WO2021138562A1 - Compositions comprenant des espèces bactériennes et procédés associés - Google Patents

Compositions comprenant des espèces bactériennes et procédés associés Download PDF

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WO2021138562A1
WO2021138562A1 PCT/US2020/067674 US2020067674W WO2021138562A1 WO 2021138562 A1 WO2021138562 A1 WO 2021138562A1 US 2020067674 W US2020067674 W US 2020067674W WO 2021138562 A1 WO2021138562 A1 WO 2021138562A1
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composition
collinsella
bacterial strain
cancer
strain
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PCT/US2020/067674
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English (en)
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Jackie Papkoff
Jun Ma
Jason HUDAK
Linh Nguyen
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Assembly Biosciences, Inc.
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Publication of WO2021138562A1 publication Critical patent/WO2021138562A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the gastrointestinal tract is a complex biological system that includes a community of many different organisms, including diverse strains of bacteria. Hundreds of different species may form a commensal community in the gastrointestinal tract and other organs in a healthy person. Moreover, microorganisms present in the gut not only play a crucial role in digestive health, but also influence the immune system. A disturbance or imbalance in a biological system, e.g ., the gastrointestinal tract, may include changes in the types and numbers of bacteria in the gut which may lead to the development of, or may be an indicator of, an unhealthy state and/or disease.
  • the disclosure relates generally to bacterial strains, bacterial strain mixtures, and compositions comprising bacterial strains of the genus Collinsella.
  • the disclosed bacterial strains, bacterial strain mixtures or compositions are useful for preventing and/or treating cancer.
  • composition for example, a pharmaceutical composition, comprising a bacterial strain of the genus Collinsella.
  • the composition further comprises an excipient, diluent and/or carrier.
  • the bacterial strain in the composition is lyophilized, freeze dried or spray dried.
  • the composition or the bacterial strain in the composition is capable of increasing production of at least one pro-inflammatory gene product, e.g., interferon gamma (IFN-g), interleukin 1 beta (IL-Ib), interleukin 12 (IL-12, e.g, IL-12p40, IL-12p70), interleukin 17A (IL-17A), interleukin 23 (IL-23), and/or tumor necrosis factor (TNF), in a human cell, e.g, a macrophage (e.g, a THEM macrophage), a monocyte, a peripheral blood mononuclear cell (PBMC), or a monocyte-derived dendritic cell.
  • a pro-inflammatory gene product e.g., interferon gamma (IFN-g), interleukin 1 beta (IL-Ib), interleukin 12 (IL-12, e.g, IL-12p40, IL-12p70), interleukin 17A (IL-17A), inter
  • the composition increases production of at least one pro- inflammatory gene product, e.g, IFN-g, IL-Ib, IL-12p40, IL-12p70, IL-17A, IL-23, and/or TNF, in a human cell, e.g, a macrophage (e.g, a THP-1 macrophage), a monocyte, a PBMC, or a monocyte-derived dendritic cell, when the human cell is contacted with the composition.
  • a pro- inflammatory gene product e.g, IFN-g, IL-Ib, IL-12p40, IL-12p70, IL-17A, IL-23, and/or TNF
  • a human cell e.g, a macrophage (e.g, a THP-1 macrophage), a monocyte, a PBMC, or a monocyte-derived dendritic cell, when the human cell is contacted with the composition.
  • the composition comprises a bacterial strain of Collinsella that comprises a 16s rRNA gene sequence with at least about 98% sequence identity to the polynucleotide sequence of SEQ ID NO: 46.
  • the Collinsella bacterial strain comprises a 16s rRNA gene sequence with at least about 98.5%, 99% or 99.5% sequence identity to the polynucleotide sequence of SEQ ID NO: 46.
  • the Collinsella bacterial strain comprises a 16s rRNA gene sequence of SEQ ID NO: 46.
  • the Collinsella bacterial strain shares at least 70% DNA-DNA hybridization with strain Collinsella sp.
  • the Collinsella bacterial strain comprises a nucleotide sequence having at least about 70% identity to any one of SEQ ID NOs: 1-45. In some embodiments, the Collinsella bacterial strain comprises a genome having at least 95% average nucleotide identity (ANI) with the genome of Collinsella ASMB strain P121-D5a, having the deposit accession number DSM 33276. In some embodiments, the Collinsella bacterial strain comprises a genome having at least 96.5% average nucleotide identity (ANI) and at least 60% alignment fraction (AF) with the genome of Collinsella ASMB strain P121-D5a, having the deposit accession number DSM 33276. In some embodiments, the Collinsella bacterial strain is Collinsella ASMB P121-D5a, having the deposit accession number DSM 33276.
  • the Collinsella bacterial strain of the composition is viable.
  • the bacterial strain is capable of at least partially colonizing an intestine of a human subject.
  • the composition is suitable for oral delivery to a subject.
  • the composition is formulated as an enteric formulation.
  • the enteric formulation is formulated as a capsule, tablet, caplet, pill, troche, lozenge, powder, or granule.
  • the composition is formulated as a suppository, suspension, emulsion, or gel.
  • the composition comprises at least lxlO 3 CFU of the bacterial strain.
  • the composition comprises a therapeutically effective amount of the bacterial strain sufficient to prevent or treat a disorder when administered to a subject in need thereof.
  • the disorder is a cancer.
  • cancers include solid tumors, soft tissue tumors, hematopoietic tumors and metastatic lesions.
  • hematopoietic tumors include, leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), e.g ., transformed CLL, diffuse large B-cell lymphomas (DLBCL), follicular lymphoma, hairy cell leukemia, myelodyplastic syndrome (MDS), a lymphoma, Hodgkin’s disease, a malignant lymphoma, non-Hodgkin’s lymphoma, Burkitt’s lymphoma, multiple myeloma, or Richter’s Syndrome (Richter’s Transformation).
  • ALL acute lymphoblastic leukemia
  • B-cell T-cell or FAB ALL
  • AML acute myeloid leukemia
  • CML chronic myelocytic leukemia
  • CLL chronic lymphocy
  • solid tumors include malignancies, e.g, sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting head and neck (including pharynx), thyroid, lung (small cell or non-small cell lung carcinoma (NSCLC)), breast, lymphoid, gastrointestinal (e.g, oral, esophageal, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), genitals and genitourinary tract (e.g, renal, urothelial, bladder, ovarian, uterine, cervical, endometrial, prostate, testicular), CNS (e.g, neural or glial cells, e.g, neuroblastoma or glioma), or skin (e.g, melanoma).
  • the cancer is colorectal cancer (CRC).
  • the composition comprises an excipient selected from the group consisting of a filler, a binder, a disintegrant, and any combination(s) thereof.
  • the excipient is selected from the group consisting of cellulose, polyvinyl pyrrolidone, silicon dioxide, stearyl fumarate or a pharmaceutically acceptable salt thereof, and any combination(s) thereof.
  • the composition further comprises a cryoprotectant.
  • the cryoprotectant is selected from the group consisting of a fructoligosaccharide, trehalose and a combination thereof.
  • the fructoligosaccharide is raftilose ® (fructooligosaccharide derived from inulin).
  • the composition is suitable for bolus administration or bolus release.
  • the composition comprises the Collinsella bacterial strain and at least one more additional bacterial strain(s).
  • a bacterial strain e.g, an isolated bacterial strain, of the genus Collinsella , wherein the bacterial strain comprises a 16s rRNA gene sequence with at least about 98% sequence identity to the polynucleotide sequence of SEQ ID NO: 46.
  • the Collinsella bacterial strain is capable of increasing production of at least one pro-inflammatory gene product, e.g, IFN-g, IL-Ib, IL-12p40, IL- 12p70, IL-17A, IL-23, and/or TNF, in a human cell, e.g, a macrophage (e.g, a THP-1 macrophage), a monocyte, a PBMC, or a monocyte-derived dendritic cell.
  • a pro-inflammatory gene product e.g, IFN-g, IL-Ib, IL-12p40, IL- 12p70, IL-17A, IL-23, and/or TNF
  • a human cell e.g, a macrophage (e.g, a THP-1 macrophage), a monocyte, a PBMC, or a monocyte-derived dendritic cell.
  • the Collinsella bacterial strain increases production of at least one pro- inflammatory gene product, e.g., IFN-g, IL-Ib, IL-6, IL-12p40, IL-12p70, IL-17A, IL-23, and/or TNF, in a human cell, e.g, a macrophage (e.g, a THP-1 macrophage), a monocyte, a PBMC, or a monocyte-derived dendritic cell, when the human cell is contacted with the Collinsella bacterial strain.
  • a pro- inflammatory gene product e.g., IFN-g, IL-Ib, IL-6, IL-12p40, IL-12p70, IL-17A, IL-23, and/or TNF
  • a human cell e.g, a macrophage (e.g, a THP-1 macrophage), a monocyte, a PBMC, or a monocyte-derived dendritic cell, when the human cell is
  • the Collinsella bacterial strain comprises a 16s rRNA gene sequence with at least about 98.5%, 99%, or 99.5% sequence identity to the polynucleotide sequence of SEQ ID NO: 46. In some embodiments, the Collinsella bacterial strain comprises a 16s rRNA gene sequence of SEQ ID NO: 46. In some embodiments, the Collinsella bacterial strain shares at least 70% DNA-DNA hybridization with strain Collinsella ASMB P121-D5a, having the deposit accession number DSM 33276. In some embodiments, the Collinsella bacterial strain comprises a nucleotide sequence having at least about 70% identity to any one of SEQ ID NOs: 1-45.
