WO2018171555A1

WO2018171555A1 - Use of gut microbiota composition in immunotherapy

Info

Publication number: WO2018171555A1
Application number: PCT/CN2018/079477
Authority: WO
Inventors: Wenqing Yang; Fei Chen; Jian Ding; Xiangchao GU; Qian Shi
Original assignee: Crown Bioscience Inc. (Taicang)
Priority date: 2017-03-20
Filing date: 2018-03-19
Publication date: 2018-09-27

Abstract

Provided are methods and compositions, e.g., kits, for determining the abundance of certain bacterial species in a cancer patient's gut and methods of using such information to predict the cancer patient's response to immunotherapy. Provided are methods and compositions for improving the efficacy of an immunotherapy in patients.

Description

USE OF GUT MICROBIOTA COMPOSITION IN IMMUNOTHERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of International Applications PCT/CN2017/077292, filed March 20, 2017, and PCT/CN2017/101853, filed September 15, 2017, both of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention generally relates to cancer diagnosis, prognosis and treatment. In particular, the present invention concerns the role of microbiota in the efficacy of cancer treatments and provides methods for determining the likelihood that a patient benefits from a cancer treatment, as well as probiotics to improve the efficacy of such a treatment in patients.

BACKGROUND

Immunotherapy has emerged as one novel cancer treatment in recent years, exampled by several FDA approved antibodies that target immune checkpoint inhibitors, e.g. Ipilimumab (anti-CTLA-4) , Pembrolizumab (anti-PD-1) , Atezolizumab (anti-PD-L1) to treat melanoma, NSCLC or colon cancer. However, like in other anticancer therapies, clinical responses to immunotherapy are restricted to a subset of patients. To maximize the efficacy of immunotherapy, there is a need for biomarkers to identify likely responders to the immunotherapy. Using the expression level of the targeted immune checkpoint alone, on the other hand, cannot effectively predict the responders. There is therefore a compelling need for additional biomarkers for predicting responders to immunotherapies.

SUMMARY OF INVENTION

The present disclosure in one aspect provides a method for predicting efficacy of an immunotherapy agent in a subject having cancer. In one embodiment, the method comprises: measuring the abundance of a microbe in the gut of the subject, wherein the microbe belongs to a genus selected from the group consisting of Akkermansia, Adlercreutzia, Bifidobacterium, Coprobacillus, Dehalobacterium, Enterococcus, Victivallis, Anaerostipes, Bacteroides, Coprococcus, Eubacterium, Holdemania, and Lactobacillu; comparing the measured abundance of the microbe to a corresponding reference level; and predicting a responsiveness of the subject to the immunotherapy agent.

In another embodiment, the method comprises: measuring a first level of a gut microbe in a first sample of the subject, wherein the gut microbe belongs to a genus selected from the group consisting of Akkermansia, Adlercreutzia and Coprococcus; administering an immunotherapy agent to the subject; measuring a second level of the gut microbe in a second sample of the subject; comparing the second level of the gut microbe to the first level of the gut microbe; and predicting a responsiveness of the subject to the immunotherapy agent.

In certain embodiments, the gut microbe is selected from the group consisting of Akkermansia muciniphila, Adlercreutzia equolifaciens, Coprococcus comes, Coprococcus eutactus and Coprococcus catus.

In some embodiments, the gut microbe belongs to the genus of Akkermansia, Adlercreutzia, Bifidobacterium, Coprobacillus, Dehalobacterium, Enterococcus, and an increase of the level of the microbe is indicative of an increased likelihood of the subject being responsive to the immunotherapy agent.

In some embodiment, the gut microbe belongs to the genus of Anaerostipes, Bacteroides, Coprococcus, Eubacterium, Holdemania, or Lactobacillu, and a decrease of the level of the microbe is indicative of an increased likelihood of the subject being responsive to the immunotherapy agent.

In some embodiments, the measuring step of the method comprises measuring the levle of DNA encoding a 16S rRNA specific to the microbe.

In certain embodiments, the level of the gut microbe is measured by an amplification assay, a hybridization assay, a sequencing assay or an array. In certain embodiments, the level of the gut microbe is measured using a feces sample of the subject.

In certain embodiments, the immunotherapy agent is an anti-CTLA-4 antibody, an anti-PD-1 antibody or an anti-PD-L1 antibody.

In certain embodiments, the cancer is colon cancer, melanoma or lung cancer.

In one embodiment, the level of the gut microbe is measured after the administration of the immunotherapy agent to the subject.

In one embodiment, the method described herein further comprises recommending the administration of the immunotherapy agent to the subject.

In another aspect, the present disclosure provides a method for treating a subject having cancer. In one embodiment, the method comprises: administering a gut microbe to the subject, wherein the microbe belongs to a genus of Akkermansia, Adlercreutzia, Bifidobacterium, Coprobacillus, Dehalobacterium, Enterococcus, or Victivallis; and administering an immunotherapy to the subject. In another embodiment, the method comprises administering an antibiotic killing a microbe belongs to genus of Coprococcus to the subject; and administering an immunotherapy to the subject.

In another embodiment, the treatment method comprises: measuring a level of a gut microbe in a sample of the subject, wherein the gut microbe belongs to a genus selected from the group consisting of Akkermansia, Adlercreutzia, Bifidobacterium, Coprobacillus, Dehalobacterium, Enterococcus, Victivallis, Anaerostipes, Bacteroides, Coprococcus, Eubacterium, Holdemania, and Lactobacillu; comparing the measured level of the gut microbe to a corresponding reference level; determining that the subject is likely to be responsive to an immunotherapy agent; and administering the immunotherapy agent to the subject.

In yet another embodiment, the treatment method comprises: measuring a first level of a gut microbe in a first sample of the subject, wherein the gut microbe belongs to a genus selected from the group consisting of Akkermansia, Adlercreutzia, Bifidobacterium, Coprobacillus, Dehalobacterium, Enterococcus, Victivallis, Anaerostipes, Bacteroides, Coprococcus, Eubacterium, Holdemania, and Lactobacillu; administering an immunotherapy agent to the subject; measuring a second level of the gut microbe in a second sample of the subject; comparing the second level of the gut microbe to the first level of the gut microbe; determining that the subject is likely to be responsive to the immunotherapy agent; and administering the immunotherapy agent to the subject.

In some embodiments, the gut microbe belongs to the genus of Akkermansia, Adlercreutzia, Bifidobacterium, Coprobacillus, Dehalobacterium, Enterococcus, or Victivallis, and the measured level of the gut microbe is higher than the corresponding reference level. In some embodiments, the gut microbe belongs to the genus of Anaerostipes, Bacteroides, Coprococcus, Eubacterium, Holdemania, or Lactobacillu, and the measured level of the gut microbe is lower than the corresponding reference level.

