WO2025064621A1

WO2025064621A1 - Compositions and methods for treating colorectal cancer

Info

Publication number: WO2025064621A1
Application number: PCT/US2024/047408
Authority: WO
Inventors: Douglas Gowers COLE; Jason Park; Sabrina Yusang YANG; Andrew John GASPARRINI; Boris Hayete
Original assignee: Flagship Pioneering Innovations VI Inc
Current assignee: Flagship Pioneering Innovations VI Inc
Priority date: 2023-09-19
Filing date: 2024-09-19
Publication date: 2025-03-27
Anticipated expiration: 2026-03-19

Abstract

The invention relates to diagnostic and therapeutic methods for colorectal cancer (CRC) and to physiologically acceptable compositions of isolated bacterial strains, lysates thereof, and supernatants therefrom for use in treating CRC.

Description

COMPOSITIONS AND METHODS FOR TREATING COLORECTAL CANCER

FIELD OF THE INVENTION

BACKGROUND

Mammals are colonized by microorganisms in the gastrointestinal (Gl) tract, on the skin, and in other epithelial and tissue niches. The gastrointestinal tract of a healthy individual harbors an abundant and diverse microbial community. It is a complex system, providing an environment or niche for a community of many different species or organisms, including diverse strains of bacteria. Hundreds of different species may form a commensal community in the Gl tract in a healthy person, and this complement of organisms evolves from the time of birth and is believed to form a functionally mature microbial population by about 3 years of age. Interactions between microbial strains in these populations and between microorganisms and the host, e.g., interactions with the host’s immune system, shape the community structure, with availability of and competition for resources affecting the distribution of microorganisms.

A healthy microbiome may provide a subject with multiple benefits, including colonization resistance to a broad spectrum of pathogens, essential nutrient biosynthesis and absorption, and immune stimulation that plays a role in maintaining a healthy gut epithelium and appropriately controlled systemic immunity. Conversely, an unhealthy (e.g., dysregulated) microbiome may be associated with a disease state.

There is a need for methods for addressing problems in healthcare through assessment and modification of the microbiome.

SUMMARY OF THE INVENTION

In one aspect, the disclosure features a method of diagnosing colorectal cancer (CRC) in a subject, the method comprising determining a level of one or more of the bacterial strains of Table 1 in a sample from the subject, wherein a level of one or more of the bacterial strains of Table 1 that is changed relative to a respective reference level for the bacterial strain of Table 1 indicates that the subject is likely to have a CRC.

In another aspect, the disclosure features a method for determining an increased risk of CRC in a subject, comprising the steps of (a) measuring a level of one or more of the bacterial strains of Table 1 in a sample collected from the subject using (i) metagenomic sequencing or (ii) amplification and one or more pairs of primers specific for a marker gene for each of the one or more of the bacterial strains of Table 1 ; and (b) using the metagenomic sequencing or amplification results to determine whether the level of one or more of the bacterial strains of Table 1 is changed relative to a respective reference level for the bacterial strain of Table 1 , thereby determining an increased risk of CRC for the subject. In some embodiments, the method comprises determining or measuring a level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Table 1 in the sample from the subject. In some embodiments, the method comprises determining or measuring a level of each of the bacterial strains of Table 1 in the sample from the subject.

In some embodiments, a level of one or more of the bacterial strains of Table 1 in the sample from the subject is changed relative to the respective reference level for the bacterial strain of Table 1 and the method further comprises administering an anti-CRC therapy to the subject.

In some embodiments, (i) the bacterial strain of Table 1 is listed in Table 2, and the change relative to a reference level is an increase in the level of the bacterial strain; or (ii) the bacterial strain of Table 1 is listed in Table 3, and the change relative to a reference level is a decrease in the level of the bacterial strain.

In another aspect, the disclosure features a method of diagnosing colorectal cancer (CRC) in a subject, the method comprising determining a level of one or more of the bacterial strains of Table 2 and/or Table 3 in a sample from the subject, wherein (i) a level of one or more of the bacterial strains of Table 2 that is increased relative to a respective reference level for the bacterial strain of Table 2 indicates that the subject is likely to have CRC; and (ii) a level of one or more of the bacterial strains of Table 3 that is decreased relative to a respective reference level for the bacterial strain of Table 3 indicates that the subject is likely to have CRC.

In another aspect, the disclosure features a method for determining an increased risk of CRC in a subject, comprising the steps of (a) measuring a level of one or more of the bacterial strains of Table 2 and/or Table 3 in a sample collected from the subject using (i) metagenomic sequencing or (ii) amplification and one or more pairs of primers specific for a marker gene for each of the one or more of the bacterial strains of Table 2 and/or Table 3; and (b) using the metagenomic sequencing or amplification results to determine whether the level of one or more of the bacterial strains of Table 2 and/or Table 3 is changed relative to a respective reference level for the bacterial strain of Table 2 and/or Table 3, wherein: (i) a level of one or more of the bacterial strains of Table 2 that is increased relative to a respective reference level for the bacterial strain of Table 2 indicates that the subject is likely to have CRC; and (ii) a level of one or more of the bacterial strains of Table 3 that is decreased relative to a respective reference level for the bacterial strain of Table 3 indicates that the subject is likely to have CRC; thereby determining an increased risk of CRC for the subject.

In some embodiments, the method comprises determining or measuring a level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Tables 2 and 3 in the sample from the subject. In some embodiments, the method comprises determining or measuring a level of each of the bacterial strains of Tables 2 and 3 in the sample from the subject.

In some embodiments, (i) a level of one or more of the bacterial strains of Table 2 is increased in the sample from the subject relative to a respective reference level for the bacterial strain of Table 2; and/or (ii) a level of one or more of the bacterial strains of Table 3 is decreased in the sample from the subject relative to a respective reference level for the bacterial strain of Table 3, and the method further comprises administering an anti-CRC therapy to the subject.

In some embodiments, the anti-CRC therapy comprises one or more of a surgery, a chemotherapy, an immunotherapy targeting vascular endothelial growth factor (VEGF), an immunotherapy targeting epidermal growth factor receptor (EGFR), an anti-BRAF therapy, a kinase inhibitor, and a checkpoint inhibitor. In some embodiments, the chemotherapy is 5-fluorouracil (5-FU), capecitabine (XELODA®), irinotecan (CAMPTOSAR®), oxaliplatin (ELOXATIN®), or trifluridine and tipiracil (LONSURF®); the immunotherapy targeting VEGF is bevacizumab (AVASTIN®), ramucirumab (CYRAMZA®), or ziv-aflibercept (ZALTRAP®); the immunotherapy targeting EGFR is cetuximab (ERBITUX®) or panitumumab (VECTIBIX®); the anti-BRAF therapy is encorafenib (BRAFTOVI®); the kinase inhibitor is regorafenib (STIVARGA®); or the checkpoint inhibitor is pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), or ipilimumab (YERVOY®).

In some embodiments, the anti-CRC therapy further comprises treatment by fecal microbiota transplant (FMT).

In some embodiments, the anti-CRC therapy comprises administration of a therapeutically effective amount of a composition comprising an isolated bacterial strain of Table 3, a lysate thereof, or a supernatant thereof. In some embodiments, the composition comprises at least two isolated bacterial strains of Table 3, lysates thereof, or supernatants thereof. In some embodiments, the composition comprises 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 isolated bacterial strains of Table 3, lysates thereof, or supernatants thereof.

In some embodiments, the composition comprises at least one isolated bacterial strain of Table 3, and the one or more isolated bacterial strains colonize the gut of the subject.

In some embodiments, the reference level is a pre-assigned level. In some embodiments, the reference level is a level in a set of samples from a reference population. In some embodiments, the reference population is a population of healthy subjects.

In some embodiments, the levels of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 of the bacterial strains of Table 2 are increased relative to a respective reference level for the bacterial strain of

Table 2.

In some embodiments, the levels of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the bacterial strains of Table 3 are decreased relative to a respective reference level for the bacterial strain of Table 3.

In some embodiments, the sample comprises a sample of the microbiota of the subject. In some embodiments, the sample is a fecal sample.

In another aspect, the disclosure features a kit for diagnosing CRC in a subject, the kit comprising (a) polypeptides or polynucleotides capable of determining the level of one or more of the bacterial strains of Table 1 in a sample from the subject; and optionally (b) instructions for use of the polypeptides or polynucleotides to determine the level of one or more of the bacterial strains of Table 1 in the sample from the subject, wherein a change in the level of one or more of the bacterial strains of Table 1 relative to a respective reference level for the bacterial strain of Table 1 indicates that the subject is likely to have CRC. In some embodiments, the kit comprises polypeptides or polynucleotides capable of determining the level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Table 1 in a sample from the subject.

In another aspect, the disclosure features a kit for diagnosing CRC in a subject, the kit comprising (a) polypeptides or polynucleotides capable of determining the level of one or more of the bacterial strains of Table 2 and/or Table 3 in a sample from the subject; and optionally (b) instructions for use of the polypeptides or polynucleotides to determine the level of one or more of the bacterial strains of Table 2 and/or Table 3 in the sample from the subject, wherein (i) a level of one or more of the bacterial strains of Table 2 that is increased relative to a respective reference level for the bacterial strain of Table 2 indicates that the subject is likely to have CRC; and (ii) a level of one or more of the bacterial strains of Table 3 that is decreased relative to a respective reference level for the bacterial strain of Table 3 indicates that the subject is likely to have CRC.

In some embodiments, the kit comprises polypeptides or polynucleotides capable of determining the level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Tables 2 and 3 in a sample from the subject.

In another aspect, the disclosure features a method of treating a subject having CRC by administering a therapy selected from the group consisting of surgery, chemotherapy, an immunotherapy targeting VEGF, an immunotherapy targeting EGFR, an anti-BRAF therapy, a kinase inhibitor, a checkpoint inhibitor, FMT, an isolated bacterial strain of Table 3, a lysate of an isolated bacterial strain of Table 3, and a supernatant of an isolated bacterial strain of Table 3, wherein the subject was diagnosed as having CRC by any of the methods provided herein.

In another aspect, the disclosure features a method of treating a subject having CRC, the method comprising the steps of (a) measuring a level of one or more of the bacterial strains of Table 1 in a sample collected from the subject, wherein a level of one or more of the bacterial strains of Table 1 that is changed relative to a respective reference level for the bacterial strain of Table 1 indicates that the subject is likely to have CRC; and (b) treating a subject who has been determined to have an increased risk of CRC by administering a therapy selected from the group consisting of surgery, chemotherapy, an immunotherapy targeting VEGF, an immunotherapy targeting EGFR, an anti-BRAF therapy, a kinase inhibitor, a checkpoint inhibitor, FMT, an isolated bacterial strain of Table 3, a lysate of an isolated bacterial strain of Table 3, and a supernatant of an isolated bacterial strain of Table 3.

In some embodiments, the levels of one or more of the bacterial strains of Table 1 in the sample collected from the subject are measured using (i) metagenomic sequencing or (ii) amplification and one or more pairs of primers specific for a marker gene for each of the one or more of the bacterial strains of Table 1 , and the metagenomic sequencing or amplification results are used to determine whether the levels of one or more of the bacterial strains of Table 1 are changed relative to a respective reference level for the bacterial strain of Table 1 .

