1 polypeptide (or XCR1 agonist), its potency, and to the extent to which the symptoms, markers, or indicators of the conditions described herein are desired to be reduced, for example, the percentage of reduction desired for symptoms of tumors and / or cancer, or, for example, to the extent to which the levels of mycospirillum genus bacteria, XCL1 polypeptide, NKT, and / or CD103+ cDC1 are desired to be increased. The dosage should not be so large as to cause adverse side effects such as sepsis or autoimmunity. In general, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of ordinary skill in the art. The dosage can also be adjusted by the individual physician in the event of any complications.

[0248] For example, the efficacy of a Musispirillum composition, a diet rich in sulfur amino acids (SAA), and / or an XCL1 polypeptide (or XCR1 agonist) as described herein, in the treatment of a condition described herein or for inducing a response as described herein, can be determined by a skilled clinician. However, treatment is considered "effective treatment" as the term is used herein if one or more of the signs or symptoms of the condition described herein change in a beneficial manner, if other clinically acceptable symptoms improve or get better, or if a desired response is induced, for example, by at least 10% after treatment according to the methods described herein. Efficacy can be evaluated, for example, by markers, indicators, symptoms, and / or incidence rates of a condition treated according to the methods described herein, or any other measurable parameter appropriate, for example, levels of Musispirillum bacteria, XCL1 polypeptide, NKT, and / or CD103+ cDC1, for example, measuring cancer indicators such as tumor size, tumor growth, and / or tumor metastasis activity. Efficacy can also be measured by the individual not worsening (i.e., the progression of the disease has stopped), as evaluated by hospitalization or the need for medical intervention. Methods for measuring these indicators are known to those of skill in the art and / or are described herein. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or animals), (1) inhibiting the disease, for example, preventing the worsening of symptoms (e.g., pain or organ damage; e.g., cancer symptoms and / or complications as further described herein; e.g., colorectal cancer symptoms and / or complications as further described herein); or (2) reducing the severity of the disease, for example, causing regression of symptoms. An effective amount for the treatment of a disease means an amount that, when administered to a subject in need thereof, is sufficient to result in an effective treatment for that disease as the term is defined herein.The effectiveness of the agent can be determined by evaluating a physical indicator of a condition or desired response (e.g., an increase in the levels of bacteria of the genus Musispirillum, XCL1 polypeptide, NKT, and / or CD103+ cDC1; e.g., a decrease in cancer indicators such as tumor size, tumor growth, and / or tumor metastasis activity). Monitoring the effectiveness of administration and / or treatment by measuring any one of such parameters, or any combination of parameters, is well within the ability of one of ordinary skill in the art. The effectiveness can be evaluated in an animal model of the treatment of a condition described herein, e.g., cancer (e.g., colorectal cancer). When using an experimental animal model, the effectiveness of the treatment is demonstrated when a statistically significant change in a marker is observed, e.g., an increase in the levels of bacteria of the genus Musispirillum, XCL1 polypeptide, NKT, and / or CD103+ cDC1, e.g., a decrease in cancer indicators such as tumor size, tumor growth, and / or tumor metastasis activity).

[0249] In vitro and animal model assays are provided herein that enable the evaluation of a composition of the genus Musispirillum, a diet rich in sulfur amino acids (SAA), and / or a given dose of an XCL1 polypeptide (or XCR1 agonist) as described herein. By way of non-limiting example, the effects of a composition of the genus Musispirillum, a diet rich in sulfur amino acids (SAA), and / or a dose of an XCL1 polypeptide (or XCR1 agonist) as described herein can be evaluated by clinical trials in human volunteers. The effectiveness of a given combination of dosages can also be evaluated in animal models including, but not limited to, the mouse models further described herein (see, e.g., Example 1).

[0250] cancer As used herein, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide without control and have the potential to invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymphatic systems. There are several main types of cancer. Carcinomas are cancers that begin in the skin or in tissues that line or cover internal organs. Sarcomas are cancers that begin in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissues. Leukemias are cancers that start in hematopoietic tissues such as the bone marrow and produce large numbers of abnormal blood cells that enter the blood. Lymphomas and multiple myelomas are cancers that begin in cells of the immune system. Cancers of the central nervous system are cancers that begin in the tissues of the brain and spinal cord.

[0251] In some aspects of any of the aspects, the cancer is a primary cancer. In some aspects of any of the aspects, the cancer is a malignant cancer. As used herein, the term "malignant" refers to a cancer in which a group of tumor cells exhibits one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., penetration and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via lymph or blood). As used herein, the term "metastasize" refers to the spread of cancer from one part of the body to another. A tumor formed by the spread cells is called a "metastatic tumor" or a "metastasis". A metastatic tumor contains cells that are like the cells in the original (primary) tumor. As used herein, the terms "benign" or "non-malignant" refer to tumors that can grow larger but do not spread to other parts of the body. Benign tumors are self-limiting and typically do not invade or metastasize.

[0252] "Cancer cell" or "tumor cell" refers to an individual cell of a cancerous growth or tissue. A tumor generally refers to a swelling or lesion formed by the abnormal growth of cells that can be benign, pre-cancerous, or malignant. Most cancer cells form tumors, but some, such as leukemia, do not necessarily form tumors. For cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.

[0253] As used herein, the term "neoplasm" refers to any new and abnormal growth of tissue, e.g., an abnormal mass of tissue where the growth exceeds and is not in harmony with that of normal tissue. Thus, a neoplasm can be a benign neoplasm, a pre-cancerous neoplasm, or a malignant neoplasm.

[0254] A subject having cancer or a tumor is a subject in which objectively measurable cancer cells are present in the subject's body. This definition includes malignant, actively growing cancers, and potentially dormant tumors or micrometastases. Cancers that migrate from their original site and seed other vital organs can ultimately lead to the death of the subject through functional deterioration of the affected organs.

[0255] Examples of cancers include carcinomas, lymphomas, blastomas, sarcomas, leukemias, basal cell carcinomas, bile duct cancers; bladder cancers; bone cancers; brain and CNS cancers; breast cancers; peritoneal cancers; cervical cancers; choriocarcinomas; colorectal cancers; connective tissue cancers; digestive system cancers; endometrial cancers; esophageal cancers; eye cancers; head and neck cancers; gastric cancers (including gastrointestinal cancers); glioblastomas (GBM); liver cancers; hepatocellular carcinomas; intraepithelial neoplasms; kidney cancers or renal cancers; laryngeal cancers; leukemias; liver cancers; lung cancers (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung); lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma; melanomas; myelomas; neuroblastomas; oral cancers (e.g., lip, tongue, mouth, and pharynx); ovarian cancers; pancreatic cancers; prostate cancers; retinoblastomas; rhabdomyosarcomas; rectal cancers; respiratory system cancers; salivary gland cancers; sarcomas; skin cancers; squamous cell carcinomas; gastric cancers; testicular cancers; thyroid cancers; uterine or endometrial cancers; urinary system cancers; vulvar cancers; and other carcinomas and sarcomas; and B cell lymphomas (including low grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; large lesion NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myelogenous leukemia; and post-transplant lymphoproliferative disorder (PTLD), and also include, but are not limited to, birthmarks, edema (such as those associated with brain tumors), and abnormal blood vessel growth associated with Meigs syndrome.

[0256] "Cancer cell" refers to any cancerous, pre-cancerous, or transformed cell, in vivo, ex vivo, or in tissue culture, that has undergone a spontaneous or induced phenotypic change, not necessarily accompanied by the uptake of new genetic material. Transformation can result from the infection with a transforming virus and the uptake of new genomic nucleic acid, or the uptake of exogenous nucleic acid, but can also occur spontaneously or following exposure to carcinogens that mutate endogenous genes. Transformation / cancer is associated with, for example, morphological changes, cell immortalization, abnormal growth control, lesion formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor-specific markers, invasion or metastasis, and tumor growth in a suitable animal host such as a nude mouse.

[0257] Definitions For convenience, the meanings of some terms and phrases used in this specification, the examples, and the appended claims are provided below. Unless otherwise stated or implied by context, the following terms and phrases include the meanings provided below. Since the scope of the present invention is limited only by the claims, the definitions are provided to assist in explaining particular embodiments and are not intended to limit the claimed invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of an apparent conflict between the usage of a term in the art and its definition provided herein, the definition provided herein shall prevail.

[0258] As used herein, the terms "colonized" or "colonization" broadly refer to the presence of an in vivo microbiota in a mammalian organism, such as in the gastrointestinal tract or on the skin, without a perceptible change other than the presence of bacteria. In contrast to transient passage through the gastrointestinal tract, colonized microbiota become non-transiently (e.g., semi-permanently) established and / or proliferate in the gastrointestinal tract, e.g., in the GI lumen, or in association with the GI epithelial or mucus layer. "Colonized" or "colonization" can also refer to the presence of microbiota on food or environmental surfaces. The terms "colonization" and "colonized" are contrasted with the terms "infection" or "infected", which are generally understood to require a perceptible adverse change as part of their definition. "Colonization" and "colonized" can also refer to the presence of bacteria in or on a human or animal without a perceptible injury, change, or disease. "Colonization" and "colonized" may be associated with a benefit to a human or animal.

[0259] As used herein, the term "isolated" refers to a bacterium, or other entity or substance that is (1) separated from at least some of the components with which it was originally associated (whether in nature, such as in human feces, or in an experimental setting, such as in a Petri dish consisting of an artificial growth medium), and / or (2) produced, prepared, purified, and / or manufactured by human hand. An isolated bacterium, protein, metabolite, or combination thereof can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which it was originally associated. In some embodiments, an isolated bacterium, protein, metabolite, or combination thereof is greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components (such as other bacterial species). The terms "purify", "purifying", and "purified" refer to a bacterium or other material that is separated from at least some of the components with which it was originally associated (whether in nature or in an experimental setting) at the time it was first produced or generated, or at any time after its initial production, as would be recognized by one of ordinary skill in the art of bacterial culture or related arts (such as chemistry). A bacterium or bacterial population can be considered purified if it is isolated from the material or environment containing the bacterium or bacterial population at the time of production or after production, and a purified bacterium or bacterial population can contain up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or greater than about 90% of other materials and still be considered "isolated". In some embodiments, purified bacteria and bacterial populations are greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure.In the case of the bacterial compositions provided herein, one or more bacterial types present in the composition can be purified independently from one or more other bacteria that are produced in and / or present in the material or environment containing that bacterial type. In some embodiments, a bacterium or population of bacteria is "isolated" when it comprises a single bacterial strain. In some embodiments, such isolated bacteria can be mixed or administered with other isolated bacteria, for example, in a defined consortium of isolated bacteria.

[0260] As used herein, "probiotics" is understood to mean live microorganisms that, when administered in a reasonable amount, confer a health benefit on the host.

[0261] As used herein, "prebiotics" is understood to mean a component that may or may not confer a benefit on the host and that enables or promotes a specific change in the composition and / or activity of the microbiota, such as the gastrointestinal microbiota.

[0262] As used herein, "medical foods" are understood to mean foods that are formulated to be consumed or administered enterally under medical supervision and whose specific nutritional requirements are established by medical evaluation based on recognized scientific principles for the purpose of special dietary management of a disease or condition.

[0263] As used herein, a "supplement" (also referred to as a dietary supplement) is understood to mean an orally administered product intended to supplement someone's diet and containing one or more ingredients (such as vitamins, minerals, amino acids, isolated microorganisms or their products as described herein) that are not considered foods. By way of non-limiting example, a supplement can be in the form of a capsule, enteric-coated capsule, tablet, caplet, pill, troche, lozenge, powder, or granule.

[0264] The term "gut" is understood to refer to the human gastrointestinal tract, also known as the alimentary canal. The gut includes the mouth, pharynx, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum and colon), and rectum.

[0265] As used herein, "bacteria" is understood as a single bacterial cell of a given species.

[0266] The terms "decrease", "reduced", "reduction", or "inhibit" are all used herein to mean a statistically significant decrease in amount. In some embodiments, "reduce", "reduction", or "decrease", or "inhibit" typically means at least a 10% decrease compared to a reference level (e.g., in the absence of a given treatment or agent), for example, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more decrease. As used herein, "reduction" or "inhibition" does not include complete inhibition or reduction compared to the reference level. "Complete inhibition" is 100% inhibition compared to the reference level. The decrease can preferably be down to a level that is acceptable within the normal range, for example, for an individual without a given disorder.

[0267] The terms "increased", "increasing", "enhanced", or "activated" are used herein to mean an increase in a statistically significant amount. In some embodiments, the terms "increased", "increasing", "enhanced", or "activated" mean an increase of at least 10% compared to a reference level, for example, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to the reference level, or an increase up to and including 100%, or any increase between 10-100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold compared to the reference level, or any increase between 2-fold and 10-fold or more. In the context of a marker or symptom, "increase" means a statistically significant increase at such levels.

[0268] As used herein, "subject" means a human or an animal. Usually, the animal is a vertebrate such as a primate, rodent, livestock, or game animal. Primates include chimpanzees, squirrel monkeys, spider monkeys, and macaque monkeys, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as salmon, catfish, and trout. In some embodiments, the subject is a mammal, such as a primate, such as a human. The terms "individual", "patient", and "subject" are used interchangeably herein.