  • the Collinsella bacterial strain comprises a genome having at least 95% average nucleotide identity (ANI) with the genome of Collinsella ASMB P121-D5a, having the deposit accession number DSM 33276. In some embodiments, the Collinsella bacterial strain comprises a genome having at least 96.5% average nucleotide identity (ANI) and at least 60% alignment fraction (AF) with the genome of Collinsella ASMB P121-D5a, having the deposit accession number DSM 33276. In some embodiments, the Collinsella bacterial strain is Collinsella ASMB P121-D5a, having the deposit accession number DSM 33276. In some embodiments, the bacterial strain is capable of at least partially colonizing an intestine of a human subject.
  • ANI average nucleotide identity
  • AF 60% alignment fraction
  • a food product comprising a Collinsella bacterial strain described herein, or a composition comprising a Collinsella bacterial strain described herein.
  • a contemplated method comprises administering to the subject an effective amount of a disclosed pharmaceutical composition, pharmaceutical unit, bacterial strain or bacterial strain mixture.
  • Also provided herein is a method of treating a dysbiosis in a subject in need thereof, the method comprising administering a Collinsella bacterial strain described herein or a composition comprising a Collinsella bacterial strain described herein (e.g a therapeutically effective amount of a Collinsella bacterial strain described herein or a composition comprising a Collinsella bacterial strain described herein) to the subject.
  • a Collinsella bacterial strain described herein or a composition comprising a Collinsella bacterial strain described herein e.g a therapeutically effective amount of a Collinsella bacterial strain described herein or a composition comprising a Collinsella bacterial strain described herein
  • Also provided herein is a method of modifying a gut microbiome in a subject, the method comprising administering a Collinsella bacterial strain described herein or a composition comprising a Collinsella bacterial strain described herein (e.g., a therapeutically effective amount of a Collinsella bacterial strain described herein or a composition comprising a Collinsella bacterial strain described herein) to the subject.
  • the method further comprises administering a prebiotic to the subject.
  • the subject is selected from the group consisting of a human, a companion animal, or a livestock animal.
  • Figure 1 depicts the effect of Collinsella ASMB P121-D5a on (A) IL-12p70; (B) IL-Ib; and (C) IL-23 production in human moDCs.
  • MoDCs were co-incubated with PBS only or the Collinsella ASMB P121-D5a strain (2 doses), and supernatants were collected and assayed for production of the indicated cytokine.
  • Each test article was evaluated in 4 replicates and results are representative of at least two independent experiments p value ⁇ 0.05 (one-way ANOVA).
  • Figure 2 depicts the effect of Collinsella ASMB P121-D5a on (A) IFN-g, (B) IL- 1b, and (C) IL-17A production in human peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • PBMCs were co-incubated with PBS only or the Collinsella ASMB P121-D5a strain (2 doses), and supernatants were collected and assayed for production of the indicated cytokine.
  • Each test article was evaluated in 4 replicates and results are representative of at least two independent experiments p value ⁇ 0.05 (one-way ANOVA).
  • Figure 3 depicts the effect of Collinsella ASMB P121-D5a on (A) IL-Ib; (B) IL-
  • THP-1 M2 macrophages were co-incubated with PBS only or the Collinsella ASMB P121-D5a strain (3 doses), and supernatants were collected and assayed for production of the indicated cytokine.
  • Each test article was evaluated in 4 replicates and results are representative of at least two independent experiments p value ⁇ 0.05 (one-way ANOVA).
  • Figure 4 depicts the effect of administration of (A) Collinsella ASMB P121-D5a; (B) an anti-PD-Ll antibody; and (C) combination of Collinsella ASMB P121-D5a and an anti-PD-Ll antibody, on tumor volume (fold-change) compared to vehicle in a murine B16F10 melanoma model.
  • Figure 5 depicts the effect of administration of (A) Collinsella ASMB P121-D5a; (B) an anti-PD-Ll antibody; and (C) combination of Collinsella ASMB P121-D5a and an anti-PD-Ll antibody, on tumor volume (mm 3 ) compared to vehicle in a murine B16F10 melanoma model.
  • Figure 6 depicts the effect of administration of an anti-PD-Ll antibody, Collinsella ASMB P121-D5a, and the combination of Collinsella ASMB P121-D5a and an anti-PD-Ll antibody, respectively, on body weight (g) compared to vehicle in a murine B16F10 melanoma model.
  • Figure 7 depicts the effect of administration of (A) Collinsella ASMB P121-D5a and (B) an anti -PD- 1 antibody, respectively, on tumor volume (fold-change) compared to vehicle in a murine CT26 colon tumor model.
  • *P ⁇ 0.05 significant difference compared to Vehicle by 2-way ANOVA with Dunnett post hoc test.
  • Figure 8 depicts the effect of administration of (A) Collinsella ASMB P121-D5a and (B) an anti -PD- 1 antibody, respectively, on tumor volume (mm 3 ) compared to vehicle in a murine CT26 colon tumor model.
  • Figure 9 depicts the effect of administration of (A) Collinsella ASMB P121-D5a; (B) a Butyricimonas faecihominis strain; and (C) combination of Collinsella ASMB P121- D5a and Butyricimonas faecihominis strains, respectively, on tumor volume (left: mm 3 ; right: fold-change) compared to vehicle in a murine CT26 colon tumor model.
  • Figure 10 depicts the effect of administration of (A) Collinsella ASMB P121-D5a; (B) a Butyricimonas faecihominis strain; (C) an anti -PD- 1 antibody; (D) combination of Collinsella ASMB P121-D5a and Butyricimonas faecihominis strains; and (E) ) combination of Collinsella ASMB P121-D5a and Butyricimonas faecihominis strains plus an anti-PD-1 antibody, respectively, on tumor volume (left: mm 3 ; right: fold-change) compared to vehicle in a murine CT26 colon tumor model.
  • A Collinsella ASMB P121-D5a
  • B a Butyricimonas faecihominis strain
  • C an anti -PD- 1 antibody
  • D combination of Collinsella ASMB P121-D5a and Butyricimonas faecihominis strains
  • Figure 11 depicts the effect of administration of a combination of Collinsella ASMB P121-D5a and Butyricimonas faecihominis P40-F2a strains plus an anti-PD-1 antibody on tumor-infiltrating CD8 + T cells in a murine CT26 colon tumor model.
  • FIG. 1 Shown are representative immunohistochemistry stainings for CD8 in formalin-fixed paraffin- embedded (FFPE) tumor sections following treatment with (A) Collinsella ASMB P121-D5a + Butyricimonas faecihominis P40-F2a (Ca + Bf); (B) Collinsella ASMB P121-D5a + Butyricimonas faecihominis P40-F2a + a-PD-1 (Ca + Bf + a-PD-1); (C) Vehicle; or (D) a- PD-1. (E) CD8-positive cells were quantified as a percentage of non-necrotic cells in whole tissue sections in blinded fashion.
  • FFPE formalin-fixed paraffin- embedded
  • Figure 12 depicts the effect of administration of a combination of Collinsella ASMB P121-D5a and Butyricimonas faecihominis P40-F2a strains with or without an anti- PD-1 antibody on tumor-infiltrating T cells in a murine CT26 colon tumor model, as determined by Nanostring RNA-based cell type profiling.
  • RNA was extracted and purified from tumor samples and analyzed using the Mouse PanCancer IO 360 Panel. T cell designation was based on expression of Cd3d, Cd3e, Cd3g, Cd6, Sh2dla and Tratl.
  • Figure 13 depicts the effect of administration of a combination of Collinsella ASMB P121-D5a and Butyricimonas faecihominis P40-F2a strains plus an anti-PD-1 antibody on IFN-g in tumors in a murine CT26 colon tumor model.
  • IFN-g concentration was determined by MSD analysis of tumor tissue homogenate and normalized to homogenate protein concentration.
  • Ca Collinsella ASMB P121-D5a
  • Bf Butyricimonas faecihominis P40-F2a.
  • a contemplated bacterial strain for example, for use in a bacterial strain mixture, pharmaceutical composition or unit, or method provided herein, includes a Collinsella species strain.
  • a Collinsella species strain includes a Collinsella species strain.
  • the genus Collinsella may undergo taxonomical reorganization.
  • contemplated Collinsella species include Collinsella species that have been renamed and/or reclassified, as well as those that may be later renamed and/or reclassified.
  • the term “species” refers to a taxonomic entity as conventionally defined by genomic sequence and phenotypic characteristics.
  • a “strain” is a particular instance of a species that has been isolated and purified according to conventional microbiological techniques. Bacterial species and/or strains described herein include those that are live and/or viable, as well as those that are killed, inactivated or attenuated. Additionally, bacterial species and/or strains described herein include vegetative forms and non-spore forming forms of bacteria.
  • a bacterial strain of Collinsella comprises a 16S rRNA gene sequence having a certain % identity to a reference sequence.
  • rRNA, 16S rDNA, 16S rRNA, 16S, 18S, 18S rRNA, and 18S rDNA refer to nucleic acids that are components of, or encode for, components of the ribosome.
  • SSU small subunit
  • LSU large subunit
  • Ribosomal RNA genes (rDNA) and their complementary RNA sequences are widely used for determination of the evolutionary relationships among organisms as they are variable, yet sufficiently conserved to allow cross organism molecular comparisons.
  • 16S rDNA sequence of the 30S SSU can be used, in embodiments, for molecular-based taxonomic assignments of prokaryotes.
  • 16S sequences may be used for phylogenetic reconstruction, as they are general highly conserved but contain specific hypervariable regions that harbor sufficient nucleotide diversity to differentiate genera and species of most bacteria.
  • 16S rDNA sequence data has been used to provide taxonomic classification, closely related bacterial strains that are classified within the same genus and species may exhibit distinct biological phenotypes.
  • a bacterial strain of Collinsella includes strains comprising a 16s rRNA gene sequence having a certain % identity to SEQ ID NO: 46.
  • the bacterial strain is a strain of the genus Collinsella comprising a 16s rRNA gene sequence with at least 98% sequence identity to the polynucleotide sequence of SEQ ID NO: 46.