In yet another aspect, the present disclosure provides a composition for treating a subject having cancer. In one embodiment, the composition comprises: an antibiotic killing a microbe belongs to genus of Anaerostipes, Bacteroides, Coprococcus, Eubacterium, Holdemania, or Lactobacillu; and an immunotherapy to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1 shows the tumor growth curve of CT-26 syngeneic model with PD-1 antibody treatment. Animal number is 8/group; aPD-1 is from BioXcell (clone RPMI-14) ; Day of cell inoculation was denoted as Day 0. At study termination, TGI (tumor growth inhibition) of PD-1 is 78.82% (p＜0.001, compared with vehicle) .

FIG. 2 shows heat map of relative abundance in genus level. Blank: day of mice arrival; Ran: day of randomization (before treatment) ; 1wk: 1 week post dose start; Ter: study termination on 15 days post dose start. The microbiota profile in genus level changed in certain genera after 1 week and 15 days of PD-1 treatment, e.g. Akkermansia, Adlercreutzia, Coprococcus. Detailed analysis was performed by t-test shown in FIGs 3A and 3B.

FIG. 3A shows bar chart presentation of relative abundance of 4 microbes in genus level. Ran, day of randomization (before treatment) ; 1wk, 1 week post dose start; Ter, study termination on 15 days post dose start. Error bar is standard error of mean; *indicates p＜0.05; **indicates p＜0.01 by student t-test for relative abundance of control and PD-1 treated groups at study termination day.

FIG. 3B shows bar chart presentation of relative abundance of 4 microbes in genus level. Ran, day of randomization (before treatment) ; 1wk, 1 week post dose start; Ter, study termination on 15 days post dose start. Error bar is standard error of mean; *indicates p＜0.05 by student t-test for relative abundance of control and PD-1 treated groups at study termination day.

DETAILED DESCRIPTION OF THE INVENTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Definitions

The following definitions are provided to assist the reader. Unless otherwise defined, all terms of art, notations and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the chemical and medical arts. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over the definition of the term as generally understood in the art.

As used herein, the singular forms “a” , “an” and “the” include plural references unless the context clearly dictates otherwise.

The term “abundance” refers to the representation of a given phylum, order, family, or genera of microbe present in the gastrointestinal tract of a subject.

The term “amount” or “level” generally refers to the quantity of a substance of interest. In the context of gut microbe, a level of the gut microbe refers to the representation of a given phylum, order, family, or genera of microbe present in a sample, e.g., a sample from the gastrointestinal tract of a subject. In the context of a polynucleotide or polypeptide, the term “level” refers to the quantity of the polynucleotide of interest or the polypeptide of interest present in a sample. Such quantity may be expressed in the absolute terms, i.e., the total quantity of the polynucleotide or polypeptide in the sample, or in the relative terms, i.e., the concentration of the polynucleotide or polypeptide in the sample.

As used herein, the term “cancer” refers to any diseases involving an abnormal cell growth and include all stages and all forms of the disease that affects any tissue, organ or cell in the body. The term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, soft tissue, or solid, and cancers of all stages and grades including pre-and post-metastatic cancers. In general, cancers can be categorized according to the tissue or organ from which the cancer is located or originated and morphology of cancerous tissues and cells. As used herein, cancer types include, without limitation, acute lymphoblastic leukemia (ALL) , acute myeloid leukemia, adrenocortical carcinoma, anal cancer, astrocytoma, childhood cerebellar or cerebral, basal-cell carcinoma, bile duct cancer, bladder cancer, bone tumor, brain cancer, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, Burkitt′s lymphoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, emphysema, endometrial cancer, ependymoma, esophageal cancer, Ewing′s sarcoma, retinoblastoma, gastric (stomach) cancer, glioma, head and neck cancer, heart cancer, Hodgkin lymphoma, islet cell carcinoma (endocrine pancreas) , Kaposi sarcoma, kidney cancer (renal cell cancer) , laryngeal cancer, leukemia, liver cancer, lung cancer, neuroblastoma, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer) , retinoblastoma, Ewing family of tumors, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, vaginal cancer.

It is noted that in this disclosure, terms such as “comprises” , “comprised” , “comprising” , “contains” , “containing” and the like have the meaning attributed in United States Patent law; they are inclusive or open-ended and do not exclude additional, un-recited elements or method steps. Terms such as “consisting essentially of” and “consists essentially of” have the meaning attributed in United States Patent law; they allow for the inclusion of additional ingredients or steps that do not materially affect the basic and novel characteristics of the claimed invention. The terms “consists of” and “consisting of” have the meaning ascribed to them in United States Patent law; namely that these terms are close ended.

The term “complementarity” refers to the ability of a nucleic acid to form hydrogen bond (s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%＞, 70%＞, 80%＞, 90%, and 100%complementary) . “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100%over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

The terms “determining, ” “assessing, ” “assaying, ” “measuring” and “detecting” can be used interchangeably and refer to both quantitative and semi-quantitative determinations. Where either a quantitative and semi-quantitative determination is intended, the phrase “determining a level” of a polynucleotide or polypeptide of interest or “detecting” a polynucleotide or polypeptide of interest can be used.

The term “hybridizing” refers to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions. The term “stringent conditions” refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences in a mixed population (e.g., a cell lysate or DNA preparation from a tissue biopsy) . A “stringent hybridization” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization (e.g., as in array, microarray, Southern or northern hybridizations) are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in, e.g., Tijssen Laboratory Techniques in Biochemistry and Molecular Bio logy-Hybridization with Nucleic Acid Probes part I, Ch. 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays, ” (1993) Elsevier, N. Y. Generally, highly stringent hybridization and wash conditions are selected to be about 5℃ lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50%of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on an array or on a filter in a Southern or northern blot is 42℃. using standard hybridization solutions (see, e.g., Sambrook and Russell Molecular Cloning: A Laboratory Manual (3rd ed. ) Vol. 1-3 (2001) Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY) . An example of highly stringent wash conditions is 0.15 M NaCl at 72℃ for about 15 minutes. An example of stringent wash conditions is a 0.2×SSC wash at 65℃ for 15 minutes. Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is l×SSC at 45℃ for 15 minutes. An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4×SSC to 6×SSC at 40℃ for 15 minutes.

The term “nucleic acid” and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA) , transfer RNA, ribosomal RNA, ribozymes, cDNA, shRNA, single-stranded short or long RNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.