[0269] Preferably, the subject is a mammal. The mammal can be, but is not limited to, a human, non-human primate, mouse, rat, dog, cat, horse, or cow. Non-human mammals can be advantageously used as subjects for animal models of cancer (e.g., colorectal cancer). The subject can be male or female.

[0270] The subject has been previously diagnosed or identified as having a condition that requires treatment (e.g., cancer) or one or more complications associated with such a condition, and optionally, can be a subject who has already received treatment for cancer or one or more complications associated with cancer. Alternatively, the subject can also be a subject who has not been previously diagnosed as having cancer or having one or more complications associated with cancer. For example, the subject can be a subject who exhibits one or more risk factors for cancer or one or more complications associated with cancer, or a subject who does not exhibit risk factors.

[0271] A "subject in need of treatment" for a particular condition can be a subject who has the condition, is diagnosed as having the condition, or is at risk of developing the condition.

[0272] As used herein, the terms "protein" and "polypeptide" are used interchangeably to designate a series of amino acid residues connected to each other by peptide bonds between the α-amino and carboxy groups of adjacent residues. The terms "protein" and "polypeptide" refer to polymers of amino acids, including modified amino acids (e.g., phosphorylation, glycosylation, glycosylation, etc.) and amino acid analogs, regardless of their size or function. The terms "protein" and "polypeptide" are often used with respect to relatively large polypeptides, whereas the term "peptide" is often used with respect to small polypeptides, but the use of these terms in the art is partially overlapping. The terms "protein" and "polypeptide" are used interchangeably herein when referring to gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the foregoing.

[0273] In various aspects described herein, it is further contemplated that any variant (naturally occurring or not) of a particular polypeptide described, alleles, homologs, conservatively modified variants, and / or conservative substitution variants are included. With respect to amino acid sequences, one of ordinary skill in the art recognizes that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that change a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants," where the modification results in a substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles that are consistent with the present disclosure.

[0274] A given amino acid can be replaced by residues having similar physicochemical characteristics. For example, one aliphatic residue can be substituted for another (e.g., Ile, Val, Leu, or Ala for one another), or one polar residue can be substituted for another (e.g., between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, e.g., substitution of an entire region having similar hydrophobic characteristics, are well known. Polypeptides containing conservative amino acid substitutions can be tested to confirm that the desired activity, e.g., the activity and specificity of the native polypeptide or a reference polypeptide (e.g., XCL1), is retained.

[0275] Amino acids can be grouped according to the similarity in the properties of their side chains (A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, the naturally occurring residues can be grouped based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging one member of these classes for another. Specific conservative substitutions include, for example, from Ala to Gly or Ser; from Arg to Lys; from Asn to Gln or His; from Asp to Glu; from Cys to Ser; from Gln to Asn; from Glu to Asp; from Gly to Ala or Pro; from His to Asn or Gln; from Ile to Leu or Val; from Leu to Ile or Val; from Lys to Arg, Gln, or Glu; from Met to Leu, Tyr, or Ile; from Phe to Met, Leu, or Tyr; from Ser to Thr; from Thr to Ser; from Trp to Tyr; from Tyr to Trp; and / or from Phe to Val, Ile, or Leu.

[0276] In some embodiments, the polypeptides described herein (or nucleic acids encoding such polypeptides) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a fragment or segment of a polypeptide that retains at least 50% of the activity of the wild-type reference polypeptide. Functional fragments can include conservative substitutions of the sequences disclosed herein.

[0277] In some embodiments, the polypeptides described herein can be variants of the polypeptide sequences described herein. In some embodiments, the variants are conservatively modified variants. Conservatively substituted variants can be obtained, for example, by mutation of the native nucleotide sequence. As referred to herein, a "variant" is a polypeptide that is substantially homologous to the native or reference polypeptide, but has an amino acid sequence different from that of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. The DNA sequence encoding the variant polypeptide includes sequences that contain one or more additions, deletions, or substitutions of nucleotides when compared to the native DNA sequence or reference DNA sequence, but encode a protein or fragment thereof that retains the activity of the native or reference polypeptide. A variety of, for example, PCR-based, site-directed mutagenesis approaches are known in the art and can be applied by those skilled in the art to generate and test artificial variants.

[0278] The amino acid sequence or DNA sequence of the variant can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to the native sequence or reference sequence. The degree of homology (percent identity) between the native sequence and the variant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0279] Variant amino acid sequences can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more similar to the native or reference sequence. As used herein, "similarity" refers to the same amino acid or a conservatively substituted amino acid as described herein. Thus, the percentage of "sequence similarity" is the percentage of amino acids that are either identical or conservatively changed; for example, "sequence similarity" = (% sequence identity) + (% conservative changes). It should be understood that a sequence having a specified percent similarity to a reference sequence will necessarily include a sequence having the same specified percent identity to that reference sequence. Those skilled in the art will be aware of various computer programs that utilize different mathematical algorithms available for determining identity or similarity between two sequences. For example, a computer program that employs the Needleman and Wunsch algorithm (Needleman et al. (1970)); the GAP program in the Accelrys GCG software package (Accelerys Inc., San Dieso U.S.A.); the algorithm of E. Meyers and W. Miller incorporated in the ALIGN program (version 2.0) (Meyers et al. (1989)); or more preferably, BLAST (Basic Local Alignment Tool using default parameters) can be used; see, for example, U.S. Patent No. 10,023,890, the contents of which are incorporated herein by reference in their entirety.

[0280] In some embodiments, the sequencing includes 16S rRNA gene sequencing, which can also be referred to as "16S ribosomal RNA sequencing", "16S rDNA sequencing", or "16s rRNA sequencing". Sequencing of the 16S rRNA gene can be used in genetic studies because it is highly conserved among different species of bacteria but does not exist in eukaryotic species. In addition to the highly conserved regions, the 16S rRNA gene also contains nine hypervariable regions (V1 - V9) that vary by species. 16S rRNA gene sequencing typically involves using multiple universal primers that bind to the conserved regions of the 16S rRNA gene, PCR amplifying the bacterial 16S rRNA gene region (including the hypervariable regions), and sequencing the amplified 16S rRNA gene using next-generation sequencing techniques as described herein (see, e.g., U.S. Patent Nos. 5,654,418; 6,344,316; and 8,889,358; and U.S. Patent Application Nos. US 2013 / 0157265 and US 2018 / 0195111, which are also incorporated by reference in their entirety).

[0281] Modification of the native amino acid sequence can be achieved by any of a number of techniques known to those of skill in the art. Mutations can be introduced at specific loci, for example, by synthesizing oligonucleotides containing the mutant sequence flanked by restriction sites that allow ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes analogs having the desired amino acid insertions, substitutions, or deletions. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide modified nucleotide sequences having specific codons altered according to the required substitutions, deletions, or insertions. A variety of site-specific mutagenesis approaches, such as the Kunkel method, cassette mutagenesis, PCR site-specific mutagenesis (e.g., traditional PCR, primer extension, or inverse PCR), whole plasmid mutagenesis, in vivo site-specific mutagenesis, CRISPR / Cas guide mutagenesis are known in the art and can be applied by those of skill in the art to introduce mutations at specific nucleic acid loci.Techniques for making such modifications are very well established and include, for example, those disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Braman, Jeff, ed. (2002) In Vitro Mutagenesis Protocols, Methods in Molecular Biology, Vol. 182 (2nd ed.); Khudyakov and Fields (2002), Artificial DNA: Methods and Applications, CRC Press; Hsu et al. (2014), Cell 157 (6): 1262-78; Cerchione et al. (2020) PLOS ONE 15 (4): e0231716; and U.S. Patent Nos. 4,518,584 and 4,737,462, the entireties of which are incorporated herein by reference. Any cysteine residues not involved in maintaining the appropriate conformation of the polypeptide can also generally be substituted with serine in order to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bonds can be added to the polypeptide to improve its stability or promote oligomerization.

[0282] In some aspects, the methods described herein relate to measuring, detecting, or determining the level of at least one marker. As used herein, the terms "detecting" or "measuring" refer to observing, for example, a signal from a probe, label, or labeled molecule that indicates the presence of an analyte in a sample. Any method known in the art for detecting a particular labeled moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescence, photochemical, biochemical, immunochemical, electrical, optical, or chemical methods. In some aspects of any of the aspects, measuring can be a quantitative observation.

[0283] In some aspects of any of the aspects, a polypeptide, nucleic acid, or cell as described herein can be manipulated. As used herein, "manipulated" refers to a situation that is being manipulated by human hands. For example, a polypeptide is considered to be "modified" if at least one aspect of the polypeptide, such as its sequence, is being manipulated by human hands such that it is different from the aspect in which it occurs in nature. As is common practice and understood by those of skill in the art, the progeny of a manipulated cell are typically still referred to as "manipulated," even if the actual manipulation was performed on a previous entity.

[0284] As used herein, "treating", "treatment", "to treat", or "ameliorate" refers to a therapeutic treatment where the purpose is to reverse, alleviate, improve, inhibit, slow down, or stop the progression or severity of a disease or disorder, such as a cancer (e.g., colorectal cancer). The term "to treat" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder associated with cancer (e.g., colorectal cancer). A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of the disease is reduced or halted. That is, "treatment" includes not only an improvement in symptoms or markers compared to what would be expected in the absence of treatment, but also the halting, or at least slowing, of the progression or worsening of symptoms. Beneficial or desirable clinical outcomes include, but are not limited to, alleviation of one or more symptoms, whether detectable or not, diminution of the extent of the disease, stabilization of the disease state (i.e., does not worsen), delay or slowing of disease progression, improvement or temporary remission of the disease state, remission (partial or complete), and / or decrease in mortality. The term "treatment" of a disease also includes providing relief from the symptoms or side effects of the disease (including palliative treatment).

[0285] As used herein, the term "pharmaceutical composition" refers to an active agent in combination with a pharmaceutically acceptable carrier, such as carriers commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to mean that within the scope of sound medical judgment, the compound, material, composition, and / or dosage form is suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problems and complications, and is commensurate with a reasonable benefit / risk ratio. In some aspects of any of the aspects, the pharmaceutically acceptable carrier can be a carrier other than water. In some aspects of any of the aspects, the pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some aspects of any of the aspects, the pharmaceutically acceptable carrier can be an artificial or engineered carrier, such as a carrier in which the active ingredient is not found to occur naturally or to exist.

[0286] As used herein, the term "administering" refers to the placement into a subject by a method or route that results in at least partial delivery of the agent of a compound as disclosed herein to a desired site. A pharmaceutical composition comprising a compound disclosed herein can be administered by any suitable route that results in an effective treatment in a subject. In some aspects, administration includes human body activities, such as injection, ingestion, application, and / or manipulation of a delivery device or machine. Such activities can be performed, for example, by a medical professional and / or the subject being treated.

[0287] The terms "statistically significant" or "significantly" refer to statistical significance and generally mean a difference of two standard deviations (2SD) or more.

[0288] Unless otherwise indicated in an operating example or separately, all numerical values expressing the amounts of ingredients or reaction conditions used herein should be understood to be modified in all cases by the term "about". When used in connection with percentages, the term "about" can mean ±1%.

[0289] As used herein, the term "comprising" means that other elements can also be present in addition to the recited defined elements. The use of "comprising" indicates inclusion rather than limitation.

[0290] The term "consisting of" refers to a composition, method, and each of its components as described herein, excluding any element not recited in the description of that embodiment.

[0291] As used herein, the term "consisting essentially of" refers to the elements required for a given embodiment. The term allows for the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the invention.

[0292] As used herein, the term "corresponding" refers to an amino acid or nucleotide at the recited position in a first polypeptide or nucleic acid, or an amino acid or nucleotide equivalent to the recited amino acid or nucleotide in a second polypeptide or nucleic acid. Equivalent recited amino acids or nucleotides can be determined by alignment of candidate sequences using homology programs known in the art, such as BLAST.

[0293] The singular terms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the suitable methods and materials are described below. The abbreviation "e.g." is derived from the Latin exempli gratia and is used herein to indicate non-limiting examples. Thus, the abbreviation "e.g." is synonymous with the term "for example".

[0294] The grouping of alternative elements or aspects of the invention disclosed herein should not be construed as limiting. Members of each group can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in or deleted from the group for reasons of convenience and / or patentability. Any such inclusion or deletion, if it occurs, is considered herein to contain the group as so modified and thus to satisfy all the Markush group descriptions used in the appended claims.

[0295] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains. It is to be understood that the invention is not limited to the specific methodologies, protocols, and reagents, etc. described herein, and thus may vary. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention, which is defined only by the claims. Definitions of common terms in cell biology, immunology, and molecular biology are incorporated herein by reference in their entirety, from The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W.Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385); Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737).

[0296] Other terms are defined in the description of the various aspects of the invention herein.