  • the bacterial strain comprises a 16s rRNA gene sequence with at least about 98.05%, about 98.1%, about 98.15%, about 98.2%, about 98.25%, about 98.3%, about 98.35%, about 98.4%, about 98.45%, about 98.5%, about 98.55%, about 98.6%, about 98.65%, about 98.7%, about 98.75%, about 98.8%, about
  • the bacterial strain comprises a 16s rRNA gene sequence identical to SEQ ID NO: 46.
  • the sequence identity referred to above is across at least about 70% of SEQ ID NO: 46.
  • sequence identity referred to above is across at least about 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% of SEQ ID NO: 46.
  • the bacterial strain of Collinsella provided herein is a bacterial strain of Collinsella ASMB.
  • the bacterial strain of Collinsella ASMB comprises a genomic sequence (e.g a whole genome sequence, or fragments or contigs thereof) having a certain % identity to one or more of SEQ ID NOs: 1- 45.
  • a Collinsella ASMB strain comprises a polynucleotide sequence selected from any one of SEQ ID NOs: 1-45, or a nucleotide sequence having at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90 %, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a polynucleotide sequence selected from any one of SEQ ID NOs: 1-45.
  • the sequence identity referred to above is across at least about 70% of the bacterial genome.
  • a Collinsella ASMB strain genome may comprise the polynucleotide sequence of each of SEQ ID NOs: 1-45, or each of a polynucleotide sequence having at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90 %, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the polynucleotide sequence of each of SEQ ID NOs: 1-45.
  • a bacterial strain of Collinsella ASMB comprises a whole genomic sequence having at least about 70% identity across at least 70% of its genome to the sum of all genomic contigs represented by SEQ ID NOs: 1-45.
  • the whole genomic sequence has at least about 75%, 80%, 85%, 90%, 95% or greater than 95% identity to the sum of all genomic contigs represented by SEQ ID NOs: 1-45.
  • the sequence identity referred to above is across at least 75%, 80%, 85%, 90%, 95% or greater than 95% of the whole genomic sequence of the bacterial strain.
  • a bacterial strain of Collinsella ASMB comprises a whole genomic sequence comprising coding regions having at least about 70% identity across at least 70% of the total coding regions in its genome to the coding regions within the sum of all genomic contigs represented by SEQ ID NOs: 1-45.
  • the coding regions within the whole genomic sequence have at least about 75%, 80%, 85%, 90%, 95% or greater than 95% identity to the coding regions within the sum of all genomic contigs represented by SEQ ID NOs: 1-45.
  • the sequence identity referred to above is across at least 75%, 80%, 85%, 90%, 95% or greater than 95% of the coding regions within the whole genomic sequence of the bacterial strain.
  • the identity of a bacterial strain of Collinsella may be determined by sequence analysis, for example, of the 16s rRNA gene sequence or a genomic sequence (e.g., a whole genome sequence, or fragments or contigs thereof) of the bacterial strain, using any sequencing methods known in the art, including, for example, Sanger sequencing.
  • An example of a sequencing technology useful for identifying strains of Collinsella is the Illumina platform.
  • the Illumina platform is based on amplification of DNA on a solid surface (e.g., flow cell) using fold-back PCR and anchored primers (e.g., capture oligonucleotides). For sequencing with the Illumina platform, bacterial DNA is fragmented, and adapters are added to terminal ends of the fragments.
  • DNA fragments are attached to the surface of flow cell channels by capturing oligonucleotides which are capable of hybridizing to the adapter ends of the fragments.
  • the DNA fragments are then extended and bridge amplified. After multiple cycles of solid-phase amplification followed by denaturation, an array of millions of spatially immobilized nucleic acid clusters or colonies of single-stranded nucleic acids are generated. Each cluster may include approximately hundreds to a thousand copies of single- stranded DNA molecules of the same template.
  • the Illumina platform uses a sequencing-by synthesis method where sequencing nucleotides comprising detectable labels (e.g., fluorophores) are added successively to a free 3' hydroxyl group.
  • a laser light of a wavelength specific for the labeled nucleotides can be used to excite the labels.
  • An image is captured and the identity of the nucleotide base is recorded. These steps can be repeated to sequence the rest of the bases. Sequencing according to this technology is described in, for example, U.S. Patent Publication Application Nos. 2011/0009278, 2007/0014362, 2006/0024681, 2006/0292611, and U.S. Pat. Nos. 7,960,120, 7,835,871, 7,232,656, and 7,115,200.
  • Another example of a sequencing technology useful for identifying strains of Collinsella is SOLiD technology by Applied Biosystems from Life Technologies Corporation (Carlsbad, Calif.).
  • bacterial DNA may be sheared into fragments, and adapters may be attached to the terminal ends of the fragments to generate a library.
  • Clonal bead populations may be prepared in microreactors containing template, PCR reaction components, beads, and primers. After PCR, the templates can be denatured, and bead enrichment can be performed to separate beads with extended primers. Templates on the selected beads undergo a 3' modification to allow covalent attachment to the slide. The sequence can be determined by sequential hybridization and ligation with several primers. A set of four fluorescently labeled di-base probes compete for ligation to the sequencing primer. Multiple cycles of ligation, detection, and cleavage are performed with the number of cycles determining the eventual read length.
  • Ion Torrent sequencing Another example of a sequencing technology useful for identifying strains of Collinsella is Ion Torrent sequencing.
  • bacterial DNA is sheared into fragments, and oligonucleotide adapters are then ligated to the terminal ends of the fragments.
  • the fragments are then attached to a surface, and each base in the fragments is resolvable by measuring the H + ions released during base incorporation.
  • This technology is described in, for example, U.S. Patent Publication Application Nos. 2009/0026082, 2009/0127589, 2010/0035252, 2010/0137143, and 2010/0188073.
  • sequence identity with a polynucleotide sequence of a Collinsella strain may be determined in various ways that are within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, BLAT (BLAST-like alignment tool), ALIGN or Megalign (DNASTAR) software.
  • BLAST Basic Local Alignment Search Tool
  • analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx Karlin et al., PROC. NATL. ACAD. SCI.
  • the default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff etal, (1992) PROC. NATL. ACAD. SCI. USA 89:10915-10919).
  • a bacterial strain of Collinsella useful for the compositions and methods provided herein is Collinsella ASMB strain P121-D5a.
  • a deposit of Collinsella ASMB strain P121-D5a was made to DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, InhoffenstraBe 7B, 38124 Brunswick, Germany) under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure on September 20, 2019. This deposit was accorded accession number DSM 33276.
  • the 16s rRNA gene sequence of Collinsella ASMB strain P121-D5a is provided herein as SEQ ID NO: 46
  • genomic sequences of Collinsella ASMB strain P121-D5a are provided herein as SEQ ID NOs: 1-45.
  • Additional bacterial strains of Collinsella provided herein include Collinsella strains having a DNA-DNA hybridization (DDH)) value of equal to or greater than about 70% with Collinsella ASMB strain P121-D5a.
  • DDH DNA-DNA hybridization
  • the Collinsella ASMB strain is one having greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% DNA-DNA hybridization with Collinsella ASMB strain P121-D5a, or any range between any of the above values.
  • Any method for determining DNA-DNA hybridization values known in the art may be used to assess the degree of DNA-DNA hybridization, including but not limited to the spectrophotometric method for determining renaturation rates described by De Ley et al. (./ Biochem 12 133-142 (1970)), slightly modified in hybridization temperature (Gavini et al, Ecology in Health and Disease 1240-45 (2001)); and those described by Grimont et ah, Curr Microbiol 4, 325-330 (1980) and Rossello-Mora, Molecular Identification, Systematics and Population Structure of Prokaryotes pp. 23-50 (2006).
  • the degree of DNA-DNA hybridization is determined by digital DNA-DNA hybridization (dDDH) analysis, for example, using the Genome-to-Genome Distance Calculator online tool (see Meier-Kolthoff et al. , BMC Bioinformatics 14:60 (2013)).
  • the Collinsella strain is one having a DDH or dDDH value of equal to or greater than about 70% with Collinsella ASMB strain P121-D5a.
  • the DDH or dDDH value is greater than about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% with Collinsella ASMB strain P121-D5a, or any range between any of the above values.
  • Additional bacterial strains of Collinsella include Collinsella strains having equal to or greater than 95% average nucleotide identity (ANI) with Collinsella ASMB strain P121-D5a.
  • the ANI is equal to or greater than about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or 100% with Collinsella ASMB strain P121-D5a, or any range between any of the above values.
  • ANI average nucleotide identity
  • the ANI between two bacterial genomes is calculated from pair-wise comparisons of all sequences shared between any two strains and can be determined, for example, using any of a number of publicly available ANI tools, including but not limited to OrthoANI with usearch (Yoon et al. Antonie van Leeuwenhoek 110:1281-1286 (2017)); ANI Calculator, JSpecies (Richter and Rossello-Mora, Proc Natl Acad Sci USA 106:19126-19131 (2009)); and JSpeciesWS (Richter et al, Bioinformatics 32:929-931 (2016)). Other methods for determining the ANI of two genomes are known in the art.
  • the ANI between two bacterial genomes can be determined using an alignment- based method, for example, by averaging the nucleotide identity of orthologous genes identified as bidirectional best hits (BBHs).
  • Protein-coding genes of a first genome (Genome A) and second genome (Genome B) are compared at the nucleotide level using a similarity search tool, for example, NSimScan (Novichkov etal, Bioinformatics 32(15): 2380-23811 (2016).
  • the results are then filtered to retain only the BBHs that display at least 70% sequence identity over at least 70% of the length of the shorter sequence in each BBH pair.
  • the ANI of Genome A to Genome B is defined as the sum of the percent identity times the alignment length for all BBHs, divided by the sum of the lengths of the BBH genes.