In some embodiments, the method comprises determining or measuring a level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Table 1 in the sample from the subject. In some embodiments, the method comprises measuring the level of each of the bacterial strains of Table 1 in the sample from the subject.

In another aspect, the disclosure features a method of treating a subject having CRC, the method comprising the steps of (a) measuring a level of one or more of the bacterial strains of Table 2 and/or Table 3 in a sample collected from the subject, wherein (i) a level of one or more of the bacterial strains of Table 2 that is increased relative to a respective reference level for the bacterial strain of Table 2 indicates that the subject is likely to have CRC; and (ii) a level of one or more of the bacterial strains of Table 3 that is decreased relative to a respective reference level for the bacterial strain of Table 3 indicates that the subject is likely to have CRC; and (b) treating a subject who has been determined to have an increased risk of CRC by administering a therapy selected from the group consisting of surgery, chemotherapy, an immunotherapy targeting VEGF, an immunotherapy targeting EGFR, an anti-BRAF therapy, a kinase inhibitor, a checkpoint inhibitor, FMT, an isolated bacterial strain of Table 3, a lysate of an isolated bacterial strain of Table 3, and a supernatant of an isolated bacterial strain of Table 3.

In some embodiments, the levels of one or more of the bacterial strains of Table 2 and/or Table 3 in the sample collected from the subject are measured using (i) metagenomic sequencing or (ii) amplification and one or more pairs of primers specific for a marker gene for each of the one or more of the bacterial strains of Table 2 and/or Table 3, and the metagenomic sequencing or amplification results are used to determine whether the levels of one or more of the bacterial strains of Table 2 and/or Table 3 are changed relative to a respective reference level for the bacterial strain of Table 2 or Table 3.

In some embodiments, the method comprises determining or measuring a level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Tables 2 and 3 in the sample from the subject. In some embodiments, the method comprises measuring the level of each of the bacterial strains of Tables 2 and 3 in the sample from the subject.

In some embodiments, the levels of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 of the bacterial strains of Table 2 are increased relative to a respective reference level for the bacterial strain of Table 2.

In some embodiments, the therapy comprises one or more of a surgery, a chemotherapy, an immunotherapy targeting vascular endothelial growth factor (VEGF), an immunotherapy targeting epidermal growth factor receptor (EGFR), an anti-BRAF therapy, a kinase inhibitor, and a checkpoint inhibitor. In some embodiments, the chemotherapy is 5-fluorouracil (5-FU), capecitabine (XELODA®), irinotecan (CAMPTOSAR®), oxaliplatin (ELOXATIN®), or trifluridine and tipiracil (LONSURF®); the immunotherapy targeting VEGF is bevacizumab (AVASTIN®), ramucirumab (CYRAMZA®), or ziv- aflibercept (ZALTRAP®); the immunotherapy targeting EGFR is cetuximab (ERBITUX®) or panitumumab (VECTIBIX®); the anti-BRAF therapy is encorafenib (BRAFTOVI®); the kinase inhibitor is regorafenib (STIVARGA®); or the checkpoint inhibitor is pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), or ipilimumab (YERVOY®).

In another aspect, the disclosure features a method of treating or preventing a colorectal cancer (CRC) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an isolated bacterial strain of Table 3, a lysate thereof, or a supernatant thereof.

In some embodiments, the composition comprises at least two isolated bacterial strains of Table 3, lysates thereof, or supernatants thereof. In some embodiments, the composition comprises 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 isolated bacterial strains of Table 3, lysates thereof, or supernatants thereof.

In some embodiments, the composition comprises one or more of said isolated bacterial strains. In some embodiments, the one or more isolated bacterial strains colonize the gut of the subject.

In some embodiments, the composition comprises a lysate of one or more of said isolated bacterial strains. In some embodiments, the composition comprises a supernatant of one or more of said isolated bacterial strains.

In some embodiments, the composition has been processed to remove endotoxin.

In some embodiments, the composition has been sterilized.

In some embodiments, the composition has been processed to remove all components having a molecular weight of more than 3 kDa.

In some embodiments, the composition has been processed to remove all components having a molecular weight of less than 3 kDa.

In another aspect, the disclosure features a composition comprising an isolated bacterial strain of Table 3, a lysate thereof, or a supernatant thereof, wherein the composition is formulated as a pharmaceutically acceptable composition, a comestible composition, or a nutraceutical.

In another aspect, the disclosure features a composition comprising at least two isolated bacterial strains of Table 3, lysates thereof, or supernatants thereof. In some embodiments, the composition is formulated as a physiologically acceptable powder, granule, capsule, or tablet. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is a comestible composition. In some embodiments, the composition is a nutraceutical. In some embodiments, the pharmaceutical composition is formulated for oral, enteral, or rectal administration.

In some embodiments, the composition comprises 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 isolated bacterial strains of Table 3, lysates thereof, or supernatants thereof.

In some embodiments, the composition comprises one or more of said isolated bacterial strains. In some embodiments, the one or more isolated bacterial strains colonize the gut of a subject.

In some embodiments, the composition comprises a lysate of one or more of said isolated bacterial strains.

In some embodiments, the composition comprises a supernatant of one or more of said isolated bacterial strains.

In some embodiments, the composition has been processed to remove endotoxin.

In some embodiments, the composition has been sterilized.

In some embodiments, the composition is for treatment of CRC.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “isolated microorganism” or “isolated bacterial strain,” as used herein, refers to a microorganism (e.g., a unicellular microorganism (e.g., a bacterium)) that is removed or purified from its natural environment. An isolated microorganism may be obtained by any method or combination of methods, e.g., through the use of cell cultures.

The term “pharmaceutical composition,” as used herein, represents a composition formulated with a pharmaceutically acceptable excipient, and for example manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of a disease, disorder, or condition in a mammal. In some examples, the pharmaceutical composition is a preapproved composition.

The term “pharmaceutical dosage form,” as used herein, represents those pharmaceutical compositions intended for administration to a subject as is, without further modification (e.g., without reconstitution).

The term “physiologically acceptable composition,” as used herein, represents a composition that, within the scope of sound medical judgment, is suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benef it/risk ratio. A physiologically acceptable composition may be a comestible composition (e.g., a food product or a nutraceutical) or a pharmaceutical composition. A nutraceutical composition may be a nutraceutical oral formulation (e.g., a tablet, powder, lozenge, sachet, cachet, elixir, suspension, emulsion, solution, syrup, or soft or hard gelatin capsule), food additive (e.g., a food additive as defined in 21 C.F.R. § 170.3), food product (e.g., food for special dietary use as defined in 21 C.F.R. § 105.3), or dietary supplement (e.g., where the active agent is a dietary ingredient (e.g., as defined in 21 U.S.C. § 321 (ff))).

The term “supernatant,” as used herein, refers to (i) the liquid portion of a composition including a live microorganism (e.g., a bacterium) or (ii) dry non-aqueous component(s) of the liquid portion (e.g., dry components obtained or isolated from the liquid portion) of a composition including a live microorganism (e.g., a bacterium). A supernatant includes microbial metabolites produced by the live organism. A supernatant can include medium in which a live microorganism (e.g., bacterium) is cultured.

“Treatment” and "treating," as used herein, refer to the medical management of a subject with the intent to improve, ameliorate, stabilize, prevent, or cure a disease, disorder, or condition. This term includes active treatment (treatment directed to improve the disease, disorder, or condition); causal treatment (treatment directed to the cause of the associated disease, disorder, or condition); palliative treatment (treatment designed for the relief of symptoms of the disease, disorder, or condition); preventative treatment (treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, disorder, or condition); and supportive treatment (treatment employed to supplement another therapy).

The term “cancer,” as used herein, refers to a disease caused by an uncontrolled division of abnormal cells in a part of the body. In one instance, the cancer is a colorectal cancer (CRC). CRC includes cancers of the colon and/or rectum, e.g., adenocarcinoma of the colon or rectum. The cancer (e.g., CRC) may be locally advanced or metastatic, e.g., may be a Stage I, Stage II, Stage III, or Stage IV cancer.

The term “changed,” as used herein, refers to an observable difference in the level of a marker in a subject (e.g., in a sample from the subject), as determined using techniques and methods known in the art for the measurement of the marker. A marker level that is changed in a subject may result in a difference of at least 1% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more than 100-fold) more or less than a reference level (e.g., a level from a healthy subject or a level prior to treatment) (e.g., up to 100% or up to 100-fold relative to the reference level). In some embodiments, the change is an increase in the level of a marker in a subject. Increasing the marker level in a subject may result in an increase of at least 1% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, or at least 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more than 100-fold) relative to the reference level (e.g., up to 100% or up to 100-fold relative to the reference level). In other embodiments, the change is a decrease the level of a marker in a subject. Decreasing the marker level in a subject may result in a decrease of at least 1% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more than 100-fold) relative to the reference level (e.g., up to 100% or up to 100-fold relative to the reference level).

In some embodiments, the change in the level of a portion of the markers analyzed is an increase, while the change in the level of another portion of the markers analyzed is a decrease. In some embodiments, the change in at least 1 , 2, 3, 4, 5, 6, 7, 8, or 9 of the markers analyzed is an increase relative to a reference level. In some embodiments, the change in at least 1 , 2, 3, 4, 5, 6, 7, 8, or 9 of the markers analyzed is a decrease relative to a reference level.

The term “subject,” as used herein, represents a human or non-human animal (e.g., a mammal). I. METHODS FOR DIAGNOSING AND TREATING COLORECTAL CANCER

The invention is based, in part, on the discovery that levels of gut microbes (e.g., bacteria) can be used to identify patients having colorectal cancer (CRC). Accordingly, the disclosure provides methods of diagnosing, treating, and monitoring subjects (e.g., human patients) based on this discovery.

Lists of bacterial strains that are associated with CRC (e.g., strains having levels that change in connection with CRC and/or exhibiting a loss-of-function in connection with CRC) are provided in Tables 1 -3, below.

Table 1. Bacterial strains associated with CRC

Table 2. Bacterial strains associated with CRC disease state

Table 3. Bacterial strains associated with CRC health state

Diagnostic methods based on assessment of levels of bacteria In one aspect, the disclosure features a method of diagnosing colorectal cancer (CRC) in a subject (e.g., a human subject), the method comprising determining a level of one or more of the bacterial strains of Table 1 in a sample from the subject, wherein a level of one or more of the bacterial strains of Table 1 that is changed relative to a respective reference level for the bacterial strain of Table 1 indicates that the subject is likely to have CRC. In another aspect, the disclosure features a method for determining an increased risk of CRC in a subject, comprising the steps of (a) measuring a level of one or more of the bacterial strains of Table 1 in a sample collected from the subject using (i) metagenomic sequencing or (ii) amplification and one or more pairs of primers specific for a marker gene for each of the one or more of the bacterial strains of Table 1 ; and (b) using the metagenomic sequencing or amplification results to determine whether the level of one or more of the bacterial strains of Table 1 is changed relative to a respective reference level for the bacterial strain of Table 1 , thereby determining an increased risk of CRC for the subject. Appropriate marker genes for each bacterial strain, and primers specific for the same, could be easily identified by one of skill in the art.