[0297] All patents and other publications, including bibliographic references, issued patents, published patent applications, and co-pending patent applications, cited throughout this application are hereby expressly incorporated by reference herein for the purpose of explaining and disclosing, for example, the methodologies described in such publications that may be used in connection with the techniques described herein. These publications are provided only for disclosures prior to the filing date of this application. None of this is to be construed as an admission that the inventors have no right to antedate such disclosure by virtue of prior invention or any other reason. All statements as to the date or content of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0298] The description of aspects of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Specific aspects and examples of the disclosure are described herein for illustrative purposes, but as will be recognized by those of ordinary skill in the relevant art, various equivalent modifications are possible within the scope of the disclosure. For example, the steps or functions of a method are presented in a given order, but alternative aspects may perform the functions in a different order or the functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied as appropriate to other procedures or methods. Various aspects described herein can be combined to provide further aspects. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions, and concepts of the above references and applications to provide further aspects of the disclosure. Additionally, some changes can be made to the protein structure without affecting the type and amount of biological and chemical actions in order to account for biological equivalents. These and other changes can be made to the disclosure in view of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0299] Specific elements of any of the foregoing aspects can be combined with or replaced by elements in other aspects. Further, while advantages associated with certain aspects of the present disclosure are described in the context of these aspects, other aspects may also exhibit such advantages, and not all aspects necessarily need to exhibit such advantages to fall within the scope of the present disclosure.

[0300] Some aspects of the technology described herein can be defined according to any of the following numbered paragraphs.

[0301] 1. A composition comprising Mucispirillum schaedleri (M. schaedleri) bacteria formulated for delivery to the intestine. 2. The composition of paragraph 1, wherein the M. schaedleri bacteria are live or inactivated. 3. The composition of paragraph 1, wherein the M. schaedleri bacteria are in a dried viable form. 4. The composition of any one of paragraphs 1 to 3, wherein the M. schaedleri bacteria are encapsulated. 5. The composition of any one of paragraphs 1 to 4, wherein the M. schaedleri bacteria are contained in enteric capsules. 6. The composition of any one of paragraphs 1 to 5, wherein the M. schaedleri bacteria are maintained in an anaerobic state in the formulation. 7. The composition of any one of paragraphs 1 to 6, wherein the M. schaedleri bacteria are in a mixture with prebiotics. 8. The composition of any one of paragraphs 1 to 7, wherein the M. schaedleri bacteria are in a mixture with sulfur-containing amino acids. 9. The composition of paragraph 8, wherein the sulfur-containing amino acids are methionine, cysteine, or derivatives thereof. 10. The composition of any one of paragraphs 1 to 9, wherein the M. schaedleri bacteria are formulated in a food composition. 11. The composition of paragraph 10, wherein the food composition is supplemented with sulfur-containing amino acids and / or prebiotics. 12. The composition of any one of paragraphs 1 to 11, further comprising 1 to 20 additional bacteria. 13. The composition of any one of paragraphs 1 to 11, comprising 20 or fewer bacteria. 14. A composition comprising live M. chadreieri bacteria, dead M. chadreieri bacteria, a conditioned M. chadreieri culture medium, or an organic solvent extract or fraction thereof of the conditioned M. chadreieri culture medium, which promotes XCL1 secretion by NKT cells, and which is formulated for delivery to the intestine. 15. The composition of paragraph 14, wherein the M. chadreieri bacteria, medium, or solvent extract is in a dried form. 16. The composition of any one of paragraphs 14 or 15, wherein the M. chadreieri bacteria, medium, or extract is encapsulated. 17. The composition of any one of paragraphs 14 to 16, wherein the M. chadreieri bacteria are contained in enteric capsules. 18. The composition of any one of paragraphs 14 to 17, wherein the M. chadreieri bacteria are maintained in an anaerobic state in the formulation. 19. The composition of any one of paragraphs 14 to 18, wherein the M. chadreieri bacteria, medium, or extract is in a mixture with prebiotics and / or sulfur-containing amino acids or derivatives thereof. 20. The composition of any one of paragraphs 14 to 19, wherein the conditioned M. chadreieri culture medium, or an organic solvent extract or fraction thereof of the conditioned M. chadreieri culture medium, contains at least one metabolite selected from Figure 4H, Figure 4I, or Figure 16. 21. The conditioned M. chadreieri culture medium, or an organic solvent extract or fraction thereof of the conditioned M. chadreieri culture medium, contains succinic acid; propionic acid; nicotinic acid; aconitic acid (cis and / or trans); pentadecanoic acid; itaconic acid; 16-hydroxyhexadecanoic acid; C8H 15NO3S; crotonic acid; myristic acid; 17-hydroxyheptadecanoic acid (C 17 H 34 O3); and at least one metabolite selected from the group consisting of 15-hydroxypentadecanoic acid (C 15 H 30 O3), a composition according to any one of paragraphs 14 to 20. 22. An adjusted M. schaedleri culture medium, or an organic solvent extract or fraction thereof of the adjusted M. schaedleri culture medium, contains succinic acid; nicotinic acid; aconitic acid (cis and / or trans); pentadecanoic acid; itaconic acid; 16-hydroxyhexadecanoic acid; and at least one metabolite selected from the group consisting of crotonic acid, a composition according to any one of paragraphs 14 to 21. 23. A food composition comprising a composition according to any one of paragraphs 14 to 22. 24. The food composition according to paragraph 23, further comprising 1 to 20 additional bacteria. 25. A method of treating cancer or promoting antitumor immune activity, comprising administering to a subject in need thereof a composition according to any one of paragraphs 1 to 24. 26. The method according to paragraph 25, wherein the cancer is colon cancer. 27. The method according to paragraph 25 or 26, further comprising administering an immune checkpoint inhibitor. 28. A method of promoting responsiveness to immune checkpoint inhibitor tumor therapy, comprising administering to a subject in need thereof a composition according to any one of paragraphs 1 to 24. 29. The method according to paragraph 28, further comprising administering an immune checkpoint inhibitor. 30. The method according to paragraph 28 or 29, wherein the subject has colon cancer. 31. The method according to any one of paragraphs 28 to 30, wherein the cancer of the subject is determined to be resistant to immune checkpoint inhibitor therapy. 32. The method according to any one of paragraphs 28 to 31, wherein the composition promotes XCL1 secretion by NKT cells. 33. A method for increasing CD103+ conventional dendritic cells (cDC1), comprising the step of administering to a subject in need thereof a composition according to any one of paragraphs 1 to 24. 34. The method of paragraph 33, wherein the cDC1 is associated with a tumor. 35. The method of paragraph 34, wherein the tumor is colon cancer. 36. The method according to any one of paragraphs 33 to 35, further comprising the step of administering a sulfur-containing amino acid. 37. The method according to any one of paragraphs 33 to 36, further comprising the step of administering an immune checkpoint inhibitor. 38. A method for increasing XCL1 secretion by NKT cells, comprising the step of administering to a subject in need thereof a composition according to any one of paragraphs 1 to 24. 39. The method of paragraph 38, wherein the subject has cancer. 40. The method of paragraph 38 or 39, wherein the subject has colon cancer. 41. A method for increasing infiltration of CD8+ T cells in a colorectal tumor, comprising the step of administering to a subject in need thereof a composition according to any one of paragraphs 1 to 24. 42. The method of paragraph 41, wherein the cDC1 is associated with a tumor. 43. The method of paragraph 42, wherein the tumor is colon cancer. 44. The method according to any one of paragraphs 41 to 43, further comprising the step of administering a sulfur-containing amino acid. 45. The method according to any one of paragraphs 41 to 44, further comprising the step of administering an immune checkpoint inhibitor. 46. A method for increasing infiltration of CD8+ T cells in a colorectal tumor, comprising the step of administering to a subject in need thereof a diet rich in sulfur-containing amino acids (SAA) or a supplement containing SAA. 47. The method of paragraph 46, wherein the diet rich in sulfur-containing amino acids contains more than 0.04 grams of SAA per kilogram of body weight per day. 48. The method according to paragraph 46 or 47, further comprising the step of administering to the subject any one of the compositions of paragraphs 13 to 20. 49. A method for establishing or maintaining a tumor-suppressive gastrointestinal environment in a subject in need thereof, said method comprising the step of administering a diet rich in sulfur-containing amino acids (SAA) or a supplement containing SAA. 50. The method according to paragraph 49, wherein the diet rich in sulfur-containing amino acids or the supplement containing SAA contains more than 0.04 grams of SAA per kilogram of body weight per day. 51. The method according to paragraph 49 or 50, further comprising the step of administering to the subject any one of the compositions of paragraphs 14 to 21. 52. A method for treating cancer, said method comprising the step of administering an XCL1 polypeptide to a subject in need thereof. 53. The method according to paragraph 52, wherein the cancer is colon cancer. 54. The method according to paragraph 52 or 53, wherein the XCL1 polypeptide is administered to the gastrointestinal tract. 55. A method for treating cancer, said method comprising the step of administering to a subject in need thereof a microorganism engineered to express an XCL1 polypeptide. 56. A method for treating cancer, said method comprising the step of administering to a subject in need thereof an agonist of XCR1, which is an XCL1 receptor. 57. The method according to paragraph 56, wherein the XCR1 agonist comprises SEQ ID NOs: 9 to 11, or an amino acid sequence that is at least 95% identical and maintains its function. 58. A method for treating cancer in a subject in need thereof, detecting the level of M. schaedleri in a sample derived from the subject; administering a cancer immunotherapeutic agent when the level of M. schaedleri is equal to or higher than a predetermined threshold; and administering a cancer immunotherapeutic agent and any one of the compositions of paragraphs 1 to 24 when the level of M. schaedleri is less than the predetermined threshold. The method as described above, including 59. A method for treating cancer in a subject in need thereof, comprising: obtaining a result from an assay that detects the level of M. shadreiri in a sample derived from the subject; administering a cancer immunotherapeutic agent when the level of M. shadreiri is equal to or higher than a predetermined threshold; and administering a cancer immunotherapeutic agent and a composition according to any one of paragraphs 1 to 24 when the level of M. shadreiri is less than the predetermined threshold The method as described above, including 60. A method for treating cancer in a subject in need thereof, comprising: detecting the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 in a sample derived from the subject; administering a cancer immunotherapeutic agent when the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 are equal to or higher than a predetermined threshold; and administering a cancer immunotherapeutic agent and a composition according to any one of paragraphs 1 to 24 when the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 are less than the predetermined threshold The method as described above, including 61. A method for treating cancer in a subject in need thereof, comprising: obtaining a result from an assay that detects the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 in a sample derived from the subject; administering a cancer immunotherapeutic agent when the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 are equal to or higher than a predetermined threshold; and administering a cancer immunotherapeutic agent and a composition according to any one of paragraphs 1 to 24 when the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 are less than the predetermined threshold The method as described above, including 62. A method for stratifying a subject for cancer treatment, comprising: detecting the level of M. schaedleri in a sample derived from the subject; and when the level of M. schaedleri is less than a predetermined threshold, classifying the subject as high risk; or when the level of M. schaedleri is greater than or equal to a predetermined threshold, classifying the subject as low risk The method as described above. 63. A method for stratifying a subject for cancer treatment, comprising: obtaining a result from an assay for detecting the level of M. schaedleri in a sample derived from the subject; and when the level of M. schaedleri is less than a predetermined threshold, classifying the subject as high risk; or when the level of M. schaedleri is greater than or equal to a predetermined threshold, classifying the subject as low risk The method as described above. 64. A method for stratifying a subject for cancer treatment, comprising: detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 in a sample derived from the subject; and when the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 is less than a predetermined threshold, classifying the subject as high risk; or when the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 is greater than or equal to a predetermined threshold, classifying the subject as low risk The method as described above. 65. A method for stratifying a subject for cancer treatment, comprising: obtaining a result from an assay for detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1 in a sample derived from the subject; and If the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 are below a predetermined threshold, classifying the subject as high-risk; or If the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 are at or above a predetermined threshold, classifying the subject as low-risk The method as described above, comprising the above steps. 66. The method according to any one of paragraphs 58 to 65, wherein the subject has colon cancer. 67. The method according to any one of paragraphs 62 to 65, further comprising the step of administering a composition according to any one of paragraphs 1 to 24. 68. The method according to any one of paragraphs 58 to 67, further comprising the step of administering a sulfur-containing amino acid. 69. The method according to any one of paragraphs 58 to 61, wherein the cancer immunotherapeutic agent is selected from the group consisting of immune checkpoint inhibitors; chemotherapy; dendritic cell vaccines; chimeric antigen receptor T cells (CAR-T); and NKT cell-based therapies. 70. The method according to any one of paragraphs 58 to 61, wherein the cancer immunotherapeutic agent comprises an immune checkpoint inhibitor. 71. The method according to any one of paragraphs 62 to 65, further comprising the step of administering an immune checkpoint inhibitor. 72. The method according to any one of paragraphs 58 to 71, further comprising the step of administering a diet rich in sulfur-containing amino acids or a supplement containing SAA. 73. The method according to any one of paragraphs 58 to 61 or 66 to 69, which results in higher treatment efficacy compared to a treatment method not initially involving the step of detecting the level of M. schaedleri; obtaining a result from an assay for detecting the level of M. schaedleri; detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1; or obtaining a result from an assay for detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1. 74. A method according to any one of paragraphs 62-72, which results in higher treatment efficacy as compared to a treatment method not initially involving stratifying the subject. 75. A method according to any one of paragraphs 58-61 or 66-72, which results in fewer treatment complications as compared to a treatment method not initially involving the step of detecting the level of M. schaedleri, obtaining a result from an assay for detecting the level of M. schaedleri, the step of detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1, or obtaining a result from an assay for detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1. 76. A method according to any one of paragraphs 62-72, which results in fewer treatment complications as compared to a treatment method not initially involving stratifying the subject. 77. An enteric delivery formulation comprising at least one metabolite selected from FIG. 4H, FIG. 4I, or FIG. 16. 78. An enteric delivery formulation comprising at least one metabolite selected from the group consisting of succinic acid; propionic acid; nicotinic acid; aconitic acid (cis and / or trans); pentadecanoic acid; itaconic acid; 16-hydroxyhexadecanoic acid; C8H 15 NO3S; crotonic acid; myristic acid; 17-hydroxyheptadecanoic acid (C 17 H 34 O3); and 15-hydroxypentadecanoic acid (C 15 H 30 O3). 79. An enteric delivery formulation comprising at least one metabolite selected from the group consisting of succinic acid; nicotinic acid; aconitic acid (cis and / or trans); pentadecanoic acid; itaconic acid; 16-hydroxyhexadecanoic acid; and crotonic acid. 80. An enteric delivery formulation according to any one of paragraphs 77-79, formulated for delivery to the intestine.