  • the ANI between two bacterial genomes can be determined using an alignment-free method, for example, FastANI, which uses alignment-free approximate sequence mapping to assess genomic relatedness. See Jain el al, Nat Commun. 9(1):5114 (2016). FastANI has been demonstrated to reveal clear genetic discontinuity between species, with 99.8% of the total 8 billion genome pairs analyzed conforming to >95% intra-species and ⁇ 83% inter-species ANI values. Accordingly, in some embodiments, a bacterial strain having a genome with equal to or greater than 95% average nucleotide identity (ANI) with the genome of Collinsella ASMB strain P121-D5a is identified as a bacterial strain of the species Collinsella ASMB.
  • ANI average nucleotide identity
  • Additional bacterial strains of Collinsella include Collinsella strains having equal to or greater than 60% alignment fraction (AF) with Collinsella ASMB strain P121-D5a.
  • the AF is equal to or greater than about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% with Collinsella ASMB strain P121-D5a, or any range between any of the above values.
  • the AF is computed by dividing the sum of the lengths of all BBH genes by the sum of the length of all the genes in Genome A. This computation is performed separately in both directions: from Genome A to genome B and from Genome B to Genome A.
  • a Collinsella strain comprises a genome having equal to or greater than about 95% ANI and equal to or greater than 60% AF with the genome of Collinsella ASMB strain P121-D5a.
  • a Collinsella strain comprises a genome having equal to or greater than about 96.5% ANI and equal to or greater than 60% AF with the genome of Collinsella ASMB strain P121-D5a.
  • Additional bacterial strains of Collinsella provided herein include Collinsella strains that having the same or approximately the same genome characteristics as Collinsella ASMB strain P121-D5a. Such genome characteristics can include, for example, genome size, G+C content, number of coding sequences, and number of tRNAs.
  • the Collinsella strain comprises a genome of about 2.3 to about 2.5 megabases (Mb) in size.
  • the Collinsella strain comprises a genome of about 2.33 to about 2.47 Mb in size.
  • the Collinsella strain comprises a genome of about 2.31, 2.32, 2.33, 2.34, 2.35, 2.36, 2.37, 2.38, 2.39, 2.40, 2.41, 2.42, 2.43, 2.44, 2.45, 2.46, 2.47, 2.48, 2.49 or about 2.5 Mb in size.
  • the Collinsella strain comprises a genome of about 2.39 Mb in size.
  • the Collinsella strain comprises a genome that comprises about 1800 to 2200 coding sequences.
  • the Collinsella strain comprises a genome that comprises about 1850 to 2150 coding sequences.
  • the Collinsella strain comprises a genome that comprises about 1860, 1870, 1880, 1890, 1900, 1910, 1920, 1930, 1940, 1950, 1960, 1970, 1980, 1990, 2000, 2010, 2020, 2030, 2040, 2050, 2060, 2070, 2080, 2090, 2100, 2110, 2120, 2130, or 2140 coding sequences.
  • the Collinsella strain comprises a genome that comprises about 2014 coding sequences.
  • the Collinsella strain comprises a genome that comprises about 45 to 65 tRNA sequences.
  • the Collinsella strain comprises a genome that comprises about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 tRNAs. In a particular embodiment, the Collinsella strain comprises a genome that comprises about 55 tRNAs. In some embodiments, the Collinsella strain comprises a genome that has a G+C content of about 50% to about 70%. In some embodiments, the Collinsella strain comprises a genome that has a G+C content of about 50.5% to about 69.5%.
  • the Collinsella strain comprises a genome that has a G+C content of about 51.0%, 51.5%, 52.0%, 52.5%, 53.0%, 53.5%, 54.0%, 54.5%, 55.0%, 55.5%, 56.0%, 56.5%, 57.0%, 57.5%, 58.0%, 58.5%, 59.0%, 59.5%, 60.0%, 60.5%, 61.0%, 61.5%, 62.0%, 62.5%, 63.0%, 63.5%, 64.0%, 64.5%, 65.0%, 65.5%, 66.0%, 66.5%, 67.0%, 67.5%, 68.0%, 68.5%, 69.0%, 69.5%, or about 70%.
  • the Collinsella strain comprises a genome that has a G+C content of about 59.61%.
  • Additional bacterial strains of Collinsella include Collinsella strains that provide the same or approximately the same pattern as Collinsella ASMB strain P121- D5a when analyzed, for example, by DNA fingerprinting techniques. Any DNA fingerprinting technique known in the art may be used to identify strains of Collinsella , including but not limited to, Pulsed Field Gel Electrophoresis (PFGE), ribotyping, Randomly Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Amplified Ribosomal DNA Restriction Analysis (ARDRA), rep-PCR (repetitive element primed PCR, directed to naturally occurring, highly conserved, repetitive DNA sequences, present in multiple copies in the genomes) including Repetitive Extragenic Palindromic PCR (REP-PCR), Enterobacterial Repetitive Intergenic Consensus Sequences-PCR (ERIC -PCR), BOX-PCR (derived from the box A element), (GTG)s-PCR, Triplicate Arbitrary Primed
  • PFGE Pulsed
  • Additional bacterial strains of Collinsella provided herein include Collinsella strains showing phenotypic similarity to Collinsella ASMB strain P121-D5a.
  • Phenotypic similarity can be based on, for example, cell shape and size, colony morphology (e.g. size, color and odor of plate colonies), Gram staining, biochemical tests, pH and temperature optima, sugar fermentation, metabolic capabilities (e.g. catalase and/or oxidase negative), chemotaxonomic analysis (e.g. polar lipid and lipoquinone composition; see Tindall etal, Int J Syst Evol Microbiol 58, 1737-1745 (2008)) and/or fatty acid methyl ester (FAME) analysis.
  • cell shape and size e.g. size, color and odor of plate colonies
  • Gram staining e.g. size, color and odor of plate colonies
  • biochemical tests e.g. size, color and odor of plate colonies
  • biochemical tests e.
  • Additional bacterial strains of Collinsella include Collinsella strains showing carbon and/or nitrogen utilization similar to Collinsella ASMB strain P121-D5a.
  • the Collinsella bacterial strain is capable of utilizing one or more carbon sources selected from the group consisting of N- Acetyl -D-glucosamine, D-Galactose, L- Proline, D-Mannose, D-Fructose, a-D-Glucose, a-D-Lactose, Lactulose, b-Methyl-D- Glucoside, Maltotriose, D-Psicose, a-Cyclodextrin, Amygdalin, Arbutin, Lactitol, b-Methyl- D-Galactoside, D-Raffinose, Stachyose, and D-Glucosamine.
  • the Collinsella bacterial strain is capable of utilizing one or more nitrogen sources selected from the group consisting of Nitrite, Nitrate, Urea, Biuret, L-Alanine, L- Arginine, L- Asparagine, L-Cysteine, N-Acetyl-D-Glucosamine, and Thymidine.
  • a contemplated bacterial strain, bacterial strain mixture, or composition may be characterized as having an effect on gene product production, e.g, IFN-g, IL-Ib, IL-12p40, IL-12p70, IL-17A, IL-23, and/or TNF production, in a cell, tissue, or subject, e.g, an immune cell, e.g, a macrophage (e.g, a THP-1 macrophage) or PBMC (including lymphocytes (T cells, B cells, NK cells) and monocytes).
  • gene product production e.g, IFN-g, IL-Ib, IL-12p40, IL-12p70, IL-17A, IL-23, and/or TNF production
  • an immune cell e.g, a macrophage (e.g, a THP-1 macrophage) or PBMC (including lymphocytes (T cells, B cells, NK cells) and monocytes).
  • cytokines include T helper cells, monocytes, macrophages and dendritic cells, however myriad immune effector cell types are capable of producing cytokines in certain contexts including B cells, cytotoxic T cells, NK cells, mast cells, and granulocytes like neutrophils and eosinophils.
  • Gene product production e.g., IFN-g, IL-Ib, IL-12p40, IL-12p70, IL-17A, IL-23, and/or TNF production, in a macrophage may, for example, be assayed as follows.
  • THP-1 human macrophages are made by culturing the THP-1 human monocyte cell line with phorbol 12-myristate 13-acetate (PMA) for 24 hours, optionally followed by IL-4 and IL-13 as described previously (Genin el al., BMC Cancer 15:577 (2015)). Abacterial strain, bacterial strain mixture, or composition is incubated with THP-1 macrophages in the presence of lipopolysaccharide (LPS) for 24 hours. Gene product production is assessed by measuring the concentration of the gene product, e.g., IFN-g, IL-Ib, IL-12p40, IL-12p70, IL-17A, IL-23, and/or TNF, in the cell culture supernatant by ELISA.
  • PMA phorbol 12-myristate 13-acetate
  • LPS lipopolysaccharide
  • Gene product production may also be assayed as described in Sudhakaran etal. , Genes Nutr., 8(6): 637-48 (2013).
  • Gene product production e.g, IFN-g, IL-Ib, IL-12p40, IL-12p70, IL-17A, IL-23, and/or TNF production, in a PBMC may, for example, be assayed as follows.
  • Primary PBMCs are isolated from blood samples of donors using a percoll gradient (Sim et al., J. Vis. Exp. (112), e54128 (2016)).
  • a bacterial strain, bacterial strain mixture, or composition is incubated with PBMCs for 24 hours.
  • Gene product production is assessed by measuring the concentration of the gene product, e.g, IFN-g, IL-Ib, IL-12p40, IL-12p70, IL-17A, IL-23, and/or TNF, in the cell culture supernatant by ELISA.
  • concentration of the gene product e.g, IFN-g, IL-Ib, IL-12p40, IL-12p70, IL-17A, IL-23, and/or TNF
  • a Collinsella e.g, Collinsella ASMB
  • a bacterial strain increases, or is capable of increasing, production of at least one pro-inflammatory gene product, e.g, a pro-inflammatory cytokine or chemokine, in a cell, tissue, or subject.