In general, a “protein” is a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds) . Proteins may include moieties other than amino acids (e.g., may be glycoproteins) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence) , or can be a functional portion thereof. Those of ordinary skill will further appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.

The term “recommending” or “suggesting” in the context of a treatment of a disease, refers to making a suggestion or a recommendation for therapeutic intervention (e.g., drug therapy, adjunctive therapy, etc. ) and/or disease management which are specifically applicable to the patient.

The terms “responsive, ” “clinical response, ” “positive clinical response, ” and the like, as used in the context of a patient’s response to a cancer therapy, are used interchangeably and refer to a favorable patient response to a treatment as opposed to unfavorable responses, i.e. adverse events. In a patient, beneficial response can be expressed in terms of a number of clinical parameters, including loss of detectable tumor (complete response, CR) , decrease in tumor size and/or cancer cell number (partial response, PR) , tumor growth arrest (stable disease, SD) , enhancement of anti-tumor immune response, possibly resulting in regression or rejection of the tumor; relief, to some extent, of one or more symptoms associated with the tumor; increase in the length of survival following treatment; and/or decreased mortality at a given point of time following treatment. Continued increase in tumor size and/or cancer cell number and/or tumor metastasis is indicative of lack of beneficial response to treatment. In a population the clinical benefit of a drug, i.e., its efficacy can be evaluated on the basis of one or more endpoints. For example, analysis of overall response rate (ORR) classifies as responders those patients who experience CR or PR after treatment with drug. Analysis of disease control (DC) classifies as responders those patients who experience CR, PR or SD after treatment with drug. A positive clinical response can be assessed using any endpoint indicating a benefit to the patient, including, without limitation, (1) inhibition, to some extent, of tumor growth, including slowing down and complete growth arrest; (2) reduction in the number of tumor cells; (3) reduction in tumor size; (4) inhibition (i.e., reduction, slowing down or complete stopping) of tumor cell infiltration into adjacent peripheral organs and/or tissues; (5) inhibition of metastasis; (6) enhancement of anti-tumor immune response, possibly resulting in regression or rejection of the tumor; (7) relief, to some extent, of one or more symptoms associated with the tumor; (8) increase in the length of survival following treatment; and/or (9) decreased mortality at a given point of time following treatment. Positive clinical response may also be expressed in terms of various measures of clinical outcome. Positive clinical outcome can also be considered in the context of an individual’s outcome relative to an outcome of a population of patients having a comparable clinical diagnosis, and can be assessed using various endpoints such as an increase in the duration of recurrence-free interval (RFI) , an increase in the time of survival as compared to overall survival (OS) in a population, an increase in the time of disease-free survival (DFS) , an increase in the duration of distant recurrence-free interval (DRFI) , and the like. Additional endpoints include a likelihood of any event (AE) -free survival, a likelihood of metastatic relapse (MR) -free survival (MRFS) , a likelihood of disease-free survival (DFS) , a likelihood of relapse-free survival (RFS) , a likelihood of first progression (FP) , and a likelihood of distant metastasis-free survival (DMFS) . An increase in the likelihood of positive clinical response corresponds to a decrease in the likelihood of cancer recurrence or relapse.

The term “reference level” as used herein refers to an amount or abundance of a gut microbe that is present is an established reference sample, e.g., a sample from a healthy human, or a sample from a cancer patient responsive to an immunotherapy. The reference level is suitable for the use of a method of the present invention, to serve as a basis for comparing the amount of a specific gut microbe that is present in a test sample. An established sample serving as a reference control provides an average amount of a specific gut microbe that is typical in a normal healthy human or a cancer patient responsive to an immunotherapy. A reference level may vary depending on the nature of the sample as well as other factors such as the gender, age, ethnicity of the subjects based on whom such a reference level is established.

As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) . A human includes pre and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient. ” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

The term “treatment, ” “treat, ” or “treating” refers to a method of reducing the effects of a cancer (e.g., breast cancer, lung cancer, ovarian cancer or the like) or symptom of cancer. Thus, in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%) , 80%) , 90%) , or 100%reduction in the severity of an cancer or symptom of the cancer. For example, a method of treating a disease is considered to be a treatment if there is a 10%reduction in one or more symptoms of the disease in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%or any percent reduction between 10 and 100%as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.

Gut Microbes Correlated with Response to Immunotherapy

The gut microbiota (formerly called gut flora or microflora) designates the population of microorganisms living in the intestine of any organism belonging to the animal kingdom (human, animal, insect, etc. ) . While each individual has a unique microbiota composition (60 to 80 bacterial species are shared by more than 50%of a sampled population on a total of 400-500 different bacterial species/individual) , it always fulfils similar main physiological functions and has a direct impact on the individual′s health: it contributes to the digestion of certain foods that the stomach and small intestine are not able to digest (mainly non-digestible fibers) ; it contributes to the production of some vitamins (B and K) ; it protects against aggressions from other microorganisms, maintaining the integrity of the intestinal mucosa; it plays an important role in the development of a proper immune system. A healthy, diverse and balanced gut microbiota is key to ensuring proper intestinal functioning.

Taking into account the major role gut microbiota plays in the normal functioning of the body and the different functions it accomplishes, it is nowadays considered as an “organ” . However, it is an “acquired” organ, as babies are born sterile; that is, intestine colonization starts right after birth and evolves afterwards.

The development of gut microbiota starts at birth. Sterile inside the uterus, the newborn′s digestive tract is quickly colonized by microorganisms from the mother (vaginal, skin, breast, etc. ) , the environment in which the delivery takes place, the air, etc. From the third day, the composition of the intestinal microbiota is directly dependent on how the infant is fed: breastfed babies′gut microbiota, for example, is mainly dominated by Bifidobacteria, compared to babies nourished with infant formulas.

The composition of the gut microbiota evolves throughout the entire life, from birth to old age, and is the result of different environmental influences. Gut microbiota′s balance can be affected during the ageing process and, consequently, the elderly have substantially different microbiota than younger adults.

While the general composition of the dominant intestinal microbiota is similar in most healthy people (4 main phyla, i.e., Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria) , composition at a species level is highly personalized and largely determined by the individuals’genetic, environment and diet. The composition of gut microbiota may become accustomed to dietary components, either temporarily or permanently. Japanese people, for example, can digest seaweeds (part of their daily diet) thanks to specific enzymes that their microbiota has acquired from marine bacteria.