[0302] The techniques described herein are further illustrated by the following examples, which should in no way be construed as being more limiting.

Example

[0303] Example 1: Diet - Microbial Interactions Enhance Anti - tumor Immunity in Colorectal Cancer Immune checkpoint inhibitors (ICIs) have transformed cancer treatment, but the response to ICIs is not universal, particularly for colorectal cancer (CRC). Certain members of the gut microbiota are associated with the response to ICI treatment in patients, but the mechanisms governing the microbial regulation of anti - tumor immunity remain poorly understood. It is contemplated herein that the metabolic capabilities of the microbiota, rather than a specific taxonomic composition, can better distinguish between ICI responders and ICI non - responders. Since the etiopathogenesis of CRC is associated with both the microbiota and dietary factors, the biochemistry affected by dietary factors was examined. The metabolic characteristics of the microbiota were examined in ICI - responsive patients. As described herein, the sulfur - amino acid (Saa) metabolic pathway and gene abundance of gut microbiota are positively correlated with ICI responsiveness, and a diet rich in Saa retarded tumor growth at a scale comparable to α - PD - 1 treatment in a xenograft mismatch repair - deficient CRC model. In a genetically engineered mouse model of mismatch - repair - proficient and ICI - resistant CRC, a high - Saa diet blunted neoplastic progression and promoted the expansion of the niche of the mucus - dwelling immunomodulatory species, Muris spiroplasma sheadleri, which is present in the colon of humans and mice. A high - Saa diet, studied herein in mice and achievable in humans via dietary supplementation, increased the secretion of XCL1 by NKT cells, expanded and activated cDC1 in tumor - draining regional lymph nodes, and ultimately tumor - infiltrating CD8 +By enhancing the number and function of T cells, M. schaedleri bloom was induced to turn "cold" CRC tumors into "warm" ones, reducing tumor growth. Collectively, diet-microbiota-host interactions are described herein, demonstrating a diet-based cancer therapy targeting the microbiota. A diet based on the microbiota analysis of immunotherapy-responsive patients that enhanced anti-tumor immunity in a CRC model is described herein in multiple aspects.

[0304] As further described herein, the abundance of sulfur amino acid (Saa) metabolism genes in the gastrointestinal microbiota correlates with improved anti-tumor immunity and ICI response, prompting testing of the effects of dietary Saa in a mouse model of CRC. A high-Saa diet retarded tumor growth in a xenograft CRC model, and such a diet increased CD8 + T cell infiltration and decreased neoplastic progression in a genetically driven CRC model. A high-Saa diet increased the abundance of Muris sp. strain Schaedleri, which increased the abundance and activation state of CD103 + conventional dendritic cells (cDC1) in the tumor-draining lymph node (TDLN) and enhanced the anti-tumor immune response. M. schaedleri-conditioned medium (CDM) induced XCL1 production by natural killer T (NKT) cells in vitro, indicating a direct causal effect of M. schaedleri on this cDC1 activation pathway. Thus, signatures that regulate diet-microbiota-host interactions and anti-tumor immunity are described herein, providing a framework for identifying and evaluating diet-based cancer adjuvant therapies targeting the microbiota.

[0305] Saa biosynthesis genes were enriched in ICI-responsive patients To quantify the microbial pathways enriched in the microbiome of ICI responders, a series of analyses were performed on the microbial metabolic capabilities in fecal metagenomics datasets from ICI responders versus non-responders in eight ICI microbiome studies; see, for example, Andrews et al. 2021, supra; Frankel et al. 2017, supra; Gopalakrishnan et al., 2018, supra; Lee et al. 2021, supra; Matson et al. 2018, supra; Routy et al. 2018, supra; Baruch et al., Science 371, 602-609 (2021); Davar et al., Science 371, 595-602 (2021), each of which is incorporated herein by reference in its entirety. The initial study focused on a cohort of patients with ICI-treated renal cell carcinoma (RCC), as this patient population has been reported to have less antibiotic (Abx) use compared to patients with non-small cell lung cancer (NSCLC) and melanoma, and Abx may act as additional modifiers of the microbiome and ICI response. Functional profiling from HUMAnN 2 (see, for example, Franzosa et al., Nat Methods 15, 962-968 (2018), which is incorporated herein by reference in its entirety; see, for example, Supplementary Methods) was used with fecal metagenomes from the RCC cohort (see, for example, Routy et al., 2018, supra), and individuals who met the criteria of the Response Evaluation Criteria in Solid Tumors (RECIST) for partial or complete response were selected, excluding those with stable disease. Profiles from these samples (n = 16) were compared to profiles from RCC patients who were ICI non-responders (n = 32) by least-squares linear model-based differential abundance analysis.

[0306] The MetaCyc superpathway for Saa biosynthesis (PWY-821) was the most enriched metabolic pathway in the ICI responder versus nonresponder microbiomes, followed by three other sulfur-related pathways: sulfate reduction 1, the superpathway for L-methionine biosynthesis (PWY-5347), and the superpathway for sulfate assimilation (see, e.g., FIG. 1A). Based on these results, an unbiased meta-analysis was performed on responder (R) versus nonresponder (NR) microbiomes from eight ICI-microbiome studies (R, n = 203, NR, n = 134) with melanoma, RCC, or non-small cell lung cancer (NSCLC). Six Saa pathways, including the superpathway PWY-821 for Saa biosynthesis, were among the top 10% of pathways enriched in ICI responders versus nonresponders (ranked by effect size, Cohen's D test) (see, e.g., FIG. 1B, Table 1). Targeted analysis of the abundance of the Saa biosynthesis superpathway within each study revealed its enrichment in the responder versus nonresponder microbiomes independently in 7 out of 8 studies (see, e.g., FIG. 1C). All analyzed ICI-microbiome cohorts were controlled for common covariates such as age, gender, and TNM stage (tumor (T), node (N), and metastasis (M)), but not all had available survival or dietary pattern data. In support of this approach, the inosine-related microbial metabolic pathway, which is produced by Lactobacillus and has been reported to enhance the response to ICI therapy in preclinical cancer models, was also enriched in the top 10% of pathways in ICI R versus NR (see, e.g., Table 1); see, e.g., Mager et al., Science 369, 1481 (2020), the content of which is hereby incorporated by reference in its entirety.

[0307] To extend MetaCyc pathway analysis, a set of 15 bacterial gene homologs was examined that included the enzyme repertoire (n = 11 genes) of the superpathway of Saa biosynthesis based on the E. coli Saa biosynthetic genes and also genes from the superpathway of methionine biosynthesis enriched in ICI responders, which are dominant in the human gut metagenome (see, e.g., Figure 1B). A bacterial pan-genome database of 15 Saa metabolic gene homologs (criteria: e-value 10 -9 , coverage 80%) was generated and fecal metagenomic sequences were aligned against this reference. Consistent with HUMAnN MetaCyc analysis, Saa metabolic genes were enriched in ICI responders versus non-responders (see, e.g., the log2-fold change shown in Figure 1C).

[0308] Enrichment of microbiota Saa metabolic gene carriage across ICI responders with various cancer types indicated a role for microbial Saa metabolism in anti-tumor immunity. This provided an opportunity to enhance anti-tumor immunity in immunologically "cold" tumors such as CRC with proficient mismatch repair. Fecal metagenomes from nine CRC cohorts (containing a total of 812 patients (610 with CRC and 202 with adenomas), as well as 639 healthy controls) were examined to profile Saa metabolism-related gene carriage in CRC patients versus controls. See, for example, Feng et al., Nat Commun 6, 6528 (2015); Gupta et al., mSystems 4, e00438-19 (2019); Hannigan et al. mBio 9, e02248-18 (2018); Vogtmann et al., PLoS One 11, e0155362 (2016); Wirbel et al., Nat Med 25, 679-689 (2019); Yachida et al., Nat Med 25, 968-976 (2019); Yu et al., Gut 66, 70-78 (2017); Zeller et al., Mol Syst Biol 10, 766 (2014); Thomas et al., Nat Med 25, 667-678 (2019), each of which is incorporated herein by reference in its entirety. All CRC cohorts selected for analysis were controlled for common covariates such as age, gender, and TNM stage, but lacked survival data. To date, there is no publicly available CRC ICI microbiota profiling. In these histopathologically, genetically, and geographically diverse CRC cohorts, the Saa superpathway and 15 Saa metabolic gene homologs were present, and were enriched in some cohorts compared to controls (see, for example, FIG. 1D).Since the abundance of these Saa genes in several CRC cohorts shared a pattern similar to that observed in many ICI responders, the idea that a proportion of CRC patients may respond to targeting the Saa pathway was proposed.

[0309] Dietary Saa and the microbiota regulated neoplastic progression in a mouse model of CRC Saa metabolic genes are enriched in the microbiome of ICI responders (see, e.g., FIG. 1A), and since diet can affect the microbiome by altering the availability of nutrient niches, there is increasing interest in foods targeted at the microbiome to modulate immune system function in cancer patients. As described herein, the Saa pathway can be targeted via dietary Saa supplementation, which can in turn affect anti-tumor immunity in CRC. To represent an extreme case of human Saa intake, two isocaloric mouse diets were devised, namely diets with low vs. high amounts of methionine and cysteine (see, e.g., TABLE 2). The low Saa diet had sufficient methionine to avoid methionine restriction, and the high diet was well below the threshold for hyperhomocysteinemia and thus corresponded to physiologically relevant human Saa intake achievable through diet modification or supplementation.

[0310] A mouse model of mismatch repair-deficient (dMMR) metastatic colon adenocarcinoma (MC38 cells) that responded to anti-PD-1 ICI treatment and was examined in microbiome meta-analysis was used (see, for example, FIGS. 1A-1C). Wild-type (WT) C57BL / 6 mice housed in an animal facility were placed on a high-Saa diet or a low-Saa diet 2 weeks prior to flank injection of MC38 cells and monitored for 12 days after injection. Mice fed the high-Saa diet had slower tumor growth, with a ~50% reduction in tumor volume and a 30% reduction in tumor weight (see, for example, FIGS. 1E-1F). Since MC38 tumors responded to anti-PD-1 antibody (Ab) treatment, we tested whether diet could exert an additive or synergistic effect in combination with anti-PD-1 Ab. The high-Saa diet was neither additive nor synergistic with anti-PD-1 Ab, but diet reduced terminal tumor volume and weight similar to levels observed with anti-PD-1 Ab treatment, indicating that the high-Saa diet has efficacy comparable to ICI therapy in this model (see, for example, FIGS. 1G-1H).

[0311] Next, we tested whether Saa dietary intervention could affect tumor progression in a genetically driven colon tumor model with proficient mismatch repair (pMMR) that did not respond to ICI. Apc flox / +Mice were mated with CDX2-Cre mice (cAPC mice) due to colonic-specific heterozygous deletion of the tumor suppressor Apc, as their pMMR status and Apc inactivation reflect two attributes observed in 85% of CRC patients; see, for example, Hinoi et al., Cancer Research 67, 9721 (2007), the content of which is hereby incorporated by reference in its entirety. At 6-8 weeks of age, cAPC mice were randomly assigned to either a 12-week low-Saa or high-Saa dietary intervention. Consistent with the findings in the MC38 CRC model, the development of progressive neoplastic lesions (i.e., adenocarcinoma) was reduced four-fold in cAPC mice fed a high-Saa diet (see, for example, Fig. 1I), and these tumors were, on average, half the weight of those from cAPC mice fed a low-Saa diet (see, for example, Fig. 1J). These data support that dietary Saa supplementation is a modulator of tumor progression in both MMR-deficient (dMMR) and pMMR mouse CRC models.