Bacterial strains of Table 1 that are associated with CRC (e.g., have increased levels in subjects having CRC) are listed in Table 2. Bacterial strains of Table 1 that are associated with health (e.g., have increased levels in healthy subjects) are listed in Table 3. Accordingly, in some embodiments, (i) the bacterial strain of Table 1 is listed in Table 2, and the change relative to a reference level is an increase in the level of the bacteria; or (ii) the bacterial strain of Table 1 is listed in Table 3, and the change relative to a reference level is a decrease in the level of the bacteria.

Accordingly, in another aspect, the disclosure features a method of diagnosing CRC in a subject, the method comprising determining a level of one or more of the bacterial strains of Table 2 and/or Table 3 in a sample from the subject, wherein (i) a level of one or more of the bacterial strains of Table 2 that is increased relative to a respective reference level for the bacterial strain of Table 2 indicates that the subject is likely to have CRC; and (ii) a level of one or more of the bacterial strains of Table 3 that is decreased relative to a respective reference level for the bacterial strain of Table 3 indicates that the subject is likely to have CRC.

In some embodiments, (i) a level of one or more of the bacterial strains of Table 2 is increased relative to a respective reference level for the bacterial strain of Table 2; and/or (ii) a level of one or more of the bacterial strains of Table 3 is decreased relative to a respective reference level for the bacterial strain of Table 3, and the method further comprises administering an anti-CRC therapy to the subject.

In some embodiments of any of the above aspects, the method comprises determining or measuring a level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Table 1 in the sample from the subject (e.g., comprises determining a level of 1 -10, 10-20, 20- 30, 30-40, or 40-50 of the bacterial strains of Table 1 in the sample from the subject).

In some embodiments, a threshold number of one or more of the bacterial strains of Table 1 is changed relative to the respective reference level for the bacterial strain of Table 1 in the sample from the subject (e.g., a number of bacterial strains of Table 1 that has been determined to indicate that the patient is likely to have CRC) in the sample from the subject is changed relative to the respective reference level for the bacterial strain of Table 1 and the method further comprises administering an anti-CRC therapy to a subject.

In some embodiments, a level of a single bacterial strain of Table 1 that is changed relative to a respective reference level for the bacterial strain of Table 1 indicates that the subject is likely to have CRC. In some embodiments, a level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Table 1 in the sample from the subject (e.g., a level of 1 -10, 10-20, 20-30, 30-40, or 40-50 bacterial strains of Table 1 ) that is changed relative to a respective reference level for the bacterial strains of Table 1 indicates that the subject is likely to have CRC. In some embodiments, a changed level of all of the bacterial strains of Table 1 relative to a respective reference level for the bacterial strain of Table 1 indicates that the subject is likely to have CRC.

In some embodiments, a level of a single bacterial strain of Table 2 that is increased relative to a respective reference level for the bacterial strain of Table 2 indicates that the subject is likely to have CRC. In some embodiments, a level of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 of the bacterial strains of Table 2 in the sample from the subject (e.g., a level of 1 -5, 5-10, or 10-15 bacterial strains of Table 2 that is increased relative to a respective reference level for the bacterial strain of Table 2) indicates that the subject is likely to have CRC. In some embodiments, an increased level of all of the bacterial strains of Table 2 relative to a respective reference level for the bacterial strain of Table 2 indicates that the subject is likely to have CRC.

In some embodiments, a level of a single bacterial strain of Table 3 that is decreased relative to a respective reference level for the bacterial strain of Table 3 indicates that the subject is likely to have CRC. In some embodiments, a level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 15, or all 20 of the bacterial strains of Table 3 in the sample from the subject (e.g., a level of 1-5, 5-10, 10-15, or 15-20 bacterial strains of Table 3 that is decreased relative to a respective reference level for the bacterial strain of Table 3) indicates that the subject is likely to have CRC. In some embodiments, a decreased level of all of the bacterial strains of Table 3 relative to a respective reference level for the bacterial strain of Table 3 indicates that the subject is likely to have CRC.

Determination of whether a detected difference (e.g., increase or decrease) in the level of a bacterial strain or a related marker is significant can be carried out using standard methods, as well as statistical analysis. In some embodiments, a significant difference is a change (e.g., increase or decrease) of at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, 100%, or more, e.g., at least 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more than 100-fold, relative to a reference level. In some embodiments, the change in a level of one or more of the bacterial strains of Table 1 (e.g., one or more bacterial strains of Table 3) in the sample from the subject is an increase relative to the respective reference level for the bacterial strain of Table 1 (e.g., an increase of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,

42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,

60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,

78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%,

96%, 97%, 98%, 99%, 100%, or more than 100%, e.g., an increase of, e.g., 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more than 100- fold, relative to the respective level; or an increase of 1 -5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 0-85%, 85- 90%, 90-95%, 95-100%, or more than 100%, relative to the respective reference level).

In some aspects, the level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Table 1 in the sample from the subject (e.g., a level of 1 -10, 10-20, 20-30, 30-40, or 40-50 bacterial strains of Table 1 ) are increased relative to a respective reference level for the bacterial strain.

In some embodiments, the change in a level of one or more of the bacterial strains of Table 1 (e.g., one or more bacterial strains of Table 3) in the sample from the subject is a decrease relative to the respective reference level for the bacterial strain of Table 1 (e.g., bacterial strain of Table 3) (e.g., a decrease of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,

35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,

53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,

71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,

89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, e.g., a decrease of, e.g., at least 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more than 100-fold) relative to the respective reference level; or a decrease of 1 -5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65- 70%, 70-75%, 75-80%, 0-85%, 85-90%, 90-95%, or 95-100% relative to the respective reference level).

In some aspects, the level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Table 1 in the sample from the subject (e.g., a level of 1 -10, 10-20, 20-30, 30-40, or 40-50 bacterial strains of Table 1 ) are decreased relative to a respective reference level for the bacterial strain. Methods of treatment based on assessment of levels of bacterial strains

In some aspects, the invention provides a method of treating or preventing CRC in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount an anti-CRC therapy (e.g., an anti-CRC therapy provided below).

For example, in one aspect, the disclosure features a method of treating a subject having CRC by administering to the subject a therapy selected from the group consisting of surgery, chemotherapy, an immunotherapy targeting VEGF, an immunotherapy targeting EGFR, an anti-BRAF therapy, a kinase inhibitor, a checkpoint inhibitor, FMT, an isolated bacteria of Table 3, a lysate of an isolated bacteria of Table 3, a supernatant of an isolated bacteria of Table 3, and a modulator of a bacterial strain of Table 1 , wherein the subject was diagnosed as having CRC by any of the methods provided herein.

In another aspect, the disclosure provides a method of treating a subject having CRC, the method comprising the steps of (a) measuring a level of one or more of the bacterial strains of Table 1 in a sample collected from the subject, wherein a level of one or more of the bacterial strains of Table 1 that is changed relative to a respective reference level for the bacterial strain of Table 1 indicates that the subject is likely to have CRC; and (b) treating a subject who has been determined to have an increased risk of CRC by administering an anti-CRC therapy (e.g., an anti-CRC therapy provided below, e.g., a therapy selected from the group consisting of surgery, chemotherapy, an immunotherapy targeting VEGF, an immunotherapy targeting EGFR, an anti-BRAF therapy, a kinase inhibitor, a checkpoint inhibitor, FMT, an isolated bacteria of Table 3, a lysate of an isolated bacteria of Table 3, a supernatant of an isolated bacteria of Table 3, and a modulator of a bacterial strain of Table 1 ).

In some aspects, the levels of one or more of the bacterial strains of Table 1 in the sample collected from the subject are measured using (i) metagenomic sequencing or (ii) amplification and one or more pairs of primers specific for a marker gene for each of the one or more of the bacterial strains of Table 1 , and the metagenomic sequencing or amplification results are used to determine whether the level of one or more of the bacterial strains of Table 1 is changed relative to a respective reference level for the bacterial strain of Table 1 . In some aspects, the method comprises determining or measuring a level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Table 1 in the sample from the subject. In some aspects, the method comprises measuring the level of each of the bacterial strains of Table 1 in the sample from the subject.

In another aspect, the disclosure provides a method of treating a subject having CRC, the method comprising the steps of (a) measuring a level of one or more of the bacterial strains of Table 2 and/or Table 3 in a sample collected from the subject, wherein (i) a level of one or more of the bacterial strains of Table 2 that is increased relative to a respective reference level for the bacterial strain of Table 2 indicates that the subject is likely to have CRC; and (ii) a level of one or more of the bacterial strains of Table 3 that is decreased relative to a respective reference level for the bacterial strain of Table 3 indicates that the subject is likely to have CRC; and (b) treating a subject who has been determined to have an increased risk of CRC by administering an anti-CRC therapy (e.g., an anti-CRC therapy provided below, e.g., a therapy selected from the group consisting of surgery, chemotherapy, an immunotherapy targeting VEGF, an immunotherapy targeting EGFR, an anti-BRAF therapy, a kinase inhibitor, a checkpoint inhibitor, FMT, an isolated bacteria of Table 3, a lysate of an isolated bacteria of Table 3, a supernatant of an isolated bacteria of Table 3, and a modulator of a bacterial strain of Table 1 ).

In some aspects, the level of one or more of the bacterial strains of Table 2 and/or Table 3 in the sample collected from the subject are measured using (i) metagenomic sequencing or (ii) amplification and one or more pairs of primers specific for a marker gene for each of the one or more of the bacterial strains of Table 2 and/or Table 3, and the metagenomic sequencing or amplification results are used to determine whether the level of one or more of the bacterial strains of Table 2 and/or Table 3 is changed relative to a respective reference level for the bacterial strain of Table 2 or Table 3. In some aspects, the method comprises determining or measuring a level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Tables 2 and 3 in the sample from the subject. In some aspects, the method comprises measuring the level of each of the bacterial strains of Tables 2 and 3 in the sample from the subject.

Anti-CRC therapies

An anti-CRC therapy may be any medicament, treatment, or combination thereof suitable for the treatment of CRC.

In some aspects, the anti-CRC therapy comprises one or more of a surgery; a chemotherapy (e.g., a chemotherapy comprising 5-fluorouracil (5-FU); capecitabine (XELODA®); irinotecan (CAMPTOSAR®); oxaliplatin (ELOXATIN®); trifluridine and tipiracil (LONSURF®); or 5-fluorouracil (FU), leucovorin (LV), and either oxaliplatin (FOLFOX) or irinotecan (FOLFIRI)); an anti-vascular endothelial growth factor (VEGF) therapy, e.g., an immunotherapy targeting VEGF (e.g., bevacizumab (AVASTIN®), ramucirumab (CYRAMZA®), or ziv-aflibercept (ZALTRAP®)); an immunotherapy targeting epidermal growth factor receptor (EGFR) (e.g., cetuximab (ERBITUX®) or panitumumab (VECTIBIX®)); an anti- BRAF therapy (e.g., encorafenib (BRAFTOVI®)); a kinase inhibitor (e.g., regorafenib (STIVARGA®)); and a checkpoint inhibitor (e.g., pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), or ipilimumab (YERVOY®)). In some aspects, the anti-CRC therapy further comprises treatment by fecal microbiota transplant (FMT).