The methods and compositions described herein are based, in part, on the discovery of certain gut microbes whose abundance is correlated with a caner patient’s response to immunotherapy. In one aspect, the present disclosure provides methods for predicting efficacy of an immunotherapy agent in a patient having cancer. In certain embodiments, the methods comprise: measuring the abundance of a microbe in a feces sample of the patient, wherein the microbe belongs to a genus selected from the group consisting of Akkermansia, Adlercreutzia, Bifidobacterium, Coprobacillus, Dehalobacterium, Enterococcus, Victivallis, Anaerostipes, Bacteroides, Coprococcus, Eubacterium, Holdemania, and Lactobacillu; comparing the measured abundance of the microbe to a corresponding reference level; and determining a likelihood of the patient being responsive to the immunotherapy agent. In some cases, the measurement is performed prior to the patient being treated with immunotherapy. In some cases, the measurement is performed during the patient being treated with immunotherapy.

The species in the Akkermansia genus includes, without limitation, Akkermansia muciniphila (Derrien M, International Journal of Systematic and Evolutionary Microbiology (2004) 54: 1474) .

The species in the Adlercreutzia genus includes, without limitation, Adlercreutzia equolifaciens (Maruo T et al., International Journal of Systematic and Evolutionary Microbiology (2008) 58: 1221-27) .

Bifidobacterium is a genus of gram-positive, nonmotile, often branched anaerobic bacteria, which is one of the major genera of bacteria that make up the colon flora in mammals, including humans. The species in the Bifidobacterium genus includes B. angulatum; B. animalis; B. asteroides; B. bifidum; B. boum; B. breve; B. catenulatum; B. choerinum; B. coryneforme; B. cuniculi; B. dentium; B. gallicum; B. gallinarum; B indicum; B. longum; B. magnum; B. merycicum; B. minimum; B. pseudocatenulatum; B. pseudolongum; B. psychraerophilum; B. pullorum; B. ruminantium; B. saeculare; B. scardovii; B. simiae; B. subtile; B. thermacidophilum; B. thermophilum; B. urinalis.

Coprobacillus is a Gram-positive, bligate anaerobic and non-motile genus, with one known species of Coprobacillus cateniformis.

The species in the Dehalobacterium genus includes Dehalobacterium formicoaceticum.

Enterococcus is a large genus of lactic acid bacteria, which is Gram-positive cocci that often occur in pairs or short chains. Two species of Enterococcus are common commensal organisms in the intestines of humans: Enterococcus faecalis and Enterococcus faecium.

Victivallis is a Gram-postive, coccus-shaped bacteria found in human digestive tract. Species in the genus of Victivallis includes Victivallis vadensis.

Anaerostipes is a Gram-positive and anaerobic bacteria genus occurs in the human gut. Species in the genus of Anaerostipes includes Anaerostipes butyraticus, Anaerostipes caccae, Anaerostipes hadrus, and Anaerostipes rhamnosivorans.

Bacteroides is a genus of Gram-negative, obligate anaerobic bacteria, which makes up a significant portion of the fecal bacterial population. Bacteroides species are nonendospore-forming bacilli and may be either motile or nonmotile.

The species in the Coprococcus genus includes, without limitation, Coprococcus catus, Coprococcus comes and Coprococcus eutacus (Holdeman and Moore, International Journal of Systematic and Evolutionary Microbiology (1974) 24: 260-77) .

Eubacterium is a genus of Gram-positive bacteria, characterized by a rigid cell wall. Eubacterium species may either be motile or nonmotile.

Holdemania is a Gram-positive, strictly anaerobic and non-spore-forming genus. Species of the genus of Holdemania includes Holdemania filiformis and Holdemania massiliensis.

Lactobacillus is a genus of Gram-positive, facultative anaerobic or microaerophilic, rod-shaped, non-spore-forming bacteria, which makes up a major portion of the lactic acid bacteria group in human, and constitute a significant component of the gut microbiota.

As used herein, the term “immunotherapy” refers to the use of the immune system to treat a disease or condition, e.g., cancer. In some cases, cancer immunotherapies actively direct the immune system to attack tumor cells by targeting tumor-associated antigens. Typically, active immunotherapies involve the removal of immune cells from the blood or from a tumor. The immune cells specific for the tumor are cultured and returned to the patient where they attack the tumor. Alternatively, immune cells can be genetically engineered to express a tumor-specific receptor, cultured and returned to the patient. In some case, cancer immunotherapies passively enhance existing anti-tumor responses and include the use of an immunotherapy agent, e.g., antibodies, lymphocytes and cytokines. In certain embodiments, the immunotherapy agents include anti-CTLA-4 antibodies, anti-PD-1 antibodies or an anti-PD-L1 antibody. In one embodiment, the immunotherapy agent is an anti-PD-1 antibody.

Methods of Measuring Level of Gut Microbes

The methods of the present disclosure include measuring the level of certain gut microbes, e.g., a species in the genus of Akkermansia, Adlercreutzia or Coprococcus, in the sample of a cancer patient being treated or to be treated with an immunotherapy. In certain embodiment, the level of the gut microbe is measured using a feces sample from the cancer patient.

Any method known to those of ordinary skill in the art can be used to measure the level of the gut microbe in the sameple of the patient. In certain embodiments, the level of the gut microbe is measured by detecting the level of microbe-specific DNA in a sample, e.g., feces sample from the gut of the patient.

In some embodiments, DNA is isolated from the feces sample. DNA can be isolated from the feces sample using a variety of methods. Standard methods for DNA extraction from tissue or cells are described in, for example, Ausubel et al., Current Protocols of Molecular Biology (1997) John Wiley &Sons, and Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3rd ed. (2001) . Commercially available kits, e.g.,

DNA Stool Mini Kit (Qiagen) can also be used to isolate DNA from a feces sample.

In certain embodiments, microbe-specific DNA is DNA encoding a 16s rRNA.

The sequences of 16s rDNA for the genus of bacteria provided herein are known in the art. For example, the sequence of 16S rDNA for the genus of Akkermansia is set forth in, e.g., NCBI Ref. Seq No. NR_042817. The sequence of 16S rDNA for the genus of Adlercreutzia is set forth in, e.g., NCBI Ref. Seq No. NR_121696. The sequence of 16S rDNA for the genus of Coprococcus comes is set forth in, e.g., NCBI Ref. Seq Nos. NR_044048, NR_044049, and NR_024750.