[0312] Dietary Saa regulated the abundance of Mus sp. strain Schaeedleri and the thickness of the mucus layer To determine whether the effect on tumor growth and progression is dependent on the gut microbiota, cAPC mice were re-derived germ-free, and the dietary intervention was repeated starting at 2 months of age in germ-free (GF) cAPC mice. Over the course of an experimental period exceeding 4 months, no colonic tumors developed in GF mice on either Saa diet (see, for example, Fig. 5A), supporting the microbiota-dependence of CRC. That is, the data suggest dietary Saa supplementation as a modulator of tumor progression in both dMMR and pMMR mouse CRC models.

[0313] Tumor development in cAPC mice was microbiota-dependent (see, e.g., Fig. 5A), so we tested whether the composition of the gut microbiota was affected by dietary Saa by performing 16S rRNA gene amplicon surveys on cecal contents from conventionally housed cAPC mice fed a low-Saa diet versus a high-Saa diet. As expected for this subtle dietary change, there were no statistically significant differences in α-diversity (see, e.g., Fig. 5B) or β-diversity separation (see, e.g., Figs. 5C–5D). Only one taxon had a significantly changed abundance; Muris spiroplum was enriched in the cecal contents of mice fed a high-Saa diet (see, e.g., Figs. 2A and 5E–5F). MaAsLin 2 can be used to analyze housing-independent changes within specific operational taxonomic units (OTUs). Akkermansia muciniphila appeared to increase in the cecal contents of low-Saa diet mice (see, e.g., Fig. 26A), but this was not statistically significant and was due to a cage effect (see, e.g., Fig. 28F).

[0314] M. schaedleri is a member of the family Deferribacteraceae that inhabits the outer mucus layer of the mouse colon; M. schaedleri has been detected in up to 42% of human colon mucosal biopsies; see, for example, Herp et al., Cell Host Microbe 25, 681-694.e8 (2019); Loy et al., mSystems 2, e00171-16 (2017); Robertson et al., Int J Syst Evol Microbiol 55, 1199-1204 (2005); Zmora et al., Cell 174, 1388-1405.e21 (2018), each of which is incorporated herein by reference in its entirety. Quantitative (q)-PCR analysis of cecal DNA from cAPC mice confirmed that M. schaedleri was increased in mice fed a high Saa diet compared to mice fed a low Saa diet (see, for example, FIG. 2B, left panel). The effect was detectable in WT mice from the animal facility (see, for example, FIG. 2B, middle panel) and was observed in modified Schaedler flora (ASF) mice harboring M. schaedleri as part of an 8-member community (see, for example, FIG. 2B, right panel).

[0315] To evaluate the human relevance of M. schaedleri, human microbiome datasets were searched for M. schaedleri-specific reads (see, e.g., methods). Analysis did not detect a reliable signal for M. schaedleri in the human fecal metagenome. Since M. schaedleri is associated with mucus, it was contemplated that searching for its presence in human mucosal tissue microbiome samples might be more informative than searching for its presence in fecal metagenome samples. The tissue-associated level of M. schaedleri was readily detectable in colon biopsy samples and was enriched in normal colon biopsy samples compared to adenomas (see, e.g., FIG. 2C). Such samples and profiling were not available from ICI-responsive patients who did not experience ICI-induced colitis.

[0316] Considering these human mucosal data (see, e.g., FIG. 2C), the localization of M. schaedleri in the mouse colon was examined using fluorescence in situ hybridization (FISH) directed against the bacterium. Since M. schaedleri localizes to the mucus layer, it was contemplated that dietary Saa might affect the abundance of M. schaedleri by regulating the thickness of the mucus layer and create a more favorable niche for M. schaedleri. Dietary amino acid supplementation (including L-cysteine) can increase colonic mucin synthesis in rats; see, e.g., Faure et al., J Nutr 136, 1558-1564 (2006), the contents of which are incorporated herein by reference in their entirety. M. schaedleri thrived in the colon mucus of mice fed a high Saa diet (see, e.g., FIG. 2D), and the mucus layer also appeared thicker (see, e.g., FIG. 2E).

[0317] To evaluate the effect of dietary Saa on mucus layer thickness and its microbiota dependence, GF mice were first fed a low-Saa diet and a high-Saa diet, and the distal colon mucus layer was measured by Alcian blue staining. In the absence of microbiota, the high-Saa diet increased the thickness of the inner mucus layer by 20% (see, e.g., FIGS. 2F and 2H). Next, this analysis was extended to examine mice with a modified Schaedler flora (ASF) microbiota (composed of a defined consortium of 8 bacterial species including M. schaedleri); see, e.g., Wymore et al., ILAR J 56, 169-178 (2015), the content of which is hereby incorporated by reference in its entirety. The high-Saa diet induced an increase of approximately 50% in mucus layer thickness (see, e.g., FIGS. 2G-2H), indicating that the microbiota amplifies the effect of the high-Saa diet on mucus thickness.

[0318] In these mice, an increase in the abundance of M. schaedleri in the mucus layer was observed in response to the high-Saa diet, as evaluated by qPCR (see, e.g., FIG. 28E). qPCR was also performed to quantify M. schaedleri levels in the microbiota context of more fully conventionally housed mice in the above dMMR metastatic CRC mice, and these results again confirmed an increase in M. schaedleri abundance in mice fed the high-Saa diet (see, e.g., FIG. 28F).

[0319] Since a reduction in colonic barrier integrity is associated with the smoldering inflammation that promotes CRC, it was also investigated whether this difference in mucus thickness alters colonic barrier integrity. However, no difference was revealed between WT mice fed a low-Saa diet and WT mice fed a high-Saa diet in the FITC-dextran intestinal permeability test (see, e.g., FIG. 5G). Collectively, these data indicate that dietary Saa levels regulate by affecting the abundance of M. schaedleri, a member of the mouse and human microbiota, in the colonic mucus layer and its viability as an ecological niche for this species.

[0320] A high-Saa diet led to an increase in the number of CD8 + T cells in cAPC tumors To identify the linkage of the mechanisms that integrate dietary Saa, microbial activity, and antitumor immunity, intratumoral cytotoxic CD8 + T cells were evaluated as important classifiers of "hot" tumors in tumors from cAPC mice fed a high-Saa or low-Saa diet. As used herein, the term "hot tumor" refers to a tumor having a "T cell inflammatory" phenotype, and the term "cold tumor" conversely refers to a tumor lacking or deficient in tumor T cell infiltration; see, for example, Bonaventura et al., Front Immunol 10, 168 (2019), the content of which is incorporated herein by reference in its entirety. In tumors from cAPC mice fed a high-Saa diet, compared to tumors from mice fed a low-Saa diet, the frequency and number of CD8 + T cells were higher (see, for example, FIG. 3A). The frequency and number of CD8 + T cells remained relatively unchanged in the adjacent normal colonic lamina propria (LP) and tumor-draining lymph nodes (TDLNs) (see, for example, FIGS. 6A-6B). Using immunofluorescence microscopy, a two-fold increase in the ratio of CD8 + T cells was also observed in tumors from cAPC mice fed a high-Saa diet (see, for example, FIGS. 3B-3C), while no difference was observed in the CD3 + CD8 + / CD3 + cell ratio in the surrounding colonic LP (see, for example, FIG. 6C). To gain functional insights into tumor-infiltrating CD8 + T cells enriched during Saa feeding, the expression of several immune co-inhibitory receptors, IFNγ, and granzyme B (GZMB) was profiled. IFNγ + CD8 + T cells and GZMB + CD8 +The proportion of T cells was significantly increased in tumors from mice fed a high-Saa diet (see, for example, Fig. 3E), while PD-1 + CD8 + The proportion of T cells and LAG-3 + CD8 + The proportion of T cells was significantly decreased (see, for example, Fig. 3D), indicating that these CD8 + T cells had a higher ability to mount an anti-tumor immune response. The expression of immune checkpoint markers TIM-3 and CTLA-4 did not differ between the two groups. No difference was observed in the expression of immune checkpoint markers in the LP or TDLN (see, for example, Figs. 6D-6E). That is, the tumor microenvironment of mice fed a high-Saa diet was more immunologically warm than that in mice fed a low-Saa diet.

[0321] Since the diversity of tumor neoantigens may contribute to the frequency and number of tumor-infiltrating T cells, TCR-Seq analysis of CD8 + T cells in tumors from cAPC mice fed a low-Saa diet versus those fed a high-Saa diet was performed. In the TCR-Seq analysis, no differences were revealed in either the clonotype composition or the abundance of CD8 + T cells in tumors from cAPC mice fed a low-Saa diet versus those fed a high-Saa diet (see, for example, Fig. 7). Therefore, dietary Saa does not affect TCR diversity in cAPC colon tumors, thus prompting an examination of antigen-presenting cells as a mechanism by which diet affects the tumor infiltration and activation of CD8 + T cells.

[0322] The abundance of Musispirillum schaedleri correlated with the frequency of CD103 + CD11b - Dendritic cells (cDC1) in the tumor draining lymph node (TDLN) M. schaedleri is thought to be a mucus-dwelling bacterium and, similar to other mucosa-associated microbes such as segmented filamentous bacteria (SFB), its interaction with host cells is increased. SFB has been extensively studied for its ability to induce a Th17 immune response, but the immunomodulatory effects and underlying mechanisms of M. schaedleri remain poorly characterized. M. schaedleri is Nod2 - / - ×Cybb - / - in mice induces T H type 1 colitis and plays a role in the generation of peripheral regulatory T cells (Tregs); see, for example, Caruso et al., Sci Immunol 4, eaaw4341 (2019); Campbell et al., Immunity 48, 1245-1257.e9 (2018), the contents of each of which are incorporated herein by reference in their entirety. To address what immunomodulatory effects can be induced by M. schaedleri in the diet-CRC model, immune cell populations in the colon LP and TDLN of cAPC mice fed a Saa diet, and in the colon LP and MLN of wild-type (WT) mice bred in-house (BIH) were profiled (for gating strategies, see, for example, FIG. 8). Analysis of the major subsets of CD4 + T cells, including Th1 cells, Th2 cells, Th17 cells, and Tregs, revealed no differences between mice fed a low or high Saa diet in either the colon LP or MLN (see, for example, FIG. 9).

[0323] In contrast, analysis of myeloid cells revealed a significant increase in the frequency and number of CD103 + CD11b - dendritic cells (referred to as cDC1) in the TDLN of cAPC mice fed a high Saa diet, while the other CD11c + cell populations remained unchanged (see, for example, FIG. 3G). CD11c in the LP and tumor +The frequency and number of cells did not change between cAPC mice fed a low-Saa diet and cAPC mice fed a high-Saa diet (see, for example, FIGS. 10A-10B). cDC1 cross-presents tumor-derived antigens in the TDLN to initiate an anti-tumor response in CD8 + T cells and is thus an initiator of anti-tumor immunity. Similar results were obtained for the myeloid cell population in the LP and MLN from WT BIH mice fed a Saa diet (see, for example, FIGS. 10C-10D). To determine whether the increase in cDC1 is dependent on microbial factors, germ-free (GF) mice were fed a low-Saa diet and a high-Saa diet, and their immune cell populations were analyzed. There was no increase in cDC1 in the MLN or LP of GF mice fed a high-Saa diet (see, for example, FIGS. 10E-10F). Using data from all conventionally housed mice (WT and cAPC), there was a significant positive correlation between the cecal abundance of M. shedleri and the frequency of the MLN / TDLN cDC1 population (Spearman ρ = 0.585, P value = 0.0002) (see, for example, FIG. 10G).

[0324] Next, three approaches were used to evaluate whether M. schaedleri contributed to cDC1 expansion. First, ASF mice harboring M. schaedleri were fed a low-Saa or high-Saa diet, and cDC1 expansion was observed only in the MLN of the group fed the high-Saa diet (see, for example, FIGS. 10H-10I); such data demonstrate that an ASF consortium of eight species, including M. schaedleri, was sufficient for cDC1 expansion in the presence of a high-Saa diet. Second, GF mice were mono-colonized with either M. schaedleri or A. muciniphila, and both groups were fed a high-Saa diet. A. muciniphila was chosen because it is also a mucus-resident bacterium with immunomodulatory properties associated with the ICI response. The frequency and number of cDC1 increased only in the MLN of mice mono-colonized with M. schaedleri, but not in those colonized with A. muciniphila, demonstrating that what was required for this observed cDC1 effect was not just the presence of gastrointestinal colonization per se (see, for example, FIGS. 3H and 10J). Third, because many bacteria secrete bioactive molecules, we tested whether cell-free M. schaedleri-conditioned medium (“CDM” or “CM”) affected cDC1 frequency in the MLN of C57BL / 6 BIH mice. Forced oral administration of M. schaedleri CDM over two weeks increased cDC1 frequency in the MLN, whereas the cell-free medium control had no effect (see, for example, FIG. 3I). These results indicate that M. schaedleri and specific factors secreted thereby were sufficient to increase the frequency and number of MLN / TDLN cDC1.