  • pro-inflammatory gene products include IFN-g, IL-Ib, IL-12p40, IL-12p70, IL-17A, IL-23, and TNF.
  • a Collinsella e.g., Collinsella ASMB
  • a bacterial strain mixture or composition comprising the Collinsella bacterial strain increases expression of one or more of IFN-g, IL-Ib, IL-12p40, IL-12p70, IL-17A, IL-23, and TNF in a cell, tissue, or subject.
  • the increased production of a pro-inflammatory gene product e.g, IFN-g, IL-Ib, IL-12p40, IL-12p70, IL- 17A, IL-23, and/or TNF, occurs in a human cell, e.g, a THP-1 macrophage, monocyte,
  • PBMC or moDC For example, contacting a human cell, e.g, a THP-1 macrophage, PBMC or moDC with the Collinsella (e.g., Collinsella ASMB) bacterial strain (or the bacterial strain mixture or composition comprising the Collinsella bacterial strain), e.g, by culturing the human cell with the Collinsella (e.g, Collinsella ASMB) bacterial strain ; or the bacterial strain mixture or composition comprising the Collinsella bacterial strain), increases production of IFN-g, IL-Ib, IL-12p40, IL-12p70, IL-17A, IL-23, and/or TNF in the cell by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 750%, at least about 1000%, from about 10% to about 20%, from about 10% to about 50%, from about
  • cultured with the Collinsella bacterial strain (or the bacterial strain mixture or composition comprising the Collinsella bacterial strain).
  • the contacting of the human cell with the Collinsella (e.g, Collinsella ASMB) bacterial strain (or the bacterial strain mixture or composition comprising the Collinsella bacterial strain) occurs in vitro.
  • the contacting of the human cell with the Collinsella (e.g., Collinsella ASMB) bacterial strain (or the bacterial strain mixture or composition comprising the Collinsella bacterial strain) occurs in vivo.
  • a Collinsella e.g, Collinsella ASMB
  • a bacterial strain mixture or composition comprising the Collinsella bacterial strain increases, or is capable of increasing, infiltration of T cells in a tumor.
  • the T cell is a CD3+ T cell.
  • the T cell is a CD8+ T cell.
  • the composition may comprise a Collinsella (e.g, Collinsella ASMB) bacterial strain and one or more non -Collinsella bacterial species.
  • the one or more non- Collinsella bacterial species includes a member of the genus Butyricimonas , for example, Butyricimonas faecihominis .
  • the Butyricimonas faecihominis is Butyricimonas faecihominis strain P40-F2a, as described in U.S. Provisional Application Nos. 62/955,951 (filed December 31, 2019), 62/959,558 (filed January 10, 2020), 62/975,827 (filed February 13, 2020) and 63/035,128 (filed June 5, 2020).
  • Butyricimonas faecihominis strain P40-F2a was made to DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, InhoffenstraBe 7B, 38124 Brunswick, Germany) under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure on January 20, 2020. This deposit was accorded accession number DSM 33411.
  • the composition comprising a Collinsella (e.g., Collinsella ASMB) bacterial strain and optionally, one or more non -Collinsella bacterial species (e.g, Butyricimonas faecihominis) also comprises an immune checkpoint inhibitor.
  • an “immune checkpoint inhibitor” refers to a molecule, such as e.g., a small molecule, a soluble receptor, or an antibody, which targets an immune checkpoint and blocks the function of said immune checkpoint.
  • an “immune checkpoint inhibitor” as used herein is a molecule, such as e.g., a small molecule, a soluble receptor, or an antibody, that is capable of inhibiting or otherwise decreasing one or more of the biological activities of an immune checkpoint.
  • an inhibitor of an immune checkpoint protein e.g., an antagonistic antibody
  • an immune checkpoint protein can, for example, act by inhibiting or otherwise decreasing the activation and/or cell signaling pathways of the cell expressing said immune checkpoint protein (e.g., a T cell), thereby inhibiting a biological activity of the cell relative to the biological activity in the absence of the antagonist.
  • immune checkpoint inhibitors include small molecule drugs, soluble receptors, and antibodies.
  • the checkpoint inhibitor may, for example, be selected from a PD-1 antagonist, PD-L1 antagonist, CTLA-4 antagonist, adenosine A2A receptor antagonist, B7-H3 antagonist, B7-H4 antagonist, BTLA antagonist, KIR antagonist, LAG3 antagonist, TIM-3 antagonist, VISTA antagonist or TIGIT antagonist.
  • the checkpoint inhibitor is a PD-1 or PD-L1 inhibitor.
  • Exemplary PD-1/PD-L1 based immune checkpoint inhibitors include antibody-based therapeutics.
  • the present disclosure encompasses derivatives of the disclosed bacterial strains.
  • derivative includes daughter strains (progeny) or stains cultured (sub-cloned) from the original but modified in some way (including at the genetic level), without negatively altering a biological activity of the strain.
  • compositions Comprising a Collinsella Strain
  • compositions for example pharmaceutical compositions, comprising a bacterial strain of Collinsella (e.g, Collinsella ASMB).
  • the compositions comprise one or more bacterial strains, including one or more bacterial strains of Collinsella.
  • a composition provided herein comprises a bacterial strain of Collinsella ASMB and does not comprise any other strains or species of bacteria.
  • the composition comprises a bacterial strain of Collinsella ASMB and at least one or more additional strains or species of bacteria.
  • the at least one additional strain or species of bacteria in the composition is a bacterial strain of the genus Collinsella.
  • the composition may comprise an additional strain of Collinsella ASMB and/or one or more strains of a Collinsella species that is not Collinsella ASMB.
  • Exemplary additional Collinsella species include C. aerofaciens,
  • the composition may comprise Collinsella ASMB and one or more non -Collinsella bacterial species.
  • the one or more non- Collinsella bacterial species includes a Butyricimonas species, for example, Butyricimonas faecihominis, for example, Butyricimonas faecihominis strain P40-F2a.
  • the composition may comprise a Collinsella strain selected from C. aerofaciens, C. stercoris, C. intestinalis, C. phocaeensis,
  • a contemplated composition or bacterial strain mixture may, e.g, comprise or consist essentially of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 bacterial strains.
  • one or more strains of the composition or bacterial strain mixture are vegetative.
  • all the strains of the composition or bacterial strain mixture are vegetative.
  • a disclosed composition or bacterial strain mixture comprises or consists essentially of 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 bacterial strains; or, for example, may comprise or consist essentially of 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5 or 3 to 4 bacterial strains; or, for example, may comprise or consist essentially of 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6 or 4 to 5 bacterial strains; or, for example, may comprise or consist essentially of 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, or 7 to 8 bacterial strains; or, for example, may comprise or consist essentially of 8 to 10 or 8 to 9 bacterial strains.
  • a disclosed composition or bacterial strain mixture comprises or consists essentially of 2 or 3 bacterial strains.
  • a composition e.g, a pharmaceutical unit provided herein, may include each bacterial strain at any appropriate ratio, measured either by total mass or by colony forming units of the bacteria.
  • a disclosed pharmaceutical composition or unit may include two strains at a ratio of 0.1:1, 0.2:1, 0.25:1, 0.5:1, 0.75:1, 1:1, 2:1, 3:1, 4:1, 5:1, or 10:1, either by total mass or by colony forming units of the bacteria.
  • a disclosed pharmaceutical composition or unit may include three strains at a ratio of 1 : 1 : 1, 1:1:2, 1:1:4, 1:2:1, 1:2:2, 1:2:4, 1:4:1, 1:4:2, 1:4:4, 2:1:1, 2:1:2, 2:1:4, 2:2:1, 2:4:1, 4:1:1, 4:1:2, 4:1:4, 4:2:1, 4:4:1, either by total mass or by colony forming units of the bacteria.
  • the composition comprises a bacterial strain of Collinsella ASMB, and optionally, one or more additional strains or species of bacteria, wherein the composition: (i) increases production of one or more pro-inflammatory gene products, for example, IFN-g, IL-Ib, IL-6, IL-12p40, IL-12p70, IL-17, IL-23, and/or TNF, in a human cell, e.g, a THP-1 macrophage or monocyte or a PBMC.
  • a pro-inflammatory gene products for example, IFN-g, IL-Ib, IL-6, IL-12p40, IL-12p70, IL-17, IL-23, and/or TNF
  • a bacterial strain of Collinsella disclosed herein may be combined with pharmaceutically acceptable excipients to form a pharmaceutical composition, which can be administered to a patient by any means known in the art.
  • pharmaceutically acceptable excipient is understood to mean one or more of a buffer, carrier, or excipient suitable for administration to a subject, for example, a human subject, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the excipient (s) should be “acceptable” in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient.
  • Pharmaceutically acceptable excipients include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration.
  • Pharmaceutically acceptable excipients also include fillers, binders, disintegrants, glidants, lubricants, and any combination(s) thereof.
  • a contemplated composition may comprise a pharmaceutical excipient selected from the group consisting of cellulose, polyvinyl pyrrolidone, silicon dioxide, stearyl fumarate or a pharmaceutically acceptable salt thereof, lactose, starch, glucose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, magnesium stearate, mannitol, sorbitol, and any combination(s) thereof.
  • excipients, carriers, stabilizers and adjuvants see, e.g ., Handbook of Pharmaceutical Excipients, 8 th Ed., Edited by P.J. Sheskey, W.G.
  • bacterial strains of Collinsella described herein may be used in any composition in stabilized form, including, for example, in a lyophilized state (with optionally one or more appropriate cryoprotectants), frozen (e.g, in a standard or super cooled freezer), spray dried, and/or freeze dried.