The level of DNA specific for a microbe described above can be detected or measured by a variety of methods including, but not limited to, an amplification assay, a hybridization assay or a sequencing assay. Non-limiting examples of such methods include quantitative real-time PCR (qRT-PCR) ; quantitative PCR, such as

Northern blotting; in situ hybridization assays; microarray analysis, e.g., microarrays from Nano String Technologies; multiplexed hybridization-based assays, e.g., QuantiGene 2.0 Multiplex Assay from Panomics; direct sequencing or pyrosequencing; massively parallel sequencing; next generation sequencing; high performance liquid chromatography (HPLC) fragment analysis; capillarity electrophoresis; and the like. Various methods involving amplification reactions and/or reactions in which probes are linked to a solid support and used to quantify DNA may be used. Alternatively, the DNA may be linked to a solid support and quantified using a probe to the sequence of interest.

In certain embodiments, the DNA specific to the microbe of interest is measured by high throughput sequencing. In certain embodiments, DNA are extracted from fecal samples to prepare 16S rDNA amplicon. The 16S rDNA amplicon is then sequenced to determine the abundance of specific microbes (see Shin J et al., Scientific Reports (2016) 6; Thomas V et al., Future Microbiol (2015) 10: 1485-504) .

In general, quantitative amplification is based on the monitoring of the signal (e.g., fluorescence of a probe) representing copies of the template in cycles of an amplification (e.g., PCR) reaction. One method for detection of amplification products is the 5 -3′exonuclease “hydrolysis” PCR assay (also referred to as the

assay) (U.S. Pat. Nos. 5,210,015 and 5,487,972; Holland et al., PNAS USA (1991) 88: 7276-7280; Lee et al, Nucleic Acids Res. (1993) 21: 3761-3766) . This assay detects the accumulation of a specific PCR product by hybridization and cleavage of a doubly labeled fluorogenic probe (the

probe) during the amplification reaction. The fluorogenic probe consists of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye. During PCR, this probe is cleaved by the 5 ′-exonuclease activity of DNA polymerase if, and only if, it hybridizes to the segment being amplified. Cleavage of the probe generates an increase in the fluorescence intensity of the reporter dye.

Another method of detecting amplification products that relies on the use of energy transfer is the “beacon probe” method described by Tyagi and Kramer, Nature Biotech. (1996) 14: 303-309, which is also the subject of U.S. Patent Nos. 5,119,801 and 5,312,728. This method employs oligonucleotide hybridization probes that can form hairpin structures. On one end of the hybridization probe (either the 5′or 3′end) , there is a donor fluorophore, and on the other end, an acceptor moiety. In the case of the Tyagi and Kramer method, this acceptor moiety is a quencher, that is, the acceptor absorbs energy released by the donor, but then does not itself fluoresce. Thus, when the beacon is in the open conformation, the fluorescence of the donor fluorophore is detectable, whereas when the beacon is in hairpin (closed) conformation, the fluorescence of the donor fluorophore is quenched. When employed in PCR, the molecular beacon probe, which hybridizes to one of the strands of the PCR product, is in “open conformation, ” and fluorescence is detected, while those that remain unhybridized will not fluoresce (Tyagi and Kramer, Nature Biotechnol. (1996) 14: 303-306) . As a result, the amount of fluorescence will increase as the amount of PCR product increases, and thus may be used as a measure of the progress of the PCR. Those of skill in the art will recognize that other methods of quantitative amplification are also available.

Various other techniques for performing quantitative amplification of nucleic acids are also known. For example, some methodologies employ one or more probe oligonucleotides that are structured such that a change in fluorescence is generated when the oligonucleotide (s) is hybridized to a target nucleic acid. For example, one such method involves a dual fluorophore approach that exploits fluorescence resonance energy transfer (FRET) , e.g., LightCycler ^TM hybridization probes, where two oligo probes anneal to the amplicon. The oligonucleotides are designed to hybridize in a head-to-tail orientation with the fluorophores separated at a distance that is compatible with efficient energy transfer. Other examples of labeled oligonucleotides that are structured to emit a signal when bound to a nucleic acid or incorporated into an extension product include: Scorpions ^TM probes (e.g., Whitcombe et al., Nature Biotechnology (1999) 17: 804-807, and U.S. Pat. No. 6,326,145) , Sunrise ^TM (or Amplifluor ^TM) probes (e.g., Nazarenko et al., Nuc. Acids Res. (1997) 25: 2516-2521, and U.S. Pat. No. 6,117,635) , and probes that form a secondary structure that results in reduced signal without a quencher and that emits increased signal when hybridized to a target (e.g., Lux probes ^TM) .

In other embodiments, intercalating agents that produce a signal when intercalated in double stranded DNA may be used. Exemplary agents include SYBR GREEN ^TM and SYBR GOLD ^TM. Since these agents are not template-specific, it is assumed that the signal is generated based on template-specific amplification. This can be confirmed by monitoring signal as a function of temperature because melting point of template sequences will generally be much higher than, for example, primer-dimers, etc.

In other embodiments, the DNA is immobilized on a solid surface and contacted with a probe, e.g., in a dot blot or Southern format. In an alternative embodiment, the probe (s) are immobilized on a solid surface and the DNA is contacted with the probe (s) , for example, in a gene chip array. A skilled artisan can readily adapt known DNA detection methods for use in detecting the level of DNA specific to a microbe of interest.

In some embodiments, gene-specific probes and/or primers are used in hybridization assays to detect the DNA of interest. The probes and/or primers may be labeled with any detectable moiety or compound, such as a radioisotope, fluorophore, chemoilluminescent agent, and enzyme.

The probes and primers necessary for practicing the present invention can be synthesized and labeled using well known techniques. Oligonucleotides used as probes and primers may be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts. (1981) 22: 1859-1862, using an automated synthesizer, as described in Needham-Van Devanter et al, Nucleic Acids Res. (1984) 12: 6159-6168.

In some embodiments, the methods further comprise detecting the level of one or more reference DNA that can be used as controls to determine the level of the gut microbe of interest. Such DNA is typically present constitutively at a high level and can act as a reference for determining accurate abundance estimates. Accordingly, a determination of the level of a gut microbe of interest may also comprise determining the levels of one or more reference DNA, i.e., a reference level. The reference level can be predetermined, determined concurrently, or determined after a sample is obtained from the subject. The reference level can be obtained in the same assay or can be a known standard from a previous assay. In the cases when the level of the DNA of interest is determined by sequencing, the level of the DNA of interest can be normalized to the total reads of the sequencing.

Methods and Compositions for Treating Cancer

The inventions disclosed herein are also based, in part, on the discovery that the level of certain gut microbes may influence a caner patient’s response to immunotherapy. Therefore, the present disclosure in another aspect provides methods and compositions for treating cancer using certain gut microbes.