[0325] Depletion of cDC1 or XCL1 abrogated the antitumor effect of the high-Saa diet CD8 +The role of cDC1 cells in T cell-mediated anti-tumor immunity was examined in the context of CRC. To determine whether cDC1 cells mediated the high-Saa diet-dependent and M. schaedleri-dependent effects on anti-tumor immunity and tumor growth as described above, cDC1 was depleted using three approaches. First, bone marrow chimeric mice were generated with hematopoietic cells from Zbtb46-DTR mice that express diphtheria toxin receptor (DTR) under the control of a cDC1-specific promoter upstream of Zbtb46, allowing for diphtheria toxin-mediated cDC1 depletion; see, for example, Meredith et al., J Exp Med 209, 1153-1165 (2012), the content of which is hereby incorporated by reference in its entirety. These cAPC Zbtb46-DTR mice were fed a high-Saa diet (which drives the expansion of M. schaedleri and subsequent increase in MLN-TDLN cDC1), and starting 4 weeks after irradiation and transplantation, were given injections of diphtheria toxin (DT) to deplete cDC1 or PBS as a control. The expected decrease in TDLN cDC1 numbers was observed in mice treated with DT, while the frequency and number of other CD11c + cells did not change (see, for example, FIG. 11A). There was no difference in the cecal abundance of M. schaedleri between the DT-treated and PBS-treated groups (see, for example, FIG. 11B). Depletion of cDC1 increased the frequency of neoplastic lesions even in the presence of a high-Saa diet (see, for example, FIG. 4A). This finding further supports the role of cDC1 as a mediator of anti-tumor immunity and neoplastic progression and demonstrates the function of cDC1 in mediating the anti-tumor effect of the high-Saa diet-M. schaedleri bloom.

[0326] As a second approach to depleting cDC1 in cAPC mice, the cAPC strain was crossed with Batf3 knockout (KO) mice to generate cAPC Batf3 - / - mice. Similar to Zbtb46, Batf3 is a transcription factor that is highly important for the development of cDC1. cAPC Batf3 - / -When mice were fed a high-Saa diet or a low-Saa diet, as expected, the frequency and number of cDC1s decreased in the TDLNs of both groups, but cDC2s did not (see, for example, Fig. 11C). The presence of higher M. schaedleri was also detected in the cecal contents of the group fed the high-Saa diet compared to mice fed the low-Saa diet (see, for example, Fig. 11D). Consistent with the findings in Fig. 4A, Batf3 deficiency resulted in the loss of the protective effect of the high-Saa diet, as there was no difference in tumor number or neoplastic progression between the two diet groups (see, for example, Fig. 4B). Because the data supported that cDC1 is an important link connecting Saa diet, M. schaedleri, and anti-tumor immunity (see, for example, Figs. 3F–3I and 4A–4B), we investigated whether M. schaedleri acted directly on cDC1. In vitro tests of cell-free M. schaedleri CM on cDC1 did not support that it directly enhanced the activation of CD8 + T cells via the increased expression of co-stimulatory molecules or cross-presentation (see, for example, Figs. 11E–11G).

[0327] To determine what drives the observed effects on cDC1, further studies focused on lymphotactin (XCL1), a factor known to activate and mobilize cDC1. In a third approach to perturb cDC1, α-XCL1 antibody (Ab) treatment was used to deplete this cDC1-specific chemokine and cytokine in the high-Saa diet setting; see, for example, Matsumoto et al., J Immunol 199, 82-90 (2017); Lei et al., Microbes Infect 14, 262-267 (2012), each of which is incorporated herein by reference in its entirety. cAPC mice treated with α-XCL1 Ab had reduced serum levels of XCL1 and had fewer cDC1 in their TDLN (see, for example, FIGS. 11H-11I). Reduction of XCL1 levels resulted in a significant increase in adenocarcinoma in cAPC mice fed a high-Saa diet (see, for example, FIG. 4C), although α-XCL1 Ab only reduced serum levels by an average of two-fold (see, for example, FIG. 11H). Collectively, these experiments confirmed the role of cDC1 in the immune control of tumor growth in cAPC mice and linked diet-microbe interactions to anti-tumor immunity.

[0328] Dietary Saa and M. shedleri induced XCL1 secretion from natural killer T (NKT) cells Observation of the increase in MLN / TDLN cDC1 in mice harboring M. shedleri fed a high-Saa diet led to an investigation of factors driving cDC1 accumulation and activation. Because XCL1 played a role in the protective effect of the high-Saa diet in cAPC mice, studies focused on XCL1 (see, for example, FIG. 4C). Furthermore, cDC1 selectively express XCR1, the XCL1 receptor. Both NK cells and NKT cells can secrete XCL1, thereby, CD8 +Indicate the role of cDC1 in orchestrating T cell-mediated antitumor immunity; see, for example, Barry et al., Nat Med 24, 1178-1191 (2018); Bottcher et al., Cell 172, 1022-1037.e14 (2018), each of which is incorporated herein by reference in its entirety. To determine whether XCL1 secretion by these cell types was affected by dietary Saa, NK cells and NKT cells were sorted from the MLN of BIH C57BL6 WT mice fed two Saa diets; NKT cells from mice fed a high Saa diet secreted higher levels of XCL1 when cultured ex vivo compared to NKT from mice fed a low Saa diet (see, for example, FIG. 4D). NK cells secreted less XCL1 than NKT cells regardless of diet (see, for example, FIG. 4D). In cAPC mice fed a high Saa diet compared to those fed a low Saa diet, the frequency of TDLN NKT cells increased, but the frequency of NK cells did not (see, for example, FIGS. 4E, 11J), indicating that the high Saa diet induced both the secretion of XCL1 from NKT cells and the increase in its frequency and number in the TDLN.

[0329] To test whether M. schaedleri can directly affect XCL1 production in NKT cells, an in vitro system using the NKT cell line GW1 (see, for example, Donado et al., Cell Rep 31, 107466 (2020), the content of which is incorporated herein by reference in its entirety) and cell-free bacterial-conditioned medium (CDM) was employed. CDM from two immunomodulatory gut bacteria, Lactobacillus plantarum and A. muciniphila, had no effect, but M. schaedleri CDM dramatically stimulated XCL1 production on a scale comparable to IL-12 treatment (see, for example, FIG. 4F). The XCL1-stimulatory activity of M. schaedleri CDM was heat-labile and was found mainly in the organic phase of the supernatant (see, for example, FIG. 4F). Since GW1 cells are a mouse cell line, the findings were then extended to human NKT cells. NKT cells were expanded from peripheral blood mononuclear cells of healthy human donors; those NKT cells were treated with M. schaedleri CDM. XCL1 increased approximately 4-fold in treated NKT cells compared to untreated NKT cells (see, for example, FIG. 4G). Together, these data indicate that NKT cell-derived XCL1 production contributed to the accumulation of activated cDC1 in the MLN / TDLN, and that dietary and microbial factors induced this NKT XCL1 production in mouse and human NKT cells.

[0330] To focus on the activity of M. sheddleri that drives NKT XCL1 secretion, non-targeted LC-MS / MS metabolomics was performed on cell-free M. sheddleri CM samples (n = 4 independent samples) and media controls without M. sheddleri (n = 2 independent samples). Of the 4,263 features detected, relatively few were enriched in CM (n = 36), had a change of more than two-fold, and had a p-value of less than 0.05 in one-way ANOVA with post hoc Tukey's HSD test (see, e.g., FIGS. 4H and 4I herein, and Table 3 of U.S. Patent Application No. 63 / 389,382). Consistent with the extraction results (see, e.g., FIG. 4F), many of the known or predicted metabolic features enriched in cell-free M. sheddleri CM are lipids and lipid components (e.g., fatty acids) (see, e.g., FIGS. 4H and 4I herein, and Table 3 of U.S. Patent Application No. 63 / 389,382). Without wishing to be bound by theory, some of the M. sheddleri metabolites may cooperate to enhance NKT secretion of XCL1, which acts on cDC1 to promote its anti-tumor immunity via the CD8 + T cell compartment, as contemplated herein.

[0331] The activation transcriptional signature in TDLN cDC1 of cAPC mice fed a high Saa diet correlated with better survival in CRC patients In the studies described herein of the ICI-responsive microbiome, high Saa diet, and M. sheddleri, cDC1 emerged as a convergence point for understanding diet-microbiome-immune-stimulatory interactions in CRC. Thus, to define the effect of dietary Saa-M. sheddleri on cDC1, single cell RNA sequencing (scRNA-Seq) was performed on FACS-sorted DCs derived from TDLN of cAPC mice fed a low Saa diet and a high Saa diet. TDLN is anti-tumor CD8 +It is a central immune regulatory site where important cDC1 cross-presentation events occur for T cells. Additionally, they are readily available for studies in mice and have not been profiled to date in human CRC scRNA-seq studies, thus corresponding to a cell population that is not fully utilized and may potentially be used for prognosis.

[0332] Approximately 12,500 CD11c + MHCII + CD64 - Cell sequencing revealed substantial cell heterogeneity with six DC clusters, as defined by manual curation inspection of known DC markers (see, for example, FIG. 12A, Table 4). The largest cluster contained CD11b+ cells corresponding to cDC2, macrophages, and monocytes, followed by clusters of migratory DCs (expressing CCR7), DC-SIGN + (CD209)+ DCs, and a cDC1 cluster identified by the expression of the Xcr1, Clec9a, and Irf8 genes (see, for example, FIGS. 12B - 12C). Fast gene set enrichment analysis (fgsea) of the Hallmark Gene Set Pathway revealed enrichment of the inflammatory response pathway (M5932) in cDC1 isolated from the TDLN of high-Saa diet-M. sherrelli (Ms) expanded mice (see, for example, FIG. 12D), while cell cycle-related pathways were enriched in cDC1 from mice fed a low-Saa diet (see, for example, FIG. 12D). + (CD209)+ DCs, and a cDC1 cluster identified by the expression of the Xcr1, Clec9a, and Irf8 genes (see, for example, FIGS. 12B - 12C). Fast gene set enrichment analysis (fgsea) of the Hallmark Gene Set Pathway revealed enrichment of the inflammatory response pathway (M5932) in cDC1 isolated from the TDLN of high-Saa diet-M. sherrelli (Ms) expanded mice (see, for example, FIG. 12D), while cell cycle-related pathways were enriched in cDC1 from mice fed a low-Saa diet (see, for example, FIG. 12D).

[0333] Genes differentially expressed were identified in cDC1s from low-Saa diet mice versus high-Saa diet mice (see, for example, Table 5). cDC1s from high-Saa diet-Ms expansion (HSME) mice expressed higher levels of the transcriptional products of Cxcl9 and Ccl4 (see, for example, Figure 4J), both of which play important roles in the anti-tumor immune response. Other transcriptional products expressed at higher levels in cDC1s from HSME mice included genes encoding the calcium-binding proteins S100A6 and S100A4, which function in efficient DC antigen presentation, co-stimulatory molecule expression, and T cell activation (see, for example, Figure 4J). Genes more highly expressed (or decreased on high-Saa diet) in cDC1s from low-Saa diet-fed mice included the thioesterase Ppt1, which balances viral resistance against T cell cross-priming (see, for example, Figure 4J, Table 5). Decreased Ppt1 expression enhances tumor clearance in mice. Overall, scRNA-Seq analysis showed that cDC1s from HSME mice exhibit higher transcription of activation and immunostimulatory genes, which is consistent with the finding that high-Saa diet restricted tumor growth and reduced neoplastic progression.

[0334] We utilized the differentially enriched genes of cDC1 to generate a cDC1 gene expression signature associated with the beneficial anti-tumor effects of high-Saa M. sheddleri dependence (see, for example, Table 5). We examined the association of this signature with survival data from bulk RNA-Seq transcriptomes of the Tissue Cancer Gene Atlas (TCGA) dataset of colon and rectal adenocarcinoma tumors (TCGA COAD-READ, N = 359). Patients with transcriptome scores similar to the HSME mouse cDC1 transcriptional state (HSME-DC; "high activation") had a statistically significant longer survival time compared to patients with transcriptome scores similar to the cDC1 state of low-Saa-M. sheddleri depletion (LSMD; "low activation") mice (Cox model, P = 0.005 for univariate analysis and P = 0.017 for stage-adjusted multivariate analysis) (see, for example, Figure 4K). For patients with tumor gene expression similar to LSMD-DC, there was an increased hazard ratio (HR) of 1.611 (1.152-2.253, 95% confidence interval (CI)) in univariate analysis and an HR of 1.521 (1.077-2.147, 95% CI) in multivariate analysis including stage. Thus, the gene expression signature detected in cDC1 from HSME cAPC mice correlated with improved survival in CRC patients.