  • Stabilized bacteria e.g. via lyophilization, freezing, spray drying or freeze drying
  • stabilized anaerobic bacteria may, in certain embodiments, possess advantageous properties over bacteria in culture with respect to administration, e.g, administration of a pharmaceutical composition provided herein.
  • lyophilizing bacterial strains involves a freeze-drying process that removes water from the bacterial cells.
  • the resulting lyophilized bacterial strains may, in certain embodiments, have enhanced stability as compared to bacterial cultures, and thus may be stored for longer periods of time (i.e. extending shelf-life).
  • dehydrated bacterial cells in stabilized form, do not grow or reproduce, but remain viable and may grow and reproduce when rehydrated.
  • viability of stabilized anaerobic Collinsella bacteria is maintained even when exposed to oxygen, thus facilitating their formulation (for example, into oral dosage forms) and use as a live biotherapeutic product that retains biological activity.
  • the bacterial strains of Collinsella described herein are stabilized (e.g.
  • Stability can be measured at a selected temperature and humidity conditions for a selected time period. Trend analysis can be used to estimate an expected shelf life before a material has actually been in storage for that time period.
  • stability may be measured as the time it takes to lose 1 log of cfu/g dry formulation under predefined conditions of temperature and/or humidity.
  • stability may be measured as the time required to measure a decrease in a particular biological function per unit of dry formulation.
  • a pharmaceutical composition or pharmaceutical unit comprising Collinsella ASMB loses at most 0.5 log cfus, 1 log cfus, 1.5 log cfus, 2 log cfus, 2.5 log cfus, 3 log cfus, 3.5 log cfus, 4 log cfus, 4.5 log cfus, 5 log cfus, 5.5 log cfus, 6 log cfus, 6.5 log cfus, 7 log cfus, 7.5 log cfus, 8 log cfus, 8.5 log cfus, 9 log cfus, 9,5 log cfus, or 10 log cfus (total, or per gram of dry formulation) of each bacterial strain present in the pharmaceutical composition or unit upon storage for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months 11, months, 12 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years,
  • a Collinsella bacteria disclosed herein may be combined with one or more cryoprotectants.
  • cryoprotectants include fructoligosaccharides (e.g., raftilose ® , fructooligosaccharide derived from inulin), trehalose, maltodextrin, sodium alginate, proline, glutamic acid, glycine (e.g, glycine betaine), mono-, di-, or polysaccharides (such as glucose, sucrose, maltose, lactose), polyols (such as mannitol, sorbitol, or glycerol), dextran, DMSO, methylcellulose, propylene glycol, polyvinylpyrrolidone, non-ionic surfactants such as Tween 80, and/or any combinations thereof.
  • fructoligosaccharides e.g., raftilose ® , fructooligosaccharide derived from inulin
  • trehalose maltod
  • the cryoprotectant comprises raftilose ® (fructooligosaccharide derived from inulin), maltodextrin, alginate, trehalose, and sucrose, or any combinations thereof.
  • a pharmaceutical composition comprising a bacterial strain of Collinsella further comprises sucrose as a cryoprotectant.
  • a pharmaceutical composition comprising a bacterial strain of Collinsella further comprises raftilose ® (fructooligosaccharide derived from inulin), maltodextrin, alginate, trehalose, and sucrose as cryoprotectants.
  • a pharmaceutical composition comprising a bacterial strain of Collinsella further comprises raftilose ® (fructooligosaccharide derived from inulin), maltodextrin, alginate, and trehalose as cryoprotectants.
  • raftilose ® fructtooligosaccharide derived from inulin
  • maltodextrin maltodextrin
  • alginate alginate
  • trehalose as cryoprotectants
  • a lyophilized powder form of a bacterial strain includes about 10% to about 80% (by weight) of one or more bacterial strains (e.g, one bacterial strain) and about 20% to about 90% (by weight) of one or more cryoprotectants and/or excipients, such as one or more cryoprotectants and/or excipients selected from the group consisting of raftilose ® (fructooligosaccharide derived from inulin), maltodextrin, sodium alginate, trehalose, sucrose, water, and/or combinations thereof.
  • raftilose ® fructtooligosaccharide derived from inulin
  • maltodextrin maltodextrin
  • sodium alginate trehalose
  • sucrose sodium alginate
  • water and/or combinations thereof.
  • 5 mg of contemplated lyophilized powder form of a bacterial strain may include about 0.5 mg to about 1.5 mg of the bacterial strain, about 1.5 mg to about 2.5 mg of the bacterial strain, about 2.5 to about 3.5 mg of the bacterial strain, or about 3.5 mg to about 4.5 mg of the bacterial strain. It can be appreciated that each lyophilized powder form of bacterial strain that may form a component of a disclosed composition may each have different excipients and/or amounts of excipients, as well as a discrete bacterial strain.
  • a pharmaceutical composition should be formulated to be compatible with its intended route of administration.
  • the bacterial compositions disclosed herein can be prepared by any suitable method and can be formulated into a variety of forms and administered by a number of different means.
  • the compositions can be administered orally, rectally, or enterally, in formulations containing conventionally acceptable carriers, adjuvants, and vehicles as desired.
  • rectal administration is understood to include administration by enema, suppository, or colonoscopy.
  • a disclosed pharmaceutical composition may, e.g., be suitable for bolus administration or bolus release.
  • a disclosed bacterial composition is administered orally.
  • Solid dosage forms for oral administration include capsules, tablets, caplets, pills, troches, lozenges, powders, and granules.
  • a capsule typically comprises a core material comprising a bacterial composition and a shell wall that encapsulates the core material.
  • the core material comprises at least one of a solid, a liquid, and an emulsion.
  • the shell wall material comprises at least one of a soft gelatin, a hard gelatin, and a polymer.
  • Suitable polymers include, but are not limited to: cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose (HPMC), methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl cellulose succinate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, such as those formed from acrylic acid, methacrylic acid, methyl acrylate, ammonio methylacrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate (e.g, those copolymers sold under the trade name "Eudragit ); vinyl polymers and copolymers such as polyvinyl pyrrolidone, polyvinyl acetate, polyvinylacetate phthalate, vinylacetate crotonic acid copolymer
  • Tablets, pills, and the like can be compressed, multiply compressed, multiply layered, and/or coated.
  • a contemplated coating can be single or multiple.
  • a contemplated coating material comprises at least one of a saccharide, a polysaccharide, and glycoproteins extracted from at least one of a plant, a fungus, and a microbe.
  • Non-limiting examples include com starch, wheat starch, potato starch, tapioca starch, cellulose, hemicellulose, dextrans, maltodextrin, cyclodextrins, inulins, pectin, mannans, gum arabic, locust bean gum, mesquite gum, guar gum, gum karaya, gum ghatti, tragacanth gum, funori, carrageenans, agar, alginates, chitosans, or gellan gum.
  • a contemplated coating material comprises a protein.
  • a contemplated coating material comprises at least one of a fat and an oil.
  • the at least one of a fat and an oil is high temperature melting. In some embodiments the at least one of a fat and an oil is hydrogenated or partially hydrogenated. In some embodiments the at least one of a fat and an oil is derived from a plant. In some embodiments the at least one of a fat and an oil comprises at least one of glycerides, free fatty acids, and fatty acid esters. In some embodiments a contemplated coating material comprises at least one edible wax.
  • a contemplated edible wax can be derived from animals, insects, or plants. Non-limiting examples include beeswax, lanolin, bayberry wax, carnauba wax, and rice bran wax. Tablets and pills can additionally be prepared with enteric or reverse-enteric coatings.
  • a contemplated food product is a drink for oral administration.
  • a suitable drink include water, fruit juice, a fruit drink, an artificially flavored drink, an artificially sweetened drink, a carbonated beverage, a sports drink, a liquid diary product, a shake, an alcoholic beverage, a caffeinated beverage, infant formula and so forth.
  • suitable means for oral administration include aqueous and nonaqueous solutions, emulsions, suspensions and solutions and/or suspensions reconstituted from non-effervescent granules, containing at least one of suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, coloring agents, and flavoring agents.
  • a pharmaceutical composition provided herein includes: (a) a Collinsella strain; and (b) a filler (e.g, microcrystalline cellulose, lactose, sucrose, mannitol, or dicalcium phosphate dihydrate), a disintegrant (e.g, polyvinyl pyrrolidone, sodium starch glycolate, starch, or carboxymethyl-cellulose), a flow-aid/glidant (e.g, talc or silica derivatives (e.g, colloidal silica such as Cab-O-Sil or Aerosil)), and a lubricant (e.g, sodium stearyl fumarate, magnesium stearate, calcium stearate, stearic acid, stearic acid salt, talc, liquid paraffin, propylene glycol (PG), PEG 6000, or magnesium/sodium lauryl sulfate).
  • a filler e.g, microcrystalline cellulose, lactose, sucrose, mannito
  • a contemplated pharmaceutical composition includes: (a) a Collinsella strain; and (b) a filler (microcrystalline cellulose), a disintegrant (polyvinyl pyrrolidone), a flow-aid/glidant (silicon dioxide), and a lubricant (sodium stearyl fumarate).
  • a contemplated pharmaceutical composition is formulated as a capsule.
  • the capsule is a hydroxypropyl methylcellulose (HPMC) capsule.
  • the capsule includes a banding polymer (e.g, hydroxypropyl methylcellulose (HPMC)), and a banding solvent (e.g, water or ethanol).
  • the capsule includes two banding solvents, water and ethanol.
  • the capsule is coated with a reverse enteric coating polymer (e.g, amino methacrylate copolymer), and comprises a surfactant (e.g, sodium lauryl sulfate), a flow- aid/glidant (e.g, silicon dioxide), a lubricant (e.g, stearic acid), an anti -tacking agent (e.g, talc), and a coating solvent (e.g, water).