In some aspects, the anti-CRC therapy comprises ALYMSYS®, MVASI®, ZIRABEV®, AVASTIN® (bevacizumab), CAMPTOSAR® (irinotecan hydrochloride), XELODA® (capecitabine), ERBITUX® (cetuximab), CYRAMZA® (ramucirumab), ELOXATIN® (oxaliplatin), 5-fluorouracil (5-FU), fluorouracil, YERVOY® (ipilimumab), KEYTRUDA® (pembrolizumab), leucovorin calcium, LONSURF® (trifluridine and tipiracil hydrochloride), OPDIVO® (nivolumab), oxaliplatin, panitumumab, ramucirumab, STIVARGA® (regorafenib), tucatinib, TUKYSA® (tucatinib), VECTIBIX® (panitumumab), or ZALTRAP® (ziv-aflibercept).

In some aspects, the anti-CRC therapy comprises a drug combination selected from CAPOX, FOLFIRI, FOLFIRI-bevacizumab, FOLFIRI-cetuximab, FOLFOX, FU-LV, XELIRI, and XELOX.

In some aspects, the anti-CRC therapy comprises an immune checkpoint-targeting antibody, e.g., an antibody targeting the T-cell receptor cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) (e.g., botensilimab) or an antibody designed to block PD-1 from interacting with PD-L1 and PD-L2 (e.g., balstilimab).

In some aspects, the anti-CRC therapy comprises a tyrosine kinase inhibitor (TKI) targeting VEGFr-1 ,2,3 (e.g., fruquintinib). In some aspects, the anti-CRC therapy comprises a KRAS G12C inhibitor (e.g., adagrasib (KRAZATI®)). In some aspects, the anti-CRC therapy comprises a dosing regimen comprising administration of encorafenib (BRAFTOVI®) and cetuximab (ERBITUX®) (e.g., in a subject whose tumor has a BRAP^W0E mutation). In some aspects, the anti-CRC therapy comprises a dosing regimen comprising administration of bevacizumab (AVASTIN®) and the combination of trifluridine and tipiracil (LONSURF®)ln some aspects, the anti-CRC therapy comprises administration of a therapeutically effective amount of a composition comprising an isolated bacterial strain of Table 3, a lysate thereof, or a supernatant thereof (e.g., an isolated bacterial strain, lysate thereof, or supernatant thereof as described in Section II herein). For example, in some embodiments, the composition comprises at least two isolated bacterial strains of Table 3, lysates thereof, or supernatants thereof, e.g., comprises 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 isolated bacterial strains of Table 3, lysates thereof, or supernatants thereof (e.g., comprises 3-, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, or 18-20 isolated bacterial strains of Table 3, lysates thereof, or supernatants thereof). In some embodiments in which the composition comprises at least one isolated bacterial strain of Table 3, one or more of the isolated bacterial strains colonize the gut of the subject.

In some aspects, the anti-CRC therapy comprises a modulator of a bacterial strain of Table 1 or comprises use of a method that modulates a bacterial strain of Table 1 ). As used herein, “modulating” refers to an observable change in the level (e.g., abundance of viable bacteria) or activity (e.g., metabolic activity, e.g., production of one or more metabolites) of a bacterial strain in a subject, e.g., as measured using techniques and methods known in the art. Modulating the level or activity of a bacterial strain in a subject may result in a change of at least 1% relative to prior to administration of the modulator or use of the modulating method (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, at least 100%, or more than 100% or more relative to prior to administration; e.g., up to 100% relative to prior to administration).

For example, in some embodiments, the bacterial strain of Table 1 is associated with health (e.g., is listed in Table 3), and modulating is increasing the level or activity of the bacterial strain in a subject. For example, the level or activity (e.g., metabolic activity, e.g., production of one or more metabolites) of the bacterial strain in the subject may be increased by at least 1% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or more than 100% more relative to prior to administration of the modulator or use of the modulating method). In some embodiments, the modulator is the bacterial strain of Table 1 (e.g., a live bacterial culture is administered).

In other embodiments, the bacterial strain of Table 1 is associated with disease (e.g., is listed in Table 2), and modulating is decreasing the level or activity of the bacterial strain in a subject. For example, the level or activity (e.g., metabolic activity, e.g., production of one or more metabolites) of the bacterial strain in the subject may be decreased by at least 1% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, at least 98%, or 100% or more relative to prior to administration of the modulator or use of the modulating method; e.g., up to 100%).

In some aspects, the modulator or modulating method increases the level or activity of two or more bacterial strains of Table 1 (e.g., increases the level or activity of two or more health-associated bacterial strains of Table 1 ); decreases the level or activity of two or more bacterial strains of Table 1 (e.g., decreases the level or activity of two or more disease-associated bacterial strains of Table 1 ); or increases the level or activity of one or more health-associated bacterial strains of Table 1 and decreases the level or activity of one or more disease-associated bacterial strains of Table 1 .

In embodiments in which the level or activity of a bacterial strain is increased or decreased in a subject following a step of administering a composition described herein, the increase or decrease may take place and/or be detectable within a range of time following the administration (e.g., within six hours, 24 hours, 3 days, a week or longer), and may take place and/or be detectable after one or more administrations (e.g., after 2, 3, 4, 5, 6, 7, 8, 9, 10, or more administrations, e.g., as part of a dosing regimen for the subject).

Any one or more of these therapies may optionally be used in any of the methods, kits, and compositions described herein as employing an anti-CRC therapy.

Sample type

The sample from which the level of one or more of the bacterial strains of Table 1 , Table 2, or Table 3 is determined may be any appropriate sample from the subject. In some embodiments, the sample comprises a sample of the microbiota (e.g., the gut microbiota) of the subject. In some embodiments, the sample is a fecal sample. In other embodiments, the sample is a colon biopsy.

Levels of bacterial strains, reference levels, and determination thereof

The presence or level of a bacterial strain (e.g., a bacterial strain of Table 1 , Table 2, or Table 3) in a sample from a subject may be determined using any method known in the art. For example, in some embodiments, the presence or level of one or more bacterial strains in a sample is determined by metagenomic sequencing. In some embodiments, the presence or level of one or more bacterial strains in a subject is determined by using one or more pairs of primers specific for a marker gene for the bacterial strain to amplify the marker gene (e.g., using polymerase chain reaction (PCR)), and detecting the presence or level of the amplification product. Appropriate marker genes for each bacterial strain, and primers specific for the same, could be easily identified by one of skill in the art. In still other embodiments, the presence or level of one or more bacterial strains in a sample is determined by a method comprising culturing or attempting to culture the bacterial strain from the sample. Additionally or alternatively, the presence or level of a bacterial strain in a sample from a subject may be determined by detecting or attempting to detect the presence or level of any appropriate molecular marker for the bacterial strain in the sample (e.g., a nucleic acid (e.g., DNA or RNA) marker, a protein or polypeptide marker, a chemical marker, or a metabolite. Methods for detecting molecular markers may include, e.g., PCR-based methods, detection of RNA levels (e.g., RT-PCR, RNA-Seq, and/or methods including the use of microarrays, as are known in the art), and detection of protein levels (e.g., immunoassay-based approaches).

In some embodiments, a bacterial strain is identified based on features including, e.g., 16S rDNA sequence analysis. The level of sequence identity used in the identification can be, e.g., at least 95%, 96%, 97%, 98%, or 99% (e.g., 100%) identity to full length, V4, or V6 region 16S rDNA sequences, as is known in the art. Determination of sequence identity can be carried out using standard approaches including, e.g., default values of the alignment programs, for example, BLAST (blast.ncbi.nlm.nih.gov).

In some embodiments of any of the above aspects, the reference level of the one or more bacterial strains of Table 1 , Table 2, or Table 3 is a pre-assigned level.

In some embodiments, the respective reference level for a bacterial strain of Table 1 is a preassigned level of the bacterial strain of Table 1 .

In some embodiments, the respective reference level for the bacterial strain of Table 1 is a level in a set of samples from a reference population, e.g., a population of healthy subjects (e.g., a population of subjects not having CRC and/or a population of subjects having a healthy gut microbiome). In some embodiments, the healthy subjects are healthy human subjects.

II. MICROORGANISMS, LYSATES THEREOF, SUPERNATANTS THEREFROM, AND RELATED METHODS OF TREATMENT

Microorganisms

In some aspects, the invention provides a composition (e.g., a composition for treating CRC) comprising an isolated bacterial strain of Table 2 (e.g., comprising 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or all 20 of the bacterial strains of Table 3).

The bacterial strain may be isolated (e.g., isolated from a culture, a sample, or a material) using techniques and methods known in the art. In some examples, the bacterial strain is obtained from a stock center, e.g., the American Type Culture Collection (ATCC®) or the German Collection of Microorganisms and Cell Cultures GmbH (DSM). In some examples, identifying information such as that provided in the tables herein can be used in obtaining the bacterial strain(s). In other examples, the bacterial strain is obtained from a biological sample. For example, in some instances, the bacterial strain is a purified population obtained from a microbiotal material such as a fecal material. In such instances, the bacterial strain can be identified based on features including, e.g., 16S rDNA sequence analysis. The level of sequence identity used in the identification can be, e.g., at least 95%, 96%, 97%, 98%, or 99% (e.g., 100%) identity to full length, V4, or V6 region 16S rDNA sequences, as is known in the art. Determination of sequence identity can be carried out using standard approaches including, e.g., default values of the alignment programs, for example, BLAST (blast.ncbi.nlm.nih.gov). Levels of identity as described herein can also be used to identify additional, related bacterial strains that can be used in the invention. In some examples, a bacterial population that is directly isolated from a fecal material does not result from any culturing or other process that results in or is intended to result in replication of the population after obtaining the fecal material. In some instances, the bacterial strain is isolated from a live bacterial culture. The live cultured bacteria may be prepared using cell culture techniques and methods known in the art. Typically, isolation of live cultured bacterial cells includes separation from the broth, e.g., by centrifuging, filtration, or decanting. The cells separated from the fermentation broth are optionally washed by water, saline (e.g., 0.9% NaCI), or with any suitable buffer. In some instances, the wet cell mass obtained is dried. The cell mass may be dried by any suitable method, e.g., dried by lyophilization.

The bacterial strain in the composition may be, e.g., live bacteria (e.g., live cultured bacteria) dead bacteria, (e.g., killed bacteria, e.g., heat-killed bacteria or irradiated bacteria), bacteria in a vegetative state, bacteria in a spore form (e.g., an endospore), or a combination thereof.

In some examples, the preparation is substantially free of contaminating bacteria (i.e., bacteria other than the bacterial strain intended to be isolated from the culture, sample, or material). In some aspects, the preparation is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% free of contaminating bacteria.

The composition comprising the bacterial strain may be further processed, e.g., sterilized and/or processed to remove endotoxin.