[0335] Discussion In the search for the biological activities of microorganisms underlying anti-tumor immunity, enrichment of the Saa pathway and genes in the gut microbiota was identified as a shared feature of ICI responsiveness across cohorts. Targeting these pathways via dietary Saa supplementation slowed tumor progression in both MMR-deficient CRC models and CRC models with proficient MMR. Methionine dependence has been reported in CRC tumor cells, and methionine restriction limits tumor growth in mouse models. Such results raised the question of why tumor growth reduction was observed in the high-Saa diet herein. Both very low-Saa diets and high-Saa diets may be tumor-suppressive for different underlying reasons; low-Saa diets limit cancer cell proliferation by metabolic dependence, and high-Saa diets enhance anti-tumor immunity as described herein, is contemplated herein. Low-Saa diets were not methionine-restrictive enough to affect tumor growth herein. Dietary sulfur takes many different forms in organic and inorganic compounds; for example, total dietary sulfur intake correlates with human colonic adenomas and CRC. Dietary Saa modulation also has pleiotropic effects on the host and microbiota, from changes in redox potential to post-translational modification of proteins, all within a dosage range that is non-toxic to the host. Considering the anti-tumor immune effects observed herein, Saa supplementation can be clinically investigated in a cohort of CRC patients. Additionally, further investigation in ICI-resistant patients can also be conducted, considering that this pathway was enriched in the microbiota of ICI non-responder melanoma patients who became ICI-responsive upon fecal microbiota transplantation.See, e.g., Gao et al., Nature 572, 397-401 (2019); Hoffman (Humana Press, 2019); Komninou et al., Nutr Cancer 54, 202-208 (2006); Tan et al., Clin Cancer Res 5, 2157-2163 (1999); Nguyen et al., Gastroenterology 158, 1313-1325 (2020); Nguyen et al., Gastroenterology 161, 1423-1432.e4 (2021); Yan et al., Journal of Biological Chemistry 285, 41525-41532 (2010); Lobel et al., Science 369, 1518-1524 (2020); Baruch et al. 2021, supra, each of which is incorporated herein by reference in its entirety.

[0336] Identifying common functional features across diverse gut microbial taxa associated with ICI response in patients that can be targeted to improve anti-tumor immunity is described herein. Also described herein is a dietary intervention targeting the Saa pathway that attenuated tumor growth in CRC, a malignancy that is generally refractory to ICI therapy and considered immunologically "cold". The dietary intervention initiated an effective mucus-M. schaedleri-anti-tumor immunity cascade (schematized in FIG. 13), indicating that foods can be engineered to improve anti-tumor immunity in CRC. Data on M. schaedleri indicated that mucus was the link between diet, microbiota, and immune function, and that the colonic mucus layer was the site of microbial activity involved in anti-tumor immunity and immune function. Additionally, the immunomodulatory effect of M. schaedleri was dependent on diet, gut microbial factors, and CD8 +NKT cells have been spotlighted as a link in the chain connecting enhanced cDC1 cell function converging on T cells. These findings confirm the role of cDC1 in anti-tumor immunity. The cDC1 depletion data described herein, combined with data depleting XCL1, a cDC1-specific chemokine and activator, demonstrate the role of cDC1 in linking enhanced immunity against Saa, M. schaedleri, and CRC.

[0337] Collectively, the data described herein not only identify the biogeography, such as the mucus layer or tumor-inflow regional lymph nodes, but also identify the CRC prognostic signature at the interface of diet, microbiome, and host immunity, highlighting the utility of the mouse model in exploring diet-microbe interactions. Together, the experiments herein elucidate how diet-microbiota interactions enhance anti-tumor immunity in a multi-step manner and how this modulates innate immune cell responses to control tumor progression in CRC. Overall, the data described herein provide an examination of the patient stool metagenomic profiles from ICI-responsive patients to determine the mechanism by which microbiota-diet interactions enhance anti-tumor immunity against immunologically cold tumors.

[0338] Materials and Methods Meta-analysis of microbial metabolic pathways in the microbiome of anti-PD-1 treatment responders versus non-responders Fecal metagenomic shotgun reads for the ICI and CRC datasets were obtained from the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI). Any metagenomic sample known to have antibiotic use as described in the available clinical metadata was excluded from the meta-analysis. The raw reads of the sequences were quality trimmed using TRIMMOMATIC (v0.39), which was configured to perform a sliding window scan with the following parameters: "ILLUMINACLIP:${adapter_library_FASTA}::2:36:7:1: keepBothReads LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36." To create an expanded collection of bacterial Saa biosynthesis reference genes from the MetaCyc pathway database, 72,380 complete and draft-level bacterial GENBANK genome assemblies with sufficient taxonomic coverage across the genera of the representative human gut microbiome were accessed. A prioritized list of 15 Saa protein sequences was incorporated for custom BLAST+ searches (options: e-value 1e-09, coverage 80), and the protein-coding regions of each assembly were repeatedly annotated via PROKKA, a rapid hierarchical genome annotation pipeline, and 484,023 bacterial Saa gene homologs were detected. See, for example, Bolger et al., Bioinformatics 30, 2114-2120 (2014); Xiao et al., Nature Biotechnology 33, 1103-1108 (2015); Seemann, Bioinformatics 30, 2068-2069 (2014), each of which is incorporated herein by reference in its entirety.

[0339] Among these Saa-utilizing gene homologs, 120,861 had non-zero abundance when reads were mapped with default parameters using BWA-MEM v0.7.17 and quantified using htseq-count of HTSeq (default union method). The gene read count matrix was constructed by aggregating the total gene abundance across the bacterial genomes (reads per kilobase; RPK). These data, along with the pathway relative abundance profiles generated using HUMAnN 2, were fit to a linear mixed effects model (LMM) with the study effect as a random covariate, as implemented in the lmerTest R / CRAN package. Effect sizes were derived from the LMM fit to pathways present in at least 20% of the samples using the emmeans R / CRAN package. To contrast biological groups of interest, a rank-based combined mean fold change guided by the interquartile range was calculated. See, for example, Li, arXiv 1303.3997v2 (2013); Anders et al., Bioinformatics 31, 166-169 (2015); Kuznetsova et al., Journal of Statistical Software 82, (2017); Russell, emmeans: Estimated Marginal Means, aka Least-Squares Means (2021); Xiao et al., Bioinformatics 30, 801-807 (2014), each of which is incorporated herein by reference in its entirety.

[0340] Mice and dietary interventions Mice (WT C57BL / 6, CDX2-Cre APC flox / + (cAPC), Zbtb46-DTR, cAPC Batf3 - / - and Ptprc a(Ly5.1, CD45.1)) were housed in a barrier facility with a constant ambient temperature of 24 °C and a 12-hour light / dark cycle. In-house born (BIH) mice were specific pathogen-free C57BL6 / J mice that were bred and conventionally housed in the animal facility of the barrier facility. All mouse strains were purchased from the JACKSON LABORATORY and then bred in-house. For the notobiotic experiments, mice were housed in semi-rigid isolators (PLASTIC CONCEPTS INC.), and the experiments were performed in individual ventilated ISOCAGEP systems (TECNIPLAST). To verify the notobiotic status (germ-free, mono-colonizing, or ASF) of the mice, routine monitoring including 16S rRNA gene amplicon sequencing, and qPCR analysis (using universal 16S rDNA primers), and Sanger sequencing were performed on fecal samples and cage swabs. For the re-derivation of cAPC mice, pregnant female mice were euthanized, and their uteri were removed aseptically in a semi-rigid isolator using a chemical sterilant (MB-10, QUIP LABS). The sterilized pups were introduced to germ-free foster mothers. Three weeks later, the germ-free status and genotype of the mice were evaluated using PCR.

[0341] A sulfur amino acid (Saa) diet was devised to correspond to edge cases of Saa intake, taking the following into account. The dietary intake of cysteine and methionine in humans is typically 0.03 - 0.06 g / kg body weight / day. Over a human diet with a range of protein intakes (44 g - 140 g / day), low levels of cysteine are 0.01 - 0.04 / g / kg / day. Very high levels of methionine or cysteine are in the range of ≥6 g / kg / day; such levels raise concerns about contributing to homocysteinemia in humans. See, for example, Elshorbagy et al., J Nutr Biochem 23, 332 - 340 (2012); Paul et al., Nature 509, 96 - 100 (2014); O'Keefe et al., J Nutr 137, 175S - 182S (2007); O'Keefe et al., Nat Commun 6, 6342 (2015); Nimni et al., Nutr Metab (Lond) 4, 24 (2007); David et al., Nature 505, 559 - 563 (2014), each of which is incorporated herein by reference in its entirety. Considering these data and with veterinary approval, two isocaloric diets were devised as used herein and manufactured by RESEARCH DIETS, INC (see, for example, Table 2 for diet formulation). For the notobiot experiment, the same irradiated formulation was ordered from TEST DIET.

[0342] At 6 - 8 weeks of age, WT mice, cAPC mice, cAPC Batf3 - / -Mice, or cAPC Zbtb46-DTR mice, were transferred to a Saa diet. After 12 weeks on the Saa diet, the mice were sacrificed, and tissues (normal, neoplastic, and adjacent normal) were collected for either histology, flow cytometry, or immunofluorescence, and cecal contents were frozen for microbiota analysis. For the notobiota experiments, GF mice, ASF mice, M. schaedleri single-colony-forming mice, or A. muciniphila single-colony-forming mice were transferred to the Saa diet at 6 - 8 weeks of age. Four weeks later, the mice were sacrificed and the tissues were analyzed by flow cytometry. GF cAPC mice were transferred to the Saa diet at weaning and maintained on that diet for 12 weeks. In the bacterial CDM feeding experiment, 100 μl of filtered culture supernatant of 5 - 7-day-old M. schaedleri or sterile mBHI medium was force-fed to mice bred in-house on a low Saa diet and WT twice every 2 days.

[0343] To generate cAPC Zbtb46-DTR mice, cAPC mice were irradiated at 10 weeks of age with a single dose of 1000 rad and then injected with 10 6 bone marrow cells from Zbtb46-DTR mice. To deplete Zbtb46-expressing cells in cAPC Zbtb46-DTR bone marrow chimeric mice, 4 weeks after irradiation, the mice were injected with 400 ng (about 20 ng / g body weight) of diphtheria toxin (DT), and then, to maintain depletion of Zbtb46-expressing cells throughout the remainder of the experiment, were injected twice weekly with 100 ng DT (about 4 ng / g body weight). For XCL1 depletion, cAPC mice were fed a high Saa diet for 8 weeks and then injected twice every 2 - 3 days for 4 weeks with 300 μg of anti-XCL1 antibody or anti-trinitrophenol (BIOXCELL), an InVivoMAb rat IgG1 isotype control. Animal studies and experiments were approved and performed in accordance with guidelines for the use and care of animals.

[0344] MC38 colon cancer flank tumor model MC38 mouse colon cancer cells (KERAFAST) were grown in RPMI GLUTAMAX medium supplemented with 10% fetal bovine serum (FBS), 500 U / ml penicillin / streptomycin, 1 mM sodium pyruvate, and 50 μM β-mercaptoethanol. At approximately 80% confluence, the cells were harvested, washed in PBS, and resuspended in a 1:1 solution of CULTREX Reduced Growth Factor Basement Membrane Extract (R&D SYSTEMS) and PBS on ice at a concentration of 3×10 6 cells / ml. C57BL / 6J mice bred in-house with WT were placed on a low Saa diet or a high Saa diet 2 weeks prior to MC38 transplantation. On the day of transplantation, the left flank of the mice was shaved and 150 μl (0.5×10 6 cells) of the MC38 cell solution was injected subcutaneously. The mice were monitored every other day and the tumor volume was calculated using the formula volume = (width 2 × length) / 2 (Equation 1). Twelve days after transplantation, the mice were sacrificed and the tumors were excised and weighed. For the anti-PD-1 experiment, the mice were treated as described above with the addition of an i.p. injection of either 250 μg per mouse of INVIVOMAB anti-mouse PD-1 (BIOXCELL) or INVIVOMAB rat IgG2a isotype control anti-trinitrophenol (BIOXCELL) on days 6, 9, and 12. The mice treated with isotype or anti-PD-1 were sacrificed on day 13 after transplantation and the tumor weights were measured. See, for example, Corbett et al., Cancer Res 35, 2434-2439 (1975); Faustino-Rocha et al., Lab Anim (NY) 42, 217-224 (2013), each of which is incorporated herein by reference in its entirety.

[0345] Histopathology After sacrifice, the colon was opened using blunt scissors and the luminal contents were removed. Cecal contents were snap-frozen in liquid N2. Tissues were fixed in 4% paraformaldehyde, processed, and paraffin-embedded using the standard protocol by the Rodent Histopathology Core. For each sample, five hematoxylin and eosin (H&E)-stained slides (five levels of sections obtained at 50-μm intervals) were blindly evaluated by a board-certified GI pathologist for neoplastic lesions - aberrant crypt foci (ACF), adenomas, or adenocarcinoma (adenoCA).

[0346] Extraction of Cecal DNA and Real-Time Quantitative PCR (RT-qPCR) Analysis Mouse cecal contents were collected in 1.5 ml tubes and immediately frozen in liquid N2. The thawed cecal contents were resuspended in 300 μl of Tris-EDTA solution (100 mM Tris and 15 mM EDTA) in a 2 ml tube along with approximately 300 μl of zirconium beads (20 micron), and 500 μl of TE-saturated phenol (SIGMA-ALDRICH) was added. The tube was placed in a bead beater for...