In certain embodiments, the composition comprises bacteria in a genus selected from the group consisting of Akkermansia, Adlercreutzia, Bifidobacterium, Coprobacillus, Dehalobacterium, Enterococcus, Victivallis and a combination thereof, for use as an adjuvant to an immunotherapy treatment administered to a cancer patient.

The above compositions can advantageously be formulated for oral administration and administered either as food supplements or as functional food. The skilled artisan knows a variety of formulas which can encompass living or killed microorganisms and which can present as food supplements (e.g., pills, tablets and the like) or as functional food such as drinks, fermented yoghurts, etc.

According to one embodiment, the composition according to the invention is administered to a patient in need thereof after the administration of an immunotherapy treatment, for example an anti-PD-1 antibody to said patient. For example, the composition can be administered the same day as an anti-PD-1 antibody dose, or after a few days of treatment. The composition can be administered daily, once every two days or once every three days. Alternatively, the composition according to the invention is administered to a patient in need thereof before the administration of an immunotherapy treatment.

In some embodiments, the immunotherapy treatment is administered in a therapeutically effective amount. The therapeutically effective amount of the immunotherapy treatment will depend on various factors known in the art, such as for example type of disease to be treated, the type of treatment, body weight, age, past medical history, present medications, state of health of the subject, immune condition and potential for cross-reaction, allergies, sensitivities and adverse side-effects, as well as the administration route and the type, the severity and development of the disease and the discretion of the attending physician or veterinarian. In certain embodiments, the immunotherapy treatment may be administered at a therapeutically effective dosage of about 0.001 mg/kg to about 100 mg/kg one or more times per day (e.g., about 0.001 mg/kg, about 0.3 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 3 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg one or more times per day) . In certain embodiments, the immunotherapy treatment is administered at a dosage of about 50 mg/kg or less, and in certain embodiments the dosage is 20 mg/kg or less, 10 mg/kg or less, 3 mg/kg or less, 1 mg/kg or less, 0.3 mg/kg or less, 0.1 mg/kg or less, or 0.01 mg/kg or less, or 0.001 mg/kg or less. In certain embodiments, the administration dosage may change over the course of treatment. For example, in certain embodiments the initial administration dosage may be higher than the subsequent administration dosages. In certain embodiments, the administration dosage may vary over the course of treatment depending on the reaction of the subject.

Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response) . In certain embodiments, the immunotherapy treatment is administered to the subject at one time or over a series of treatments. In certain embodiments, the immunotherapy treatment is administered to the subject by one or more separate administrations, or by continuous infusion depending on the type and severity of the disease.

The immunotherapy treatment may be administered by any route known in the art, such as for example parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

In some embodiments, the compositions for treating cancer described herein can be used in combination with monitoring the level of certain gut microbes of the patient. In general, the level of the gut microbe can be measured before or during the immunotherapy. When the level of the gut microbe reaches or exceeds a certain reference level, or the change of the level of the gut microbe reaches a threshold, the immunotherapy is administered to the patient. Otherwise, the probiotic composition described herein may be administered to the patient to increase the efficacy of the immunotherapy.

In one embodiment, the treatment method comprises: measuring a level of the gut microbe provided herein in a sample of the subject; comparing the measured level of the gut microbe to a corresponding reference level; determining that the subject is likely to be responsive to an immunotherapy agent; and administering the immunotherapy agent to the subject.

In yet another embodiment, the treatment method comprises: measuring a first level of a gut microbe in a first sample of the subject; administering an immunotherapy agent to the subject; measuring a second level of the gut microbe in a second sample of the subject; comparing the second level of the gut microbe to the first level of the gut microbe; determining that the subject is likely to be responsive to the immunotherapy agent; and administering the immunotherapy agent to the subject.

Another aspect of the present invention is the use of a combination of an immunotherapy agent and of an antibiotic composition which decreases the abundance of Anaerostipes, Bacteroides, Coprococcus, Eubacterium, Holdemania, or Lactobacillu in the gut microbiota of an individual when administered to said individual, for treating a cancer.

As used herein, the term “combination” refers to the use of more than one agent. The use of the term “combination” does not restrict the order in which the therapeutic agents are administered to the patient, although it is preferable to administer the antibiotic prior to or simultaneously with the immunotherapy agent. Advantageously, the antibiotic composition is administered before administration of an immunotherapy agent, in order to modulate the patient′s gut microbiota to optimize the effect of said immunotherapy agent (such as anti-PD-1 antibody) .

Examples

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention.

EXAMPLE 1

This example shows the identification of the gut microbiota changes in CT-26 syngeneic colon cancer model after PD-1 antibody treatment.

Establishment of CT-26 syngeneic colon cancer model and treatment with PD- 1 antibody

The CT-26 tumor cells (purchased from ATCC) were maintained in vitro as a monolayer culture in RPMI1640 medium supplemented with 10%fetal bovine serum at 37℃ in an atmosphere of 5%CO ₂ in air. The cells in an exponential growth phase were harvested and counted for tumor inoculation.

7-8 week female BALB/c mice were inoculated subcutaneously at the right flank region with 5 x 10 ⁵ CT-26 cells in 0.1 ml of PBS for tumor development. PD-1 antibody or PBS vehicle treatments was started when the mean tumor size reached approximately 90 mm ³, at 10mg/kg twice per week for 5 doses in total.

Feces collection and 16S rRNA sequencing

The mouse feces from each mouse were collected in sterile circumstance by handling stimulation or pushing its stomach at 4 time points, mouse arrival, randomization (before treatment) , 1 week post dose start and study termination (15 days post dose start) . The feces samples were snap frozen immediately and kept at -80 degree.

All feces samples were sent to Annoroad Genomics, LTD for DNA extraction, 16S rDNA sequencing and data analysis.

Data analysis and results description

The relative abundance for each microbe was calculated. The taxonomy profile at genus level was drawn by listing top 25 genera and the hierarchical clustering was plotted. The relative abundance of 9 genera was presented by bar chart and the student t-test was used to compare the difference of control and PD-1 treated groups.

The results and findings are shown in FIG. 1, FIG. 2, FIG. 3 and Table 1.

As shown in FIG. 1, mice treated with PD-1 antibody demonstrated tumor growth inhibition (TGI) of 78.82% (p＜0.001, compared with vehicle) .

FIG. 2 shows that the microbiota profile in genus level changed in certain genera after 1 week and 15 days of PD-1 treatment, e.g. Akkermansia, Adlercreutzia, Coprococcus. Detailed analysis were performed by t-test shown in FIGs 3A and 3B. Ran: day of randomization (before treatment) ; 1wk: 1 week post dose start; Ter: study termination on 15 days post dose start. Error bar is standard error of mean; *indicates p＜0.05; **indicates p＜0.01 by student t-test for relative abundance of control and PD-1 treated groups at study termination day.