Microbial lysates

In some aspects, the invention provides a composition (e.g., a composition for treating CRC) comprising a lysate of an isolated bacterial strain of Table 3 (e.g., comprising a lysate of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or all 20 of the bacterial strains of Table 3). The bacteria may be lysed prior to or after their incorporation into the composition (e.g., physiologically acceptable composition). Lysis of bacteria may be performed using techniques and methods known in the art for cell lysis. In one exemplary method for preparing a microbial lysate, spun-down cells are suspended in 20 mL MeOH and sonicated for 20 minutes. Cell debris is then spun down, and collected supernatant is dried under vacuum using a rotary evaporator. The dried extract is resuspended in MeOH/H2O(1 :1 ) and filtered to remove components having a molecular weight of more than 3kDa.

In some examples, the preparation comprising the lysed bacteria is substantially free of non-lysed bacteria, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% free of non-lysed bacteria.

The composition comprising the lysate of the isolated bacteria may be further processed, e.g., sterilized and/or processed to remove endotoxin.

In some examples, the composition comprising the lysate is processed to remove compounds having a particular molecular weight, e.g., is processed to remove compounds having a molecular weight above or below a threshold. The processing may comprise, e.g., filtration.

For example, in some instances, the composition comprising the lysate of the isolated bacteria is processed (e.g., filtered) to remove components having a molecular weight of more than 100 kDa, more than 50 kDa, more than 25 kDa, more than 20 kDa, more than 15 kDa, more than 10 kDa, more than 9 kDa, more than 8 kDa, more than 7 kDa, more than 6 kDa, more than 5 kDa, more than 4 kDa, more than 3.5 kDa, more than 3 kDa, more than 2.5 kDa, more than 2 kDa, more than 1 .5 kDa, more than 1 kDa, more than 0.5 kDa, or more than 0.25 kDa. In some examples, the composition has been processed to remove all components having a molecular weight of more than 3 kDa.

In some instances, the composition comprising the lysate of the isolated bacteria is processed (e.g., filtered) to remove components having a molecular weight of less than 100 kDa, less than 50 kDa, less than 25 kDa, less than 20 kDa, less than 15 kDa, less than 10 kDa, less than 9 kDa, less than 8 kDa, less than 7 kDa, less than 6 kDa, less than 5 kDa, less than 4 kDa, less than 3.5 kDa, less than 3 kDa, less than 2.5 kDa, less than 2 kDa, less than 1 .5 kDa, less than 1 kDa, less than 0.5 kDa, or less than 0.25 kDa. In some examples, the composition has been processed to remove all components having a molecular weight of less than 3 kDa.

Microbial supernatants

In some aspects, the invention provides a composition (e.g., a composition for treating CRC) comprising a supernatant of a culture of an isolated bacterial strain of Table 3 (e.g., comprises a supernatant of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or all 20 of the bacterial strains of Table 3), e.g., comprises a liquid medium in which the bacterial strain has been cultured or a fraction thereof.

The supernatant may contain molecules secreted or otherwise produced by the bacterial strain, e.g., may contain polypeptides, lipids, nucleic acids, and/or small molecules secreted or otherwise produced by the bacterial strain. The bacterial strain may be cultured, and the supernatant may be produced using methods known in the art. For example, the supernatant may be produced by centrifuging the culture and separating the supernatant from pelleted bacteria. In other examples, the supernatant is produced by filtering the culture medium to remove bacteria.

In some examples, the supernatant is substantially free of bacteria, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% free of bacteria (e.g., live bacteria).

The supernatant from the isolated bacteria may be further processed, e.g., sterilized and/or processed to remove endotoxin.

In some examples, the supernatant is processed to remove compounds having a particular molecular weight, e.g., is processed to remove compounds having a molecular weight above or below a threshold. The processing may comprise, e.g., filtration.

For example, in some instances, the supernatant is processed (e.g., filtered) to remove components having a molecular weight of more than 100 kDa, more than 50 kDa, more than 25 kDa, more than 20 kDa, more than 15 kDa, more than 10 kDa, more than 9 kDa, more than 8 kDa, more than 7 kDa, more than 6 kDa, more than 5 kDa, more than 4 kDa, more than 3.5 kDa, more than 3 kDa, more than 2.5 kDa, more than 2 kDa, more than 1 .5 kDa, more than 1 kDa, more than 0.5 kDa, or more than 0.25 kDa. In some examples, the supernatant has been processed to remove all components having a molecular weight of more than 3 kDa. In some instances, the supernatant is processed (e.g., filtered) to remove components having a molecular weight of less than 100 kDa, less than 50 kDa, less than 25 kDa, less than 20 kDa, less than 15 kDa, less than 10 kDa, less than 9 kDa, less than 8 kDa, less than 7 kDa, less than 6 kDa, less than 5 kDa, less than 4 kDa, less than 3.5 kDa, less than 3 kDa, less than 2.5 kDa, less than 2 kDa, less than 1 .5 kDa, less than 1 kDa, less than 0.5 kDa, or less than 0.25 kDa. In some examples, the supernatant has been processed to remove all components having a molecular weight of less than 3 kDa.

Combinations of bacteria, supernatants, and lysates

In some instances, the composition comprises two or all three of a bacterial strain, a lysate of a bacterial strain, and a supernatant of a culture of an isolated bacterial strain of Table 3. In some instances, the composition comprises two or all three of a bacterial strain, a lysate of the same bacterial strain, and a supernatant from the same bacterial strain. In other instances, the composition comprises two or all three of a bacterial strain, a lysate, and a supernatant, wherein the bacterial strain in the composition, the bacterial strain from which the lysate is derived, and the bacterial strain from which the supernatant is derived are different bacterial strains.

Formulations and compositions

Compositions (e.g., physiologically acceptable compositions) described herein may be prepared using techniques and methods known in the art.

The active agents (e.g., bacterial strains, lysates thereof, and supernatants therefrom) disclosed herein may be formulated into physiologically acceptable compositions (e.g., pharmaceutical or nutraceutical compositions) for administration to human subjects in a biologically compatible form suitable for administration in vivo. Pharmaceutical and nutraceutical compositions typically include an active agent as described herein and a physiologically acceptable excipient (e.g., a pharmaceutically acceptable excipient). The active agents useful in physiologically acceptable compositions described herein include isolated bacteria, lysates thereof, and supernatants therefrom.

The active agents (e.g., bacterial strains, lysates thereof, and supernatants therefrom) described herein may be administered, for example, by oral, rectal (e.g., by enema, ointment, or suppository), enteral, parenteral, buccal, sublingual, nasal, patch, pump, or transdermal administration, and the pharmaceutical or nutraceutical compositions formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.

For human use, an active agent disclosed herein can be administered alone or in admixture with a pharmaceutical or nutraceutical carrier selected regarding the intended route of administration and standard pharmaceutical practice. Pharmaceutical and nutraceutical compositions for use in accordance with the present invention thus can be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries that facilitate processing of active agents disclosed herein into preparations which can be used pharmaceutically.

This disclosure also includes pharmaceutical and nutraceutical compositions which can contain one or more physiologically acceptable carriers. In making the pharmaceutical or nutraceutical compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semisolid, or liquid material (e.g., normal saline), which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of, e.g., tablets, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, and soft and hard gelatin capsules. As is known in the art, the type of diluent can vary depending upon the intended route of administration. The resulting compositions may include additional agents, e.g., preservatives. Nutraceutical compositions may be administered enterally (e.g., orally). A nutraceutical composition may be a nutraceutical oral formulation (e.g., a tablet, powder, lozenge, sachet, cachet, elixir, suspension, emulsion, solution, syrup, or soft or hard gelatin capsule), a food additive (e.g., a food additive as defined in 21 C.F.R. § 170.3), a food product (e.g., food for special dietary use as defined in 21 C.F.R. § 105.3), or a dietary supplement (e.g., where the active agent is a dietary ingredient (e.g., as defined in 21 U.S.C. § 321 (ff))). Active agents (e.g., bacteria, lysates thereof, and supernatants therefrom) can be used in nutraceutical applications and as food additive or food products. Non-limiting examples of compositions including an active agent of the invention are a bar, drink, shake, powder, additive, gel, or chew.

The excipient or carrier is selected on the basis of the mode and route of administration. Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed., Lippincott Williams & Wilkins (2005), a well-known reference text in this field, and in the USP/NF (United States Pharmacopeia and the National Formulary). Examples of suitable excipients are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include lubricating agents, e.g., talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents, e.g., methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. Other exemplary excipients are described in Handbook of Pharmaceutical Excipients, 6th Edition, Rowe et al., Eds., Pharmaceutical Press (2009).

These pharmaceutical and nutraceutical compositions can be manufactured in a conventional manner, e.g., by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Methods well known in the art for making formulations are found, for example, in Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed., Lippincott Williams & Wilkins (2005), and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York. Proper formulation is dependent upon the route of administration chosen. The formulation and preparation of such compositions is well-known to those skilled in the art of pharmaceutical and nutraceutical formulation. In preparing a formulation, the active agents can be milled to provide the appropriate particle size prior to combining with the other ingredients. If the active agent is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If the active agent is substantially water soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, e.g., about 40 mesh.

In instances in which the composition comprises a bacterial strain, the physiologically acceptable compositions described herein may be formulated such that a single unit dose contains at least about 1 x10⁴ colony forming units (cfu) of the bacteria, and a single consumed unit may contain, e.g., about 1 x10⁴, 1 x10⁵, 1 x10⁶, 1 x10⁷, 1 x10⁸, 1 x10⁹, 1 x10¹°, 1 x10¹¹ , 1 x10¹², 1 x10¹³, 1 x10¹⁴, 1 x10¹⁵, or greater than 1 x10¹⁵ cfu of the bacteria; or contain at least 1 x10⁴, 1 x10⁵, 1 x10⁶, 1 x10⁷, 1 x10⁸, 1 x10⁹, 1 x10¹°, 1 x10¹¹ , 1 x10¹², 1 x10¹³, 1 x10¹⁴, 1 x10¹⁵, or greater than 1 x10¹⁵ cfu. The concentration of bacteria of a given species is e.g., 1 x10⁴, 1 x10⁵, 1 x10⁶, 1 x10⁷, 1 x10⁸, 1 x10⁹, 1 x10¹°, 1 x10¹¹ , 1 x10¹², 1 x10¹³, 1 x10¹⁴, 1 x10¹⁵, or greater than 1 x10¹⁵ viable bacteria per gram of the physiologically acceptable composition. The bacteria are administered, for example, as live cultured bacteria, in vegetative or spore form or as a combination of vegetative and spore forms. Alternatively, the bacteria are provided as purified populations obtained from a microbiotal material such as a fecal material.

The amount of bacteria contained in a unit dose of a physiologically acceptable composition may be about 0.001 mg to about 1 mg, about 0.5 mg to about 5 mg, about 1 mg to about 1000 mg, about 2 mg to about 200 mg, about 2 mg to about 100 mg, about 2 mg to about 50 mg, about 4 mg to about 25 mg, about 5 mg to about 20 mg, about 10 mg to about 15 mg, about 50 mg to about 200 mg, about 200 mg to about 1000 mg, or about 1 , 2, 3, 4, 5 or more than 5 g per dose or composition; or 0.001 mg to 1 mg, 0.5 mg to 5 mg, 1 mg to 1000 mg, 2 mg to 200 mg, or 2 mg to 100 mg, or 2 mg to 50 mg, or 4 mg to 25 mg, or 5 mg to 20 mg, or 10 mg to 15 mg, or 50 mg to 200 mg, or 200 mg to 1000 mg, or 1 , 2, 3, 4, 5 or more than 5 g per dose or composition.

A physiologically acceptable composition described herein may be a food product, e.g., a medical food product. A food product may be, e.g., a dairy product, an infant food product, a fruit-containing food product, a vegetable-containing food product, or a sports nutrition food product. The food products can be applied in infant diets, adult diets and special diets, including diets requiring medical foods or functional foods. The bacterial compositions can be incorporated in powder or in liquid form in foods used by the general population, particularly milk and milk-derived products, especially fermented milk and cheeses; cereals and derivatives, including bread, bread doughs, cakes, cookies, crackers, extruded snacks; soups and other similar products in dehydrated form; fermented meat products; fruit and vegetable derivatives, juices and soft drinks; foods for specific nutritional uses, including infant milk, infant cereals, ready-to-eat infant foods, etc. They can also be found in food supplements and special formulas for oral and enteral nutrition for clinical use. In other embodiments, the foodstuff is yogurt, kefir, yakult, miso, natto, tempeh, kimchee, sauerkraut, water, coffee, tea, beer, wine, liquor, alcoholic mixed drinks, soups, frozen desserts, fried foods, pasta products, potato products, rice products, corn products, wheat products, dairy products, confectioneries, hard candies, nutritional bars, and breakfast cereals.

Generally, the bacterial population includes bacteria present in the foodstuff in an amount from about 10⁴ to about 10¹² cfu per gram of foodstuff, e.g., from 10⁴ to 10¹² cfu per gram of foodstuff. Additionally, the bacterial population may be stabilized to prevent spoilage of the foodstuff. Generally, the bacterial population is present in the liquid in an amount from about 10⁴ to about 10¹² cfu per gram of liquid, e.g., from 10⁴ to 10¹² cfu per gram of liquid. The beverage may be hot, warm, room temperature, cool, or cold.

Physiologically acceptable compositions described herein may be prepared using methods known in the art and those described herein. Typically, preparation of a physiologically acceptable composition involves combining an effective amount of the active agent (e.g., a bacteria composition and/or microbial metabolite) with a carrier. For comestible compositions, e.g., orally administered pharmaceutical composition and food products (foodstuffs and beverages), an effective amount of the bacterial population is a population containing an amount of bacteria such that the population is at least partially retained in the gastrointestinal tract of a subject that consumes the composition. For example, the bacterial population contains bacteria present in the carrier in an amount from about 10⁴ to about 10¹² cfu per gram of carrier, e.g., from 10⁴ to 10¹² cfu per gram of carrier.

A comestible composition may be provided in a unit dose or serving of 5-500 g, e.g., 5-15 g, 15- 50 g, 25-75 g, 50-100 g, 100-200 g, 200-300 g, 300-400 g, or 400-500 g. For example, a yogurt composition can be about 4, 6, 8, 10 or 12 ounces, or a quarter, half, three-quarters or whole cup. In one embodiment, a typical serving size for a beverage product such as a fluid is about 10-500 ml, e.g., 10-25 ml, 25-50 ml, 50-75 ml, 75-100 ml, 100-150 ml, 150-200 ml, 250-300 ml, 300-400 ml, or 400-500 ml.

A physiologically acceptable composition may be a pharmaceutical composition, e.g., for administration in solid, semi-solid, micro-emulsion, gel, or liquid form. Examples of such dosage forms include tablet forms disclosed in U.S. Pat. Nos. 3,048,526, 3,108,046, 4786505, 4,919,939, and 4,950,484; gel forms disclosed in U.S. Pat. Nos. 4,904,479, 6,482,435, 6,572,871 , and 5,013,726; capsule forms disclosed in U.S. Pat. Nos. 4,800,083, 4,532,126, 4935243, and 6,258,380; or liquid forms disclosed in U.S. Pat. Nos. 4,625,494, 4,478,822, and 5,610,184; each of which is incorporated herein by reference in its entirety. Forms of the compositions that can be used orally include tablets, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with binders (e.g., povidone, gelatin, hydroxypropylmethyl cellulose), inert diluents, preservative, antioxidant, disintegrant (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) or lubricating, surface active or dispersing agents. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets can optionally be coated or scored and can be formulated so as to provide slow or controlled release of the active ingredient therein. Tablets can optionally be provided with an enteric coating, to provide release in stomach or in parts of the gut (e.g., colon, lower intestine) other than the stomach. All formulations for oral administration can be in dosages suitable for such administration. The push-fit capsules can contain the active ingredients in admixture with filler, such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds (prebiotics or probiotics) can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions can be used, which can optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water soluble carrier such as polyethylene glycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil. Oral liquid preparations can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions syrups or elixirs, or can be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations can contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, acacia; nonaqueous vehicles (which can include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavoring or coloring agents.

A physiologically acceptable composition may be a softgel formulation. A softgel can contain a gelatin-based shell that surrounds a liquid fill. The shell can be made of gelatin, plasticizer (e.g., glycerin and/or sorbitol), modifier, water, color, antioxidant, or flavor. The shell can be made with starch or carrageenan. The outer layer can be enteric coated. In one embodiment, a softgel formulation can include a water or oil soluble fill solution, or suspension of a composition, for example, a prebiotic composition, covered by a layer of gelatin.

An enteric coating can control the location of where a physiologically acceptable composition is absorbed in the digestive system. For example, an enteric coating can be designed such that a physiologically acceptable composition does not dissolve in the stomach but rather travels to the small intestine, where it dissolves. An enteric coating can be stable at low pH (e.g., in the stomach) and can dissolve at higher pH (e.g., in the small intestine). Material that can be used in enteric coatings includes, for example, alginic acid, cellulose acetate phthalate, plastics, waxes, shellac, and fatty acids (e.g., stearic acid, palmitic acid). Enteric coatings are described, for example, in U.S. Pat. Nos. 5,225,202, 5,733,575, 6,139,875, 6,420,473, 6,455,052, and 6,569,457, all of which are herein incorporated by reference in their entirety. The enteric coating can be an aqueous enteric coating. Examples of polymers that can be used in enteric coatings include, for example, shellac, cellulose acetate phthalate, polyvinylacetate phthalate, and methacrylic acid. Enteric coatings can be used to (1 ) prevent the gastric juice from reacting with or destroying the active substance, (2) prevent dilution of the active substance before it reaches the intestine, (3) ensure that the active substance is not released until after the preparation has passed the stomach, and (4) prevent live bacteria contained in the preparation from being killed because of the low pH-value in the stomach. In one embodiment a bacterial composition or the bacterial component of a food or beverage is provided as a tablet, capsule, or caplet with an enteric coating. In one embodiment the enteric coating is designed to hold the tablet, capsule, or caplet together when in the stomach. The enteric coating is designed to hold together in acid conditions of the stomach and break down in non-acid conditions and therefore release the drug in the intestines. Softgel delivery systems can also incorporate phospholipids or polymers or natural gums to entrap a composition, for example, a prebiotic composition, in the gelatin layer with an outer coating to give desired delayed/control release effects, such as an enteric coating.

A physiologically acceptable composition may be provided in a dosage form which includes an effective amount of an active agent (e.g., an isolated bacteria population or a microbial metabolite) and one or more release controlling excipients as described herein. Suitable modified release dosage vehicles include, but are not limited to, hydrophilic or hydrophobic matrix devices, water-soluble separating layer coatings, enteric coatings, osmotic devices, multi-particulate devices, and combinations thereof. In one embodiment the dosage form is a tablet, caplet, capsule or lollipop. In another embodiment, the dosage form is a liquid, oral suspension, oral solution, or oral syrup. In yet another embodiment, the dosage form is a gel capsule, soft gelatin capsule, or hard gelatin capsule. In another embodiment, a composition including an active agent is provided in effervescent dosage forms. The compositions can also include non-release controlling excipients.

A physiologically acceptable composition (e.g., a physiologically acceptable composition formulated for oral administration) may be provided in the form of enteric-coated pellets, enteric-coated tablet, enteric-coated dragee, or enteric-coated capsules. The compositions can further include glyceryl monostearate 40-50, hydroxypropyl cellulose, hypromellose, magnesium stearate, methacrylic acid copolymer type C, polysorbate 80, sugar spheres, talc, and triethyl citrate. In one embodiment a composition including a bacterial population is provided in the form of enteric-coated granules, for oral administration. The compositions can further include carnauba wax, crospovidone, diacetylated monoglycerides, ethylcellulose, hydroxypropyl cellulose, hypromellose phthalate, magnesium stearate, mannitol, sodium hydroxide, sodium stearyl fumarate, talc, titanium dioxide, and yellow ferric oxide.

Physiologically acceptable compositions can be formulated in various dosage forms for oral administration. The compositions can also be formulated as a modified release dosage form, including immediate-, delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, extended, accelerated-, fast-, targeted-, programmed-release, and gastric retention dosage forms. These dosage forms can be prepared according to known methods and techniques (see, Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed., Lippincott Williams & Wilkins (2005); Modified-Release Drug Delivery Technology, Rathbone et al., Eds., Drugs and the Pharmaceutical Science, Marcel Dekker, Inc.: New York, N.Y., 2002; Vol. 126, which are incorporated herein by reference in their entirety). In one embodiment, the compositions are in one or more dosage forms. For example, a composition can be administered in a solid or liquid form. Examples of solid dosage forms include but are not limited to discrete units in capsules or tablets, as a powder or granule, or present in a tablet conventionally formed by compression molding. Such compressed tablets can be prepared by compressing in a suitable machine the three or more agents and a pharmaceutically acceptable carrier. The molded tablets can be optionally coated or scored, having indicia inscribed thereon and can be so formulated as to cause immediate, substantially immediate, slow, controlled or extended release of a composition including a prebiotic. Furthermore, dosage forms can include acceptable carriers or salts known in the art, such as those described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986), incorporated by reference herein in its entirety.

Physiologically acceptable compositions described herein can be in liquid form. The liquid formulations can include, for example, an agent in water-in-solution and/or suspension form; and a vehicle including polyethoxylated castor oil, alcohol, and/or a polyoxyethylated sorbitan mono-oleate with or without flavoring. Each dosage form includes an effective amount of an active agent and can optionally include pharmaceutically inert agents, such as conventional excipients, vehicles, fillers, binders, disintegrants, pH adjusting substances, buffer, solvents, solubilizing agents, sweeteners, coloring agents, and any other inactive agents that can be included in pharmaceutical dosage forms for oral administration. Examples of such vehicles and additives can be found in Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed., Lippincott Williams & Wilkins (2005).

Methods of treatment

Methods of treating or preventing CRC

In some aspects, the invention provides a method of treating or preventing CRC in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an isolated bacterial strain of Table 3, a lysate thereof, or a supernatant thereof.

Exemplary bacterial strains, lysates, and supernatants that may be used in the invention, and compositions and formulations comprising such agents, are described in Section II herein.

The composition may be administered to the subject in a single dose or in multiple doses. When multiple doses are administered, the doses may be separated from one another by, for example, 1 -24 hours, 1 -7 days, 1 -4 weeks, 1 month, two months, six months, or more than six months. In some examples, the composition is administered according to a schedule; in other examples, the composition is administered without a predetermined schedule. It is to be understood that, for any particular subject, specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

In examples of the method comprising multiple doses, the duration of treatment may vary. For example, in some embodiments, doses of the composition comprising the bacteria, lysate thereof, or supernatant of a culture thereof are administered to a subject over a time period that is 1 -7 days (e.g., administered daily or every 2, 3, 4, 5, 6, or 7 days or administered more frequently for a duration of 1 -7 days); 1 -12 weeks (e.g., administered weekly or every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks or administered more frequently for a duration of 2-12 weeks); or 1 -3 months (e.g., administered every 1 , 2, or 3 months or administered more frequently for a duration of 1 -3 months). In other embodiments, the active agents are administered to the subject over a time period that is, for example, 4-11 months (e.g., administered every 4, 5, 6, 7, 8, 9, 10, or 11 months or administered more frequently for a duration of 4- 11 months) or 1 -30 years (e.g., administered every 1 , 2, 3, 4, 5, 10, 15, 20, 25, or 30 years or administered more frequently for a duration of 1 -30 years). In yet other embodiments, the active agents disclosed herein are administered to a subject at the onset of symptoms. In any of these embodiments, the amount of the active agent that is administered may vary during the time period of administration. When an active agent is administered daily, administration may occur, for example, 1 , 2, 3, or 4 times per day.

In embodiments in which a parameter is increased or decreased (or reduced) in a subject following a step of administering a composition described herein, the increase or decrease may take place and/or be detectable within a range of time following the administration (e.g., within six hours, 24 hours, 3 days, a week or longer), and may take place and/or be detectable after one or more administrations (e.g., after 2, 3, 4, 5, 6, 7, 8, 9, 10 or more administrations, e.g., as part of a dosing regimen for the subject).

In some embodiments of methods in which the composition comprises one or more isolated bacteria, the one or more isolated bacteria may colonize the gut of a subject. Colonization of the gut by a bacteria may be determined by detecting the bacteria in a sample from the gut (e.g., by detecting one or more nucleotide sequences that may be used to identify the bacteria) of the subject following the administration of the composition comprising the bacteria, e.g., detecting the bacteria in a sample from the gut of the subject at least 1 day, at least 5 days, at least 1 week, at least 2 weeks, at least one month, at least two months, or more than two months following administration of the composition comprising the bacteria.

III. ARTICLES OF MANUFACTURE AND KITS

In another aspect of the invention, an article of manufacture or kit containing materials useful for the diagnosis, prognostic assessment, and/or treatment of individuals is provided.

For example, in one aspect, the disclosure features a kit for diagnosing CRC in a subject, the kit comprising (a) polypeptides or polynucleotides capable of determining the level of one or more of the bacterial strains of Table 1 in a sample from the subject; and optionally (b) instructions for use of the polypeptides or polynucleotides to determine the level of one or more of the bacterial strains of Table 1 in the sample from the subject, wherein a change in the level of one or more of the bacterial strains of Table 1 relative to a respective reference level for the bacterial strain of Table 1 indicates that the subject is likely to have CRC.

In some embodiments, the kit comprises polypeptides or polynucleotides capable of determining the level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Table 1 in a sample from the subject.

In another aspect, the disclosure features a kit for diagnosing CRC in a subject, the kit comprising (a) polypeptides or polynucleotides capable of determining the level of one or more of the bacterial strains of Table 2 and/or Table 3 in a sample from the subject; and optionally (b) instructions for use of the polypeptides or polynucleotides to determine the level of one or more of the bacterial strains of Table 2 and/or Table 3 in the sample from the subject, wherein: (i) a level of one or more of the bacterial strains of Table 2 that is increased relative to a respective reference level for the bacterial strain of Table 2 indicates that the subject is likely to have CRC; and (ii) a level of one or more of the bacterial strains of Table 3 that is decreased relative to a respective reference level for the bacterial strain of Table 3 indicates that the subject is likely to have CRC.

For polynucleotide-based articles of manufacture or kits, the article of manufacture or kit may include, for example: (1 ) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a protein, (2) a pair of primers useful for amplifying a nucleic acid molecule, or (3) a microarray comprising multiple oligonucleotide probes. For protein-based articles of manufacture or kits, the article of manufacture or kit may include, for example, one or more antibodybased reagents. The article of manufacture or kit can also include, e.g., a buffering agent, a preservative, or a protein-stabilizing agent. The article of manufacture or kit can further include components necessary for detecting the detectable label (e.g., an enzyme or a substrate). The article of manufacture or kit can further include components necessary for analyzing the sequence of a sample (e.g., a restriction enzyme or a buffer). The article of manufacture or kit can also contain a control sample or a series of control samples that can be assayed and compared to the test sample (e.g., a reference sample, as described herein). Each component of the article of manufacture or kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

OTHER EMBODIMENTS

Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

Other embodiments are in the claims.

Claims

WHAT IS CLAIMED IS:

1 . A method of diagnosing colorectal cancer (CRC) in a subject, the method comprising determining a level of one or more of the bacterial strains of Table 1 in a sample from the subject, wherein a level of one or more of the bacterial strains of Table 1 that is changed relative to a respective reference level for the bacterial strain of Table 1 indicates that the subject is likely to have a CRC.

2. A method for determining an increased risk of CRC in a subject, comprising the steps of:

(a) measuring a level of one or more of the bacterial strains of Table 1 in a sample collected from the subject using (i) metagenomic sequencing or (ii) amplification and one or more pairs of primers specific for a marker gene for each of the one or more of the bacterial strains of Table 1 ; and

(b) using the metagenomic sequencing or amplification results to determine whether the level of one or more of the bacterial strains of Table 1 is changed relative to a respective reference level for the bacterial strain of Table 1 , thereby determining an increased risk of CRC for the subject.

3. The method of claim 1 or 2, wherein the method comprises determining or measuring a level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Table 1 in the sample from the subject.

4. The method of claim 3, wherein the method comprises determining or measuring a level of each of the bacterial strains of Table 1 in the sample from the subject.

5. The method of any one of claims 1 -4, wherein a level of one or more of the bacterial strains of Table 1 in the sample from the subject is changed relative to the respective reference level for the bacterial strain of Table 1 and the method further comprises administering an anti-CRC therapy to the subject.

6. The method of any one of claims 1 -5, wherein:

(i) the bacterial strain of Table 1 is listed in Table 2, and the change relative to a reference level is an increase in the level of the bacterial strain; or

(ii) the bacterial strain of Table 1 is listed in Table 3, and the change relative to a reference level is a decrease in the level of the bacterial strain.

7. A method of diagnosing colorectal cancer (CRC) in a subject, the method comprising determining a level of one or more of the bacterial strains of Table 2 and/or Table 3 in a sample from the subject, wherein:

(i) a level of one or more of the bacterial strains of Table 2 that is increased relative to a respective reference level for the bacterial strain of Table 2 indicates that the subject is likely to have CRC; and (ii) a level of one or more of the bacterial strains of Table 3 that is decreased relative to a respective reference level for the bacterial strain of Table 3 indicates that the subject is likely to have CRC.

8. A method for determining an increased risk of CRC in a subject, comprising the steps of:

(a) measuring a level of one or more of the bacterial strains of Table 2 and/or Table 3 in a sample collected from the subject using (i) metagenomic sequencing or (ii) amplification and one or more pairs of primers specific for a marker gene for each of the one or more of the bacterial strains of Table 2 and/or Table 3; and

(b) using the metagenomic sequencing or amplification results to determine whether the level of one or more of the bacterial strains of Table 2 and/or Table 3 is changed relative to a respective reference level for the bacterial strain of Table 2 and/or Table 3, wherein:

(i) a level of one or more of the bacterial strains of Table 2 that is increased relative to a respective reference level for the bacterial strain of Table 2 indicates that the subject is likely to have CRC; and

(ii) a level of one or more of the bacterial strains of Table 3 that is decreased relative to a respective reference level for the bacterial strain of Table 3 indicates that the subject is likely to have CRC; thereby determining an increased risk of CRC for the subject.

9. The method of claim 7 or 8, wherein the method comprises determining or measuring a level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Tables 2 and 3 in the sample from the subject.

10. The method of claim 9, wherein the method comprises determining or measuring a level of each of the bacterial strains of Tables 2 and 3 in the sample from the subject.

11 . The method of any one of claims 7-10, wherein:

(i) a level of one or more of the bacterial strains of Table 2 is increased in the sample from the subject relative to a respective reference level for the bacterial strain of Table 2; and/or

(ii) a level of one or more of the bacterial strains of Table 3 is decreased in the sample from the subject relative to a respective reference level for the bacterial strain of Table 3, and the method further comprises administering an anti-CRC therapy to the subject.

12. The method of claim 5 or 11 , wherein the anti-CRC therapy comprises one or more of a surgery, a chemotherapy, an immunotherapy targeting vascular endothelial growth factor (VEGF), an immunotherapy targeting epidermal growth factor receptor (EGFR), an anti-BRAF therapy, a kinase inhibitor, and a checkpoint inhibitor.

13. The method of claim 12, wherein the chemotherapy is 5-fluorouracil (5-FU), capecitabine (XELODA®), irinotecan (CAMPTOSAR®), oxaliplatin (ELOXATIN®), or trifluridine and tipiracil (LONSURF®); the immunotherapy targeting VEGF is bevacizumab (A VASTIN®), ramucirumab (CYRAMZA®), or ziv-aflibercept (ZALTRAP®); the immunotherapy targeting EGFR is cetuximab (ERBITUX®) or panitumumab (VECTIBIX®); the anti-BRAF therapy is encorafenib (BRAFTOVI®); the kinase inhibitor is regorafenib (STIVARGA®); or the checkpoint inhibitor is pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), or ipilimumab (YERVOY®).

14. The method of any one of claims 5 and 11 -13, wherein the anti-CRC therapy further comprises treatment by fecal microbiota transplant (FMT).

15. The method of any one of claims 5 and 11 -14, wherein the anti-CRC therapy comprises administration of a therapeutically effective amount of a composition comprising an isolated bacterial strain of Table 3, a lysate thereof, or a supernatant thereof.

16. The method of claim 15, wherein the composition comprises at least two isolated bacterial strains of Table 3, lysates thereof, or supernatants thereof.

17. The method of claim 16, wherein the composition comprises 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 isolated bacterial strains of Table 3, lysates thereof, or supernatants thereof.

18. The method of any one of claims 15-17, wherein the composition comprises at least one isolated bacterial strain of Table 3, and the one or more isolated bacterial strains colonize the gut of the subject.

19. The method of any one of claims 1 -18, wherein the reference level is a pre-assigned level.

20. The method of any one of claims 1 -19, wherein the reference level is a level in a set of samples from a reference population.

21 . The method of claim 20, wherein the reference population is a population of healthy subjects.

22. The method of any one of claims 1 -21 , wherein the levels of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12,

13, 14, or 15 of the bacterial strains of Table 2 are increased relative to a respective reference level for the bacterial strain of Table 2.

23. The method of any one of claims 1 -22, wherein the levels of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12,

13, 14, 15, 16, 17, 18, 19, or 20 of the bacterial strains of Table 3 are decreased relative to a respective reference level for the bacterial strain of Table 3.

24. The method of any one of claims 1 -23, wherein the sample comprises a sample of the microbiota of the subject.

25. The method of any one of claims 1 -24, wherein the sample is a fecal sample.

26. A kit for diagnosing CRC in a subject, the kit comprising:

(a) polypeptides or polynucleotides capable of determining the level of one or more of the bacterial strains of Table 1 in a sample from the subject; and optionally

(b) instructions for use of the polypeptides or polynucleotides to determine the level of one or more of the bacterial strains of Table 1 in the sample from the subject, wherein a change in the level of one or more of the bacterial strains of Table 1 relative to a respective reference level for the bacterial strain of Table 1 indicates that the subject is likely to have CRC.

27. The kit of claim 26, wherein the kit comprises polypeptides or polynucleotides capable of determining the level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Table 1 in a sample from the subject.

28. A kit for diagnosing CRC in a subject, the kit comprising:

(a) polypeptides or polynucleotides capable of determining the level of one or more of the bacterial strains of Table 2 and/or Table 3 in a sample from the subject; and optionally

(b) instructions for use of the polypeptides or polynucleotides to determine the level of one or more of the bacterial strains of Table 2 and/or Table 3 in the sample from the subject, wherein:

(ii) a level of one or more of the bacterial strains of Table 3 that is decreased relative to a respective reference level for the bacterial strain of Table 3 indicates that the subject is likely to have CRC.

29. The kit of claim 28, wherein the kit comprises polypeptides or polynucleotides capable of determining the level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Tables 2 and 3 in a sample from the subject.

30. A method of treating a subject having CRC by administering a therapy selected from the group consisting of surgery, chemotherapy, an immunotherapy targeting VEGF, an immunotherapy targeting EGFR, an anti-BRAF therapy, a kinase inhibitor, a checkpoint inhibitor, FMT, an isolated bacterial strain of Table 3, a lysate of an isolated bacterial strain of Table 3, and a supernatant of an isolated bacterial strain of Table 3, wherein the subject was diagnosed as having CRC by a method of any one of claims 1 - 25.

31 . A method of treating a subject having CRC, the method comprising the steps of:

(a) measuring a level of one or more of the bacterial strains of Table 1 in a sample collected from the subject, wherein a level of one or more of the bacterial strains of Table 1 that is changed relative to a respective reference level for the bacterial strain of Table 1 indicates that the subject is likely to have CRC; and

(b) treating a subject who has been determined to have an increased risk of CRC by administering a therapy selected from the group consisting of surgery, chemotherapy, an immunotherapy targeting VEGF, an immunotherapy targeting EGFR, an anti-BRAF therapy, a kinase inhibitor, a checkpoint inhibitor, FMT, an isolated bacterial strain of Table 3, a lysate of an isolated bacterial strain of Table 3, and a supernatant of an isolated bacterial strain of Table 3.

32. The method of claim 31 , wherein the levels of one or more of the bacterial strains of Table 1 in the sample collected from the subject are measured using (i) metagenomic sequencing or (ii) amplification and one or more pairs of primers specific for a marker gene for each of the one or more of the bacterial strains of Table 1 , and the metagenomic sequencing or amplification results are used to determine whether the levels of one or more of the bacterial strains of Table 1 are changed relative to a respective reference level for the bacterial strain of Table 1 .

33. The method of claim 31 or 32, wherein the method comprises determining or measuring a level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Table 1 in the sample from the subject.

34. The method of claim 33, wherein the method comprises measuring the level of each of the bacterial strains of Table 1 in the sample from the subject.

35. A method of treating a subject having CRC, the method comprising the steps of:

(a) measuring a level of one or more of the bacterial strains of Table 2 and/or Table 3 in a sample collected from the subject, wherein:

(ii) a level of one or more of the bacterial strains of Table 3 that is decreased relative to a respective reference level for the bacterial strain of Table 3 indicates that the subject is likely to have CRC; and

36. The method of claim 35, wherein the levels of one or more of the bacterial strains of Table 2 and/or Table 3 in the sample collected from the subject are measured using (i) metagenomic sequencing or (ii) amplification and one or more pairs of primers specific for a marker gene for each of the one or more of the bacterial strains of Table 2 and/or Table 3, and the metagenomic sequencing or amplification results are used to determine whether the levels of one or more of the bacterial strains of Table 2 and/or Table 3 are changed relative to a respective reference level for the bacterial strain of Table 2 or Table 3.

37. The method of claim 35 or 36, wherein the method comprises determining or measuring a level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 20, at least 30, at least 40, or at least 50 of the bacterial strains of Tables 2 and 3 in the sample from the subject.

38. The method of claim 37, wherein the method comprises measuring the level of each of the bacterial strains of Tables 2 and 3 in the sample from the subject.

39. The method of any one of claims 30-38, wherein the reference level is a pre-assigned level.

40. The method of any one of claims 30-39, wherein the reference level is a level in a set of samples from a reference population.

41 . The method of claim 40, wherein the reference population is a population of healthy subjects.

42. The method of any one of claims 30-41 , wherein the levels of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 of the bacterial strains of Table 2 are increased relative to a respective reference level for the bacterial strain of Table 2.

43. The method of any one of claims 30-42, wherein the levels of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the bacterial strains of Table 3 are decreased relative to a respective reference level for the bacterial strain of Table 3.

44. The method of any one of claims 30-43, wherein the sample comprises a sample of the microbiota of the subject.

45. The method of any one of claims 30-44, wherein the sample is a fecal sample.

46. The method of any one of claims 30-45, wherein the therapy comprises one or more of a surgery, a chemotherapy, an immunotherapy targeting vascular endothelial growth factor (VEGF), an immunotherapy targeting epidermal growth factor receptor (EGFR), an anti-BRAF therapy, a kinase inhibitor, and a checkpoint inhibitor.

47. The method of claim 46, wherein the chemotherapy is 5-fluorouracil (5-FU), capecitabine (XELODA®), irinotecan (CAMPTOSAR®), oxaliplatin (ELOXATIN®), or trifluridine and tipiracil (LONSURF®); the immunotherapy targeting VEGF is bevacizumab (A VASTIN®), ramucirumab (CYRAMZA®), or ziv-aflibercept (ZALTRAP®); the immunotherapy targeting EGFR is cetuximab (ERBITUX®) or panitumumab (VECTIBIX®); the anti-BRAF therapy is encorafenib (BRAFTOVI®); the kinase inhibitor is regorafenib (STIVARGA®); or the checkpoint inhibitor is pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), or ipilimumab (YERVOY®).

48. The method of any one of claims 30-47, wherein the anti-CRC therapy further comprises treatment by fecal microbiota transplant (FMT).

49. The method of any one of claims 30-48, wherein the anti-CRC therapy comprises administration of a therapeutically effective amount of a composition comprising an isolated bacterial strain of Table 3, a lysate thereof, or a supernatant thereof.

50. The method of claim 49, wherein the composition comprises at least two isolated bacterial strains of Table 3, lysates thereof, or supernatants thereof.

51 . The method of claim 50, wherein the composition comprises 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 isolated bacterial strains of Table 3, lysates thereof, or supernatants thereof.

52. The method of any one of claims 49-51 , wherein the composition comprises at least one isolated bacterial strain of Table 3, and the one or more isolated bacterial strains colonize the gut of the subject.

53. A method of treating or preventing a colorectal cancer (CRC) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an isolated bacterial strain of Table 3, a lysate thereof, or a supernatant thereof.

54. The method of claim 53, wherein the composition comprises at least two isolated bacterial strains of Table 3, lysates thereof, or supernatants thereof.

55. The method of claim 54, wherein the composition comprises 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 isolated bacterial strains of Table 3, lysates thereof, or supernatants thereof.

56. The method of any one of claims 53-55, wherein the composition comprises one or more of said isolated bacterial strains.

57. The method of claim 56, wherein the one or more isolated bacterial strains colonize the gut of the subject.

58. The method of any one of claims 53-57, wherein the composition comprises a lysate of one or more of said isolated bacterial strains.

59. The method of any one of claims 53-58, wherein the composition comprises a supernatant of one or more of said isolated bacterial strains.

60. The method of claim 58 or 59, wherein the composition has been processed to remove endotoxin.

61 . The method of any one of claims 58-60, wherein the composition has been sterilized.

62. The method of any one of claims 58-61 , wherein the composition has been processed to remove all components having a molecular weight of more than 3 kDa.

63. The method of any one of claims 58-61 , wherein the composition has been processed to remove all components having a molecular weight of less than 3 kDa.

64. A composition comprising an isolated bacterial strain of Table 3, a lysate thereof, or a supernatant thereof, wherein the composition is formulated as a pharmaceutically acceptable composition, a comestible composition, or a nutraceutical.

65. A composition comprising at least two isolated bacterial strains of Table 3, lysates thereof, or supernatants thereof.

66. The composition of claim 64 or 65, wherein the composition is formulated as a physiologically acceptable powder, granule, capsule, or tablet.

67. The composition of any one of claims 64-66, wherein the composition is a pharmaceutical composition.

68. The composition of any one of claims 64-67, wherein the composition is a comestible composition.

69. The composition of any one of claims 64-68, wherein the composition is a nutraceutical.

70. The composition of claim 67, wherein the pharmaceutical composition is formulated for oral, enteral, or rectal administration.

71 . The composition of claim any one of claims 64-70, wherein the composition comprises 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 isolated bacterial strains of Table 3, lysates thereof, or supernatants thereof.

72. The composition of any one of claims 64-71 , wherein the composition comprises one or more of said isolated bacterial strains.

73. The composition of claim 72, wherein the one or more isolated bacterial strains colonize the gut of a subject.

74. The composition of any one of claims 64-73, wherein the composition comprises a lysate of one or more of said isolated bacterial strains.

75. The composition of any one of claims 64-74, wherein the composition comprises a supernatant of one or more of said isolated bacterial strains.

76. The composition of claim 74 or 75, wherein the composition has been processed to remove endotoxin.

77. The composition any one of claims 74-76, wherein the composition has been sterilized.

78. The composition of any one of claims 74-77, wherein the composition has been processed to remove all components having a molecular weight of more than 3 kDa.

79. The composition of any one of claims 74-77, wherein the composition has been processed to remove all components having a molecular weight of less than 3 kDa.

80. The composition of any one of claims 74-79, wherein the composition is for treatment of CRC.