  • a reverse enteric coating polymer e.g, amino methacrylate copolymer
  • a surfactant e.g, sodium lauryl sulfate
  • a flow- aid/glidant e.g, silicon dioxide
  • a lubricant e.g, stearic acid
  • an anti -tacking agent e.g, talc
  • a coating solvent e.g, water
  • the capsule is coated with an enteric coating polymer (e.g, poly (methacrylic acid-co-methyl methacrylate)), and further includes a plasticizer (e.g., tri ethyl citrate), an anti -tacking agent (e.g, talc), a pH adjuster (e.g, ammonia solution), and a coating solvent (e.g, purified water and isopropyl alcohol).
  • an enteric coating polymer e.g, poly (methacrylic acid-co-methyl methacrylate)
  • a plasticizer e.g., tri ethyl citrate
  • an anti -tacking agent e.g, talc
  • a pH adjuster e.g, ammonia solution
  • a coating solvent e.g, purified water and isopropyl alcohol
  • a contemplated capsule is a capsule-in-capsule dosage form, which includes an inner capsule and an outer capsule.
  • the inner capsule includes one or more lyophilized bacterial strains, a filler (e.g, microcrystalline cellulose, lactose, sucrose, mannitol, dicalcium phosphate dihydrate, or starch), a disintegrant (e.g, polyvinyl pyrrolidone, sodium starch glycolate, or carboxymethyl-cellulose), a flow- aid/glidant (e.g, silicon dioxide, talc, or colloidal silica), and a lubricant (e.g, sodium stearyl fumarate, magnesium stearate, calcium stearate, stearic acid, stearic acid salt, talc, liquid paraffin, propylene glycol (PG), PEG 6000, or magnesium/sodium lauryl sulfate).
  • a filler e.g, microcrystalline cellulose, lactose, sucrose
  • the outer capsule includes one or more lyophilized bacterial strains, a filler (e.g, microcrystalline cellulose, lactose, sucrose, mannitol, dicalcium phosphate dihydrate, or starch) a disintegrant (e.g, polyvinyl pyrrolidone, sodium starch glycolate, or carboxymethyl-cellulose), a flow-aid/glidant (e.g, silicon dioxide, talc, or colloidal silica), and a lubricant (e.g, sodium stearyl fumarate, magnesium stearate, calcium stearate, stearic acid, stearic acid salt, talc liquid paraffin, propylene glycol (PG), PEG 6000, or magnesium/sodium lauryl sulfate).
  • a filler e.g, microcrystalline cellulose, lactose, sucrose, mannitol, dicalcium phosphate dihydrate, or starch
  • a disintegrant e.g,
  • a contemplated capsule is a capsule-in-capsule dosage form, which includes an inner capsule and an outer capsule.
  • the inner capsule includes one or more lyophilized bacterial strains, a filler (microcrystalline cellulose), a disintegrant (polyvinyl pyrrolidone), a flow-aid/glidant (silicon dioxide), and a lubricant (sodium stearyl fumarate).
  • the outer capsule includes one or more lyophilized bacterial strains, a filler (microcrystalline cellulose), a disintegrant (polyvinyl pyrrolidone), a flow-aid/glidant (silicon dioxide), and a lubricant (sodium stearyl fumarate).
  • a filler microcrystalline cellulose
  • a disintegrant polyvinyl pyrrolidone
  • a flow-aid/glidant silicon dioxide
  • a lubricant sodium stearyl fumarate
  • a disclosed pharmaceutical unit comprises a dual component capsule.
  • a dual component capsule may comprise an inner capsule, wherein the inner capsule has a reverse enteric polymeric coating, and an outer capsule encapsulating the inner capsule, wherein the outer capsule has an enteric polymeric coating.
  • a contemplated inner and/or outer capsule may comprise a bacterial strain or a bacterial strain mixture.
  • a dual component capsule may comprise an inner capsule having an inner composition comprising a bacterial strain or bacterial strain mixture and one or more pharmaceutical excipients, wherein the inner capsule has a reverse enteric polymeric coating, and an outer capsule encapsulating the inner capsule and an outer composition comprising a bacterial strain or bacterial strain mixture and one or more pharmaceutical excipients, wherein the outer capsule has an enteric polymeric coating.
  • a contemplated inner and/or outer composition may, e.g., comprise a Collinsella strain, and optionally one or more additional strains. The inner composition and the outer composition may be the same or different.
  • a contemplated dual component capsule may include a total of about 5 mg to about 60 mg of the inner and outer composition, e.g. , a total of about 5 mg to about 50 mg of the inner and outer composition, a total of about 5 mg to about 15 mg of the inner and outer composition, a total of about 5 mg to about 25 mg of the inner and outer composition, or a total of about 25 mg to about 50 mg of the inner and outer composition.
  • a contemplated dual component capsule may include a total of about 50 mg to about 120 mg of the inner and outer composition, e.g.
  • a total of about 50 mg to about 75 mg of the inner and outer composition a total of about 60 mg to about 85 mg of the inner and outer composition, a total of about 50 mg to about 95 mg of the inner and outer composition, or a total of about 25 mg to about 110 mg of the inner and outer composition.
  • a disclosed dual component capsule includes an inner capsule with a reverse enteric polymeric coating, and an outer capsule with an enteric polymeric coating.
  • Each respective coating allows for biphasic release of the capsule’s contents (including bacterial strains) at distinct sites along the gastrointestinal tract.
  • contents including bacterial strains
  • the GI tract has several regions sharply demarcated by local pH ranging from 1 to 8.2.
  • the normal pH profile of the GI tract rises and falls between the stomach and the colon with pH ranges of 1-4 in the stomach, 5.5-6.4 in the duodenum, 6.8-8.2 in the ileum, and 5.5-6.5 in the colon.
  • the enteric polymeric coating of the outer capsule solubilizes in a pH of about 7 to 8, allowing for release in the ileum, and the reverse enteric polymeric coating of the inner capsule solubilizes in a pH of about 6.2 to 6.5, allowing for subsequent release in the colon.
  • the outer capsule maintains integrity (e.g ., absence of splits, cracks, or rupture of capsule shell) for about 2 hours at pH 1.2 and 37°C. In certain embodiments, the outer capsule maintains integrity (e.g., absence of splits, cracks, or rupture of capsule shell) for about 2 hours at pH 5.5 and 37°C. In certain embodiments, the outer capsule disintegrates within about 1 hour at pH 7.4 and 37°C. In certain embodiments, the inner capsule maintains integrity (e.g, absence of splits, cracks, or rupture of capsule shell) for up to 1 hour at pH 7.4 and 37°C. In certain embodiments, the inner capsule disintegrates within 2 hours at pH 6.5 and 37°C.
  • the inner and/or outer capsule coating is comprised of poly(dl-lactide-co-glycolide, chitosan (Chi) stabilized with PVA (poly-vinylic alcohol), a lipid, an alginate, carboxymethylethylcellulose (CMEC), cellulose acetate trimellitiate (CAT), hydroxypropylmethyl cellulose phthalate (HPMCP), hydroxypropylmethyl cellulose, ethyl cellulose, food glaze, mixtures of hydroxypropylmethyl cellulose and ethyl cellulose, polyvinyl acetate phthalate (PVAP), cellulose acetate phthalate (CAP), shellac, copolymers of methacrylic acid and ethyl acrylate, or copolymers of methacrylic acid and ethyl acrylate to which a monomer of methylacrylate has been added during polymerization.
  • Eudragit LI 00 and Eudragit SI 00 (anionic copolymers based on methacrylic acid and methyl methacrylate) can be used, either alone or in combination.
  • Eudragit LI 00 dissolves at about pH 6 and upwards and comprises between 46.0% and 50.6% methacrylic acid units per g dry substance;
  • Eudragit SI 00 dissolves at about pH 7 and upwards and comprises between 27.6% and 30.7% methacrylic acid units per g dry substance.
  • Another exemplary group of encapsulating polymers are the polyacrylic acids Eudragit® L and Eudragit® S which optionally may be combined with Eudragit RL or RS (copolymers of ethyl acrylate, methyl methacrylate and a low content of methacrylic acid ester with quaternary ammonium groups). These modified acrylic acids are useful since they can be made soluble at a pH of 6 to 7.5, depending on the particular Eudragit chosen, and on the proportion of Eudragit S to
  • Eudragit L Eudragit L, RS, and RL used in the formulation.
  • (S) coating of the inner capsule is comprised of Eudragit EPO ReadyMix.
  • a contemplated coating of the outer capsule is comprised of Eudragit L100
  • a contemplated capsule is suitable for extended or timed release.
  • a contemplated inner and/or outer capsule coating further comprises a band/seal, e.g ., hypromellose, an opacifier, e.g. , titanium dioxide, a plasticizer, e.g. tri ethyl citrate (TEC) or an anti-tacking agent, e.g. talc.
  • a band/seal e.g ., hypromellose
  • an opacifier e.g. , titanium dioxide
  • TEC tri ethyl citrate
  • an anti-tacking agent e.g. talc.
  • compositions comprising a Collinsella strain disclosed herein can be presented in a unit dosage form, i.e., a pharmaceutical unit.
  • a composition, e.g. , a pharmaceutical unit provided herein, may include any appropriate amount of one or more bacterial strains, measured either by total mass or by colony forming units of the bacteria.
  • a disclosed pharmaceutical composition or unit may include from about 10 3 cfus to about 10 12 cfus, about 10 6 cfus to about 10 12 cfus, about 10 7 cfus to about 10 12 cfus, about 10 8 cfus to about 10 12 cfus, about 10 9 cfus to about 10 12 cfus, about 10 10 cfus to about 10 12 cfus, about 10 11 cfus to about 10 12 cfus, about 10 3 cfus to about 10 11 cfus, about 10 6 cfus to about 10 11 cfus, about 10 7 cfus to about 10 11 cfus, about 10 8 cfus to about 10 11 cfus, about 10 9 cfus to about 10 11 cfus, about 10 10 cfus to about 10 11 cfus, about 10 3 cfus to about 10 10 cfus, about 10 6 cfus to about 10 12 cfus, about 10 7
  • a disclosed pharmaceutical composition or unit may include from about 10 3 cfus to about 10 12 cfus, about 10 6 cfus to about 10 12 cfus, about 10 7 cfus to about 10 12 cfus, about 10 8 cfus to about 10 12 cfus, about 10 9 cfus to about 10 12 cfus, about 10 10 cfus to about 10 12 cfus, about 10 11 cfus to about 10 12 cfus, about 10 3 cfus to about 10 11 cfus, about 10 6 cfus to about 10 11 cfus, about 10 7 cfus to about 10 11 cfus, about 10 8 cfus to about 10 11 cfus, about 10 9 cfus to about 10 11 cfus, about 10 10 cfus to about 10 11 cfus, about 10 3 cfus to about 10 10 cfus, about 10 6 cfus to about 10 11 cfus, about 10 6
  • a provided pharmaceutical unit comprises at least 1 x 10 3 colony forming units of each bacterial strain (e.g ., vegetative bacterial strain), or, at least 1 x
  • each bacterial strain e.g, vegetative bacterial strain
  • at least 1 x 10 7 colony forming units of each bacterial strain e.g. vegetative bacterial strain
  • at least 1 x 10 8 colony forming units of each bacterial strain e.g, vegetative bacterial strain
  • at least 1 x 10 9 colony forming units of each bacterial strain e.g, vegetative bacterial strain
  • compositions e.g, a pharmaceutical unit such as e.g., a capsule
  • a pharmaceutical unit such as e.g., a capsule
  • a pharmaceutical unit may comprise a total of about 30 mg to about 70 mg, about 30 mg to about 60 mg, about 30 mg to about 50 mg, about 30 mg to about 40 mg, about 40 mg to about 70 mg, about 40 mg to about 60 mg, about40 mg to about 50 mg, about 50 mg to about 70 mg, about 50 mg to about 60 mg, about 80 mg to about 100 mg, about 90 mg to about 110 mg, about 100 mg to about 120 mg, or about 110 mg to about 150 mg of lyophilized powder forms of the bacterial strain.
  • the pharmaceutical unit comprises a total of about 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 100 mg, 120 mg, 130 mg, 140 mg, or 150 mg of lyophilized powder form of the bacterial strain.
  • a disclosed composition such as a disclosed pharmaceutical unit may include about 5 to about 50 mg of each lyophilized powder form of a bacterial strain, for example, about 5 to about 45 mg, about 5 to about 40 mg, about 5 to about 35 mg, about 5 to about 30 mg, about 5 to about 25 mg, about 5 to about 15 mg, about 5 to about 10 mg, about 10 to about 50 mg, about 10 to about 35 mg of each lyophilized powder form of a bacterial strain (e.g ., a vegetative bacterial strain), about 10 to about 20 mg, about 10 to about 15 mg, or about 15 to about 45 mg of each lyophilized powder form of a bacterial strain (e.g., a vegetative bacterial strain).
  • a bacterial strain e.g ., a vegetative bacterial strain
  • a disclosed pharmaceutical unit comprises about 5, about 10, about 15, about 20, about 25, or about 30 mg of each lyophilized powder form of a bacterial strain (e.g, a vegetative bacterial strain).
  • a disclosed pharmaceutical unit includes about 25 to about 50 mg of a lyophilized powder form of one bacterial strain (e.g, vegetative bacterial strain) and about 5 mg to about 10 mg of the remaining lyophilized powder forms of bacterial strains (e.g, vegetative bacterial strains), or about 5 to about 15 mg of one lyophilized powder form of bacterial strain (e.g, vegetative bacterial strain) and about 5 to 10 mg of the remaining lyophilized powder forms of bacterial strains (e.g, vegetative bacterial strains), for example, about 15 mg of one lyophilized powder form of bacterial strain (e.g, vegetative bacterial strain) and about 5 mg of the remaining lyophilized powder forms of bacterial strains (e.g, vegetative bacterial strains), or about 15 mg to about
  • a pharmaceutical composition or unit may include, or may be administered in combination with a prebiotic, i.e., a compound or composition which modifies the growth, maintenance, activity and/or balance of the intestinal micro flora (e.g, can allow for specific changes in the composition and/or activity of the microbiome).
  • a prebiotic i.e., a compound or composition which modifies the growth, maintenance, activity and/or balance of the intestinal micro flora (e.g, can allow for specific changes in the composition and/or activity of the microbiome).
  • Exemplary prebiotics include complex carbohydrates, complex sugars, resistant dextrins, resistant starch, amino acids, peptides, nutritional compounds, biotin, polydextrose, fructooligosaccharide (FOS), galactooligosaccharides (GOS), inulin, lignin, psyllium, chitin, chitosan, chitosanoligosaccharides, lacitol, gums ( e.g ., guar gum), high amylose cornstarch (HAS), cellulose, b-glucans, hemi-celluloses, lactulose, mannooligosaccharides, mannan oligosaccharides (MOS), oligofructose-enriched inulin, oligofructose, oligodextrose, tagatose, trans-galactooligosaccharide, pectin, resistant starch, isomaltoligosaccharides, and x
  • Prebiotics can be found in foods (e.g., acacia gum, guar seeds, brown rice, rice bran, barley hulls, chicory root, Jerusalem artichoke, dandelion greens, garlic, leek, onion, asparagus, wheat bran, oat bran, baked beans, whole wheat flour, and banana), and breast milk. Prebiotics can also be administered in other forms (e.g, a capsule or dietary supplement).
  • compositions and methods disclosed herein can be used to treat various forms of inflammatory disorders, gastrointestinal disorders, and/or dysbiosis in a subject.
  • the disclosure provides a method of treating a gastrointestinal disorder, inflammatory disorder, and/or dysbiosis in a subject.
  • a contemplated method comprises administering to the subject an effective amount of a pharmaceutical composition and/or pharmaceutical unit comprising a Collinsella bacterial strain disclosed herein (and optionally one or more additional bacterial strains), either alone or in a combination with another therapeutic agent to treat the gastrointestinal disorder, inflammatory disorder, and/or dysbiosis in the subject.
  • treat means the treatment of a disease in a subject, e.g. , in a human. This includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease, i.e., causing regression of the disease state.
  • subject and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals, e.g. , human, a companion animal (e.g, dog, cat, or rabbit), or a livestock animal (for example, cow, sheep, pig, goat, horse, donkey, and mule, buffalo, oxen, or camel)).
  • the exact dosage of a pharmaceutical unit, pharmaceutical composition, or bacterial strain is chosen by an individual physician in view of the patient to be treated, in general, dosage and administration are adjusted to provide an effective amount of the bacterial agent to the patient being treated.
  • the “effective amount” refers to the amount necessary to elicit a beneficial or desired biological response.
  • An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
  • the effective amount of a pharmaceutical unit, pharmaceutical composition, or bacterial strain may vary depending on such factors as the desired biological endpoint, the drug to be delivered, the target tissue, the route of administration, etc. Additional factors which may be taken into account include the severity of the disease state; age, weight and gender of the patient being treated; diet, time and frequency of administration; drug combinations; reaction sensitivities; and tolerance/response to therapy.
  • a disclosed bacterial strain, bacterial strain mixture, or composition may not require colonization of the gut, e.g., an intestine, of the subject and/or persistence in the subject in order elicit a beneficial or desired biological response.
  • a bacterial strain, bacterial strain mixture, or composition colonizes or partially colonizes the gut of the subject and/or persists in the subject after administration.
  • a bacterial strain, bacterial strain mixture, or composition does not colonize the gut of the subject and/or persist in the subject after administration.
  • compositions and methods disclosed herein can also be used to treat cancer in a subject.
  • a contemplated method comprises administering to the subject an effective amount of a pharmaceutical composition and/or pharmaceutical unit comprising a Collinsella bacterial strain disclosed herein (and optionally one or more additional bacterial strains), either alone or in a combination with another therapeutic agent to treat the cancer in the subject.
  • a pharmaceutical composition and/or pharmaceutical unit comprising a Collinsella bacterial strain disclosed herein (and optionally one or more additional bacterial strains), either alone or in a combination with another therapeutic agent to treat the cancer in the subject.
  • cancers include solid tumors, soft tissue tumors, hematopoietic tumors and metastatic lesions.
  • hematopoietic tumors include, leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), e.g.
  • transformed CLL diffuse large B-cell lymphomas (DLBCL), follicular lymphoma, hairy cell leukemia, myelodyplastic syndrome (MDS), a lymphoma, Hodgkin’s disease, a malignant lymphoma, non-Hodgkin’s lymphoma, Burkitt’s lymphoma, multiple myeloma, or Richter’s Syndrome (Richter’s Transformation).
  • DLBCL diffuse large B-cell lymphomas
  • MDS myelodyplastic syndrome
  • a lymphoma Hodgkin’s disease
  • malignant lymphoma a malignant lymphoma
  • non-Hodgkin’s lymphoma a malignant lymphoma
  • Burkitt non-Hodgkin’s lymphoma
  • multiple myeloma multiple myeloma
  • Richter’s Syndrome Richter’s Syndrome
  • solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting head and neck (including pharynx), thyroid, lung (small cell or non-small cell lung carcinoma (NSCLC)), breast, lymphoid, gastrointestinal (e.g, oral, esophageal, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), genitals and genitourinary tract (e.g ., renal, urothelial, bladder, ovarian, uterine, cervical, endometrial, prostate, testicular), CNS (e.g., neural or glial cells, e.g. , neuroblastoma or glioma), or skin (e.g, melanoma).
  • the cancer is colorectal