Claims

1. A composition comprising Mucispirillum schaedleri (M. schaedleri) bacteria formulated for delivery to the intestine.

2. The composition according to claim 1, wherein the M. schaedleri bacteria are alive or inactivated.

3. The composition according to claim 1, wherein the M. schaedleri bacteria are in a dried viable form.

4. The composition according to claim 1, wherein the M. schaedleri bacteria are encapsulated.

5. The composition according to claim 1, wherein the M. schaedleri bacteria are contained in enteric capsules.

6. The composition according to claim 1, wherein the M. schaedleri bacteria are maintained in an anaerobic state in the formulation.

7. The composition according to claim 1, wherein the M. schaedleri bacteria are in a mixture with prebiotics.

8. The composition according to claim 1, wherein the M. schaedleri bacteria are in a mixture with sulfur-containing amino acids.

9. The composition according to claim 8, wherein the sulfur-containing amino acids are methionine, cysteine, or derivatives thereof.

10. The composition according to claim 1, wherein the M. schaedleri bacteria are formulated in a food composition.

11. The composition according to claim 10, wherein the food composition is supplemented with sulfur-containing amino acids and / or prebiotics.

12. The composition according to claim 1, further comprising 1 to 20 additional bacteria.

13. The composition according to claim 1, comprising 20 or fewer bacteria.

14. A composition comprising live M. schaedleri bacteria, dead M. schaedleri bacteria, a conditioned M. schaedleri culture medium, or an organic solvent extract or fraction thereof of the conditioned M. schaedleri culture medium, which promotes XCL1 secretion by NKT cells, and is formulated for delivery to the intestine.

15. The composition according to claim 14, wherein the M. schaedleri bacteria, medium, or solvent extract is in a dried form.

16. The composition according to claim 14, wherein the M. schaedleri bacteria, medium, or extract is encapsulated.

17. The composition according to claim 14, wherein the M. schaedleri bacteria are contained in enteric capsules.

18. The composition according to claim 14, wherein the M. schaedleri bacteria are maintained in an anaerobic state in the formulation.

19. The composition according to claim 14, wherein the M. sheddleri bacteria, culture medium, or extract is in a mixture with prebiotics and / or sulfur-containing amino acids or derivatives thereof.

20. The composition according to claim 14, wherein the adjusted M. sheddleri culture medium, or an organic solvent extract or fraction thereof of the adjusted M. sheddleri culture medium, contains at least one metabolite selected from FIG. 4H, FIG. 4I, or FIG.

16.

21. The adjusted M. sherdleri culture medium, or an organic solvent extract or fraction thereof of the adjusted M. sherdleri culture medium, contains succinic acid; propionic acid; nicotinic acid; aconitic acid (cis and / or trans); pentadecanoic acid; itaconic acid; 16-hydroxyhexadecanoic acid; C 8 H 15 NO 3 S; crotonic acid; myristic acid; 17-hydroxyheptadecanoic acid (C 17 H 34 O 3 ); and 15-hydroxypentadecanoic acid (C 15 H 30 O 3 ), and the composition according to claim 14, comprising at least one metabolite selected from the group consisting of

22. The composition according to claim 14, wherein the adjusted M. sheddleri culture medium, or an organic solvent extract or fraction thereof of the adjusted M. sheddleri culture medium, contains at least one metabolite selected from the group consisting of succinic acid; nicotinic acid; aconitic acid (cis and / or trans); pentadecanoic acid; itaconic acid; 16-hydroxyhexadecanoic acid; and crotonic acid.

23. A food composition comprising the composition according to claim 14.

24. The food composition according to claim 23, further comprising 1 to 20 additional bacteria.

25. A method of treating cancer or promoting anti-tumor immune activity, the method comprising administering the composition according to claim 1 to a subject in need thereof.

26. The method according to claim 25, wherein the cancer is colon cancer.

27. The method according to claim 25, further comprising administering an immune checkpoint inhibitor.

28. A method of promoting responsiveness to immune checkpoint inhibitor tumor therapy, the method comprising administering the composition according to claim 1 to a subject in need thereof.

29. The method according to claim 28, further comprising administering an immune checkpoint inhibitor.

30. The method according to claim 28, wherein the subject has colon cancer.

31. The method according to claim 28, wherein the subject's cancer is determined to be resistant to immune checkpoint inhibitor therapy.

32. The method according to claim 28, wherein the composition promotes XCL1 secretion by NKT cells.

33. A method of increasing CD103+ conventional dendritic cells (cDC1), the method comprising administering the composition according to claim 1 to a subject in need thereof.

34. The method according to claim 33, wherein the cDC1 is associated with a tumor.

35. The method according to claim 34, wherein the tumor is colon cancer.

36. The method according to claim 33, further comprising the step of administering a sulfur-containing amino acid.

37. The method according to claim 33, further comprising the step of administering an immune checkpoint inhibitor.

38. A method for increasing XCL1 secretion by NKT cells, comprising the step of administering the composition according to claim 1 to a subject in need thereof.

39. The method according to claim 38, wherein the subject has cancer.

40. The method according to claim 38, wherein the subject has colon cancer.

41. A method for increasing infiltration of CD8+ T cells in a colorectal tumor, comprising the step of administering the composition according to claim 1 to a subject in need thereof.

42. The method according to claim 41, wherein cDC1 is associated with the tumor.

43. The method according to claim 42, wherein the tumor is colon cancer.

44. The method according to claim 41, further comprising the step of administering a sulfur-containing amino acid.

45. The method according to claim 41, further comprising the step of administering an immune checkpoint inhibitor.

46. A method for increasing infiltration of CD8+ T cells in a colorectal tumor, comprising the step of administering a diet rich in sulfur-containing amino acids (SAA) or a supplement containing SAA to a subject in need thereof.

47. The method according to claim 46, wherein the diet rich in sulfur-containing amino acids contains more than 0.04 grams of SAA per kilogram of body weight per day.

48. The method according to claim 46, further comprising the step of administering the composition according to any one of claims 13 to 20 to the subject.

49. A method for establishing or maintaining a tumor-suppressive gastrointestinal environment in a subject in need thereof, comprising the step of administering a diet rich in sulfur-containing amino acids (SAA) or a supplement containing SAA.

50. The method according to claim 49, wherein the diet rich in sulfur-containing amino acids or the supplement containing SAA contains more than 0.04 grams of SAA per kilogram of body weight per day.

51. The method according to claim 49, further comprising the step of administering the composition according to any one of claims 14 to 21 to the subject.

52. A method for treating cancer, comprising the step of administering an XCL1 polypeptide to a subject in need thereof.

53. The method according to claim 52, wherein the cancer is colon cancer.

54. The method according to claim 52, wherein the XCL1 polypeptide is administered to the digestive tract.

55. A method of treating cancer, comprising administering to a subject in need thereof a microorganism engineered to express an XCL1 polypeptide.

56. A method of treating cancer, comprising administering to a subject in need thereof an agonist of XCR1, which is an XCL1 receptor.

57. The method according to claim 56, wherein the XCR1 agonist comprises SEQ ID NOs: 9-11, or an amino acid sequence that is at least 95% identical and maintains its function.

58. A method of treating cancer in a subject in need thereof, detecting the level of M. sherrelli in a sample from the subject; administering a cancer immunotherapeutic agent when the level of M. sherrelli is equal to or greater than a predetermined threshold; and administering a cancer immunotherapeutic agent and the composition according to claim 1 when the level of M. sherrelli is less than a predetermined threshold comprising the above method.

59. A method of treating cancer in a subject in need thereof, obtaining a result from an assay for detecting the level of M. sherrelli in a sample from the subject; administering a cancer immunotherapeutic agent when the level of M. sherrelli is equal to or greater than a predetermined threshold; and administering a cancer immunotherapeutic agent and the composition according to claim 1 when the level of M. sherrelli is less than a predetermined threshold comprising the above method.

60. A method of treating cancer in a subject in need thereof, detecting the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 in a sample from the subject; administering a cancer immunotherapeutic agent when the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 are equal to or greater than a predetermined threshold; and administering a cancer immunotherapeutic agent and the composition according to claim 1 when the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 are less than a predetermined threshold comprising the above method.

61. A method of treating cancer in a subject in need thereof, A step of obtaining results from an assay for detecting the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 in a sample derived from the subject; When the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 are equal to or higher than a predetermined threshold, a step of administering a cancer immunotherapeutic agent; and When the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 are less than a predetermined threshold, a step of administering a cancer immunotherapeutic agent and the composition according to claim 1 The method comprising the above.

62. A method for stratifying a subject for cancer treatment, comprising: A step of detecting the level of M. schaedleri in a sample derived from the subject; and When the level of M. schaedleri is less than a predetermined threshold, a step of classifying the subject as high risk; or When the level of M. schaedleri is equal to or higher than a predetermined threshold, a step of classifying the subject as low risk The method comprising the above.

63. A method for stratifying a subject for cancer treatment, comprising: A step of obtaining results from an assay for detecting the level of M. schaedleri in a sample derived from the subject; and When the level of M. schaedleri is less than a predetermined threshold, a step of classifying the subject as high risk; or When the level of M. schaedleri is equal to or higher than a predetermined threshold, a step of classifying the subject as low risk The method comprising the above.

64. A method for stratifying a subject for cancer treatment, comprising: A step of detecting the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 in a sample derived from the subject; and When the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 are less than a predetermined threshold, a step of classifying the subject as high risk; or When the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 are equal to or higher than a predetermined threshold, a step of classifying the subject as low risk The method comprising the above.

65. A method for stratifying a subject for cancer treatment, comprising: A step of obtaining results from an assay for detecting the levels of XCL1 polypeptide, NKT, and / or CD103+ cDC1 in a sample derived from the subject; and If the levels of the XCL1 polypeptide, NKT, and / or CD103+ cDC1 are below a predetermined threshold, classifying the subject as high risk; or If the levels of the XCL1 polypeptide, NKT, and / or CD103+ cDC1 are at or above a predetermined threshold, classifying the subject as low risk The method, comprising.

66. The method according to any one of claims 58 to 65, wherein the subject has colon cancer.

67. The method according to any one of claims 62 to 65, further comprising administering the composition according to any one of claims 1 to 24.

68. The method according to any one of claims 58 to 65, further comprising administering a sulfur-containing amino acid.

69. The method according to any one of claims 58 to 61, wherein the cancer immunotherapeutic agent is selected from the group consisting of an immune checkpoint inhibitor; chemotherapy; dendritic cell vaccine; chimeric antigen receptor T cell (CAR-T); and NKT cell-based therapy.

70. The method according to any one of claims 58 to 61, wherein the cancer immunotherapeutic agent comprises an immune checkpoint inhibitor.

71. The method according to any one of claims 62 to 65, further comprising administering an immune checkpoint inhibitor.

72. The method according to any one of claims 58 to 65, further comprising administering a diet rich in sulfur-containing amino acids or a supplement containing SAA.

73. The method according to any one of claims 58 to 61 or 66 to 65, which results in higher treatment efficacy compared to a method of treating without first involving detecting the level of M. schaedleri; obtaining a result from an assay for detecting the level of M. schaedleri; detecting the levels of the XCL1 polypeptide, NKT, and / or CD103+ cDC1; or obtaining a result from an assay for detecting the levels of the XCL1 polypeptide, NKT, and / or CD103+ cDC1.

74. The method according to any one of claims 62 to 65, which results in higher treatment efficacy compared to a method of treating without first involving stratifying the subject.

75. A method according to any one of claims 58 to 61, which results in fewer treatment complications as compared to a method of treatment not initially involving the step of detecting the level of M. schadeletti, obtaining a result from an assay for detecting the level of M. schadeletti, the step of detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1, or obtaining a result from an assay for detecting the level of XCL1 polypeptide, NKT, and / or CD103+ cDC1.

76. A method according to any one of claims 62 to 65, which results in fewer treatment complications as compared to a method of treatment not initially involving stratifying a subject.

77. An enteric delivery formulation comprising at least one metabolite selected from Figure 4H, Figure 4I, or Figure 16.

78. Succinic acid; Propionic acid; nicotinic acid; aconitic acid (cis and / or trans); pentadecanoic acid; itaconic acid; 16-hydroxyhexadecanoic acid; C 8 H 15 NO 3 S; crotonic acid; myristic acid; 17-hydroxyheptadecanoic acid (C 17 H 34 O 3 ); and 15-hydroxypentadecanoic acid (C 15 H 30 O 3 ), an enteric delivery formulation comprising at least one metabolite selected from the group consisting of

79. An enteric delivery formulation comprising at least one metabolite selected from the group consisting of succinic acid; nicotinic acid; aconitic acid (cis and / or trans); pentadecanoic acid; itaconic acid; 16-hydroxyhexadecanoic acid; and crotonic acid.

80. An enteric delivery formulation according to any one of claims 77 to 79, formulated for delivery to the intestine.

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