Table 1. Gut microbiota changed in CT-26 syngeneic colon cancer model after PD-1 antibody treatment.

Genus	Response to aPD-1 Treatment
Akkermansia	Up-regulated
Adlercreutzia	Up-regulated
Bifidobacterium	Up-regulated
Coprobacillus	Up-regulated
Dehalobacterium	Up-regulated
Enterococcus	Up-regulated
Victivallis	Up-regulated
Anaerostipes	Down-regulated
Bacteroides	Down-regulated
Coprococcus	Down-regulated
Eubacterium	Down-regulated
Holdemania	Down-regulated
Lactobacillus	Down-regulated

EXAMPLE 2

This example illustrates the cause-effect relationship between the responsiveness to immunotherapy and the gut microbiota.

Female BALB/c mice (7-8 week) are treated with an antibiotic solution containing ampicillin (1 mg/ml) , streptomycin (5 mg/ml) and colistin (1 mg/ml) added to the sterile drinking water of mice. Solutions and bottles are changed 3 times and once weekly respectively.

After 3 days of antibiotic treatment, the mice are used for fecal microbial transplantation. To conduct fecal microbial transplantation, fecal materials are collected from the mice used in Example 1: feces are collected from 4 mice treated with PD-1 antibody (NT group) and 4 mice treated with PD-1 antibody (T group) . The feces collected are suspended in sterile water, and 200 uL of the suspension is transferred by oral gavage into each antibiotic pretreated mouse. The mice with fecal microbial transplantation are maintained in germ free environment.

After two weeks of the fecal microbial transplantation, the mice are inoculated subcutaneously at the right flank region with 5 x 10 ⁵ CT-26 cells in 0.1 ml of PBS for tumor development. PD-1 antibody or PBS vehicle treatments is started when the mean tumor size reached approximately 90 mm ³, at 10mg/kg twice per week for 5 doses in total. Normal mice and antibiotic treated mice without fecal microbial transplantation are also inoculated with CT-26 cells and are used as control.

The results show that antibiotic treated mice are significantly less responsive to PD-1 treatment compared to normal mice. In contrast, mice transplanted with fecal microbial from T group demonstrate delayed tumor growth when treated with PD-1 antibody. On the other hand, mice transplanted with fecal microbial from NT group do not demonstrate delayed tumor growth when treated with PD-1 antibody. In addition, neither mice transplanted with fecal material from T group nor those transplanted with fecal material from NT group demonstrate delayed tumor growth if not treated with PD-1 antibody. These results indicate that fecal microbial material influence a cancer patient’s responsiveness to PD-1 antibody treatment.

EXAMPLE 3

This example illustrates that increased abundance of certain gut microbe species is sufficient to increase a subject’s responsiveness to immunotherapy.

Female BALB/c mice (7-8 week) are treated with the antibiotic solution added to the sterile drinking water of mice as described in Example 2.

After 3 days of antibiotic treatment, the mice are transferred to normal environment for natural recolonization. Three days later, the mice are supplemented with 5 oral gavages of Akkermansia muciniphila (100 uL of suspension containing 1×10 ⁸ bacteria) .

After two weeks of recolonization, the mice are inoculated subcutaneously at the right flank region with 5 x 10 ⁵ CT-26 cells in 0.1 ml of PBS for tumor development. PD-1 antibody or PBS vehicle treatments is started when the mean tumor size reached approximately 90 mm ³, at 10mg/kg twice per week for 5 doses in total. Antibiotic treated mice without recolonization and recolonized mice without Akkermansia muciniphila supplementation are also inoculated with CT-26 cells and are used as control.

The results show that recolonized mice with Akkermansia muciniphila supplementation restores responsiveness to PD-1 treatment.

EXAMPLE 4

After 3 days of antibiotic treatment, the mice are transplanted with fecal microbial material from NT group as described in Example 2. Three days later, the mice are supplemented with 5 oral gavages of Akkermansia muciniphila (100 uL of suspension containing 1×10 ⁸ bacteria) .

After two weeks of the fecal microbial transplantation, the mice are inoculated subcutaneously at the right flank region with 5 x 10 ⁵ CT-26 cells in 0.1 ml of PBS for tumor development. PD-1 antibody or PBS vehicle treatments is started when the mean tumor size reached approximately 90 mm ³, at 10mg/kg twice per week for 5 doses in total. Mice transplanted with fecal microbial from NT group but received no Akkermansia muciniphila supplementation are also inoculated with CT-26 cells and are used as control.

The results show that Akkermansia muciniphila supplementation significantly increases the mice’s responsiveness to PD-1 treatment.

While the disclosure has been particularly shown and described with reference to specific embodiments (some of which are preferred embodiments) , it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.

Claims

A method for predicting efficacy of an immunotherapy agent in a subject having cancer, the method comprising:

measuring a level of a gut microbe in a sample of the subject, wherein the gut microbe belongs to a genus selected from the group consisting of Akkermansia, Adlercreutzia, Bifidobacterium, Coprobacillus, Dehalobacterium, Enterococcus, Victivallis, Anaerostipes, Bacteroides, Coprococcus, Eubacterium, Holdemania, and Lactobacillus;

comparing the measured level of the gut microbe to a corresponding reference level; and

predicting a responsiveness of the subject to the immunotherapy agent.
The method of claim 1, wherein the gut microbe belongs to the genus of Akkermansia, Adlercreutzia, Bifidobacterium, Coprobacillus, Dehalobacterium, Enterococcus, or Victivallis, and wherein an increase of the level of the gut microbe is indicative of an increased likelihood of the subject being responsive to the immunotherapy agent.
The method of claim 1, wherein the gut microbe belongs to the genus of Anaerostipes, Bacteroides, Coprococcus, Eubacterium, Holdemania, or Lactobacillu, and wherein a decrease of the level of the gut microbe is indicative of an increased likelihood of the subject being responsive to the immunotherapy agent.
The method of any one of claims 1-3, wherein the measuring step comprises measuring a level of DNA encoding a 16S rRNA specific to the gut microbe.
The method of any one of claims 1-4, wherein the level of the gut microbe is measured by an amplification assay, a hybridization assay, a sequencing assay or an array.
The method of any one of claims 1-5, wherein the sample is a feces sample of the subject.
The method of any one of claims 1-6, wherein the immunotherapy agent is an anti-CTLA-4 antibody, an anti-PD-1 antibody or an anti-PD-L1 antibody.
The method of any one of claims 1-7, wherein the cancer is colon cancer, melanoma or lung cancer.
The method of any one of claims 1-8, wherein the level of the gut microbe is measured after the administration of the immunotherapy agent to the subject.
The method of any one of claims 1-9, further comprising recommending the administration of the immunotherapy agent to the subject.
A method for predicting efficacy of an immunotherapy agent in a subject having cancer, the method comprising:

measuring a first level of a gut microbe in a first sample of the subject, wherein the gut microbe belongs to a genus selected from the group consisting of Akkermansia, Adlercreutzia, Bifidobacterium, Coprobacillus, aehalobacterium, Enterococcus, Victivallis, Anaerostipes, Bacteroides, Coprococcus, Eubacterium, Holdemania, and Lactobacillu;

administering an immunotherapy agent to the subject;

measuring a second level of the gut microbe in a second sample of the subject;

comparing the second level of the gut microbe to the first level of the gut microbe; and

predicting a responsiveness of the subject to the immunotherapy agent.
The method of claim 11, wherein the gut microbe belongs to the genus of Akkermansia, Adlercreutzia, Bifidobacterium, Coprobacillus, Dehalobacterium, Enterococcus, or Victivallis, and wherein an increase of the level of the gut microbe is indicative of an increased likelihood of the subject being responsive to the immunotherapy agent.
The method of claim 11, wherein the gut microbe belongs to the genus of Anaerostipes, Bacteroides, Coprococcus, Eubacterium, Holdemania, or Lactobacillu, and wherein a decrease of the level of the gut microbe is indicative of an increased likelihood of the subject being responsive to the immunotherapy agent.
The method of any one of claims 11-13, wherein the measuring step comprises measuring a level of DNA encoding a 16S rRNA specific to the gut microbe.
The method of any one of claims 11-14, wherein the level of the gut microbe is measured by an amplification assay, a hybridization assay, a sequencing assay or an array.
The method of any one of claims 11-15, wherein the sample is a feces sample of the subject.
The method of any one of claims 11-16, wherein the immunotherapy agent is an anti-CTLA-4 antibody, an anti-PD-1 antibody or an anti-PD-L1 antibody.
The method of any one of claims 11-17, wherein the cancer is colon cancer, melanoma or lung cancer.
The method of any one of claims 11-18, wherein the level of the gut microbe is measured after the administration of the immunotherapy agent to the subject.
The method of any one of claims 11-19, further comprising recommending the administration of the immunotherapy agent to the subject.
A method for treating a subject having cancer, the method comprising:

administering a gut microbe to the subject, wherein the gut microbe belongs to a genus of Akkermansia, Adlercreutzia, Bifidobacterium, Coprobacillus, Dehalobacterium, Enterococcus, or Victivallis; and

administering an immunotherapy agent to the subject.
The method of claim 21, wherein the immunotherapy agent is an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody.
The method of claim 21 or 22, wherein the cancer is colon cancer, melanoma or lung cancer.
A method for treating a subject having cancer, the method comprising:

administering an antibiotic killing a microbe belongs to genus of Anaerostipes, Bacteroides, Coprococcus, Eubacterium, Holdemania, or Lactobacillu to the gut of the patient; and

administering an immunotherapy to the patient.
The method of claim 24, wherein the immunotherapy agent is an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody.
The method of claims 24 or 25, wherein the cancer is colon cancer, melanoma or lung cancer.
A method for treating a subject having cancer, the method comprising:

measuring a level of a gut microbe in a sample of the subject, wherein the gut microbe belongs to a genus selected from the group consisting of Akkermansia, Adlercreutzia, Bifidobacterium, Coprobacillus, Dehalobacterium, Enterococcus, Victivallis, Anaerostipes, Bacteroides, Coprococcus, Eubacterium, Holdemania, and Lactobacillu;

comparing the measured level of the gut microbe to a corresponding reference level;

determining that the subject is likely to be responsive to an immunotherapy agent; and

administering the immunotherapy agent to the subject.
The method of claim 31, wherein the gut microbe belongs to the genus of Akkermansia, Adlercreutzia, Bifidobacterium, Coprobacillus, Dehalobacterium, Enterococcus, or Victivallis, and wherein the measured level of the gut microbe is higher than the corresponding reference level.
The method of claim 28, wherein the gut microbe belongs to the genus of Anaerostipes, Bacteroides, Coprococcus, Eubacterium, Holdemania, or Lactobacillu, and wherein the measured level of the gut microbe is lower than the corresponding reference level.
The method of claims 28 or 29, wherein the immunotherapy agent is an anti-CTLA-4 antibody, an anti-PD-1 antibody or an anti-PD-L1 antibody.
The methods of any one of claims 28-30, wherein the cancer is colon cancer, melanoma or lung cancer.
The method of any one of claims 28-31, wherein the sample is a feces sample of the subject.
The method of any one of claims 28-32, wherein the level of the microbe is measured after the administration of the immunotherapy agent to the subject.
A method of treating a subject having cancer, the method comprising:

measuring a first level of a gut microbe in a first sample of the subject, wherein the gut microbe belongs to a genus selected from the group consisting of Akkermansia, Adlercreutzia, Bifidobacterium, Coprobacillus, Dehalobacterium, Enterococcus, Victivallis, Anaerostipes, Bacteroides, Coprococcus, Eubacterium, Holdemania, and Lactobacillu;

administering an immunotherapy agent to the subject;

measuring a second level of the gut microbe in a second sample of the subject;

comparing the second level of the gut microbe to the first level of the gut microbe;

determining that the subject is likely to be responsive to the immunotherapy agent; and

administering the immunotherapy agent to the subject.
The method of claim 34 wherein the gut microbe belongs to the genus of Akkermansia, Adlercreutzia, Bifidobacterium, Coprobacillus, Dehalobacterium, Enterococcus, or Victivallis, and wherein the second level of the gut microbe is higher than the second level of the microbe.
The method of claim 34, wherein the gut microbe belongs to the genus of Anaerostipes, Bacteroides, Coprococcus, Eubacterium, Holdemania, or Lactobacillu, and wherein the second level of the gut microbe is lower than the first level of the gut microbe.
The method of any one of claims 34-36, wherein the immunotherapy agent is an anti-CTLA-4 antibody, an anti-PD-1 antibody or an anti-PD-L1 antibody.
The method of any one of claims 34-37, wherein the cancer is colon cancer, melanoma or lung cancer.
The method of any one of claims 34-38, wherein the sample is a feces sample of the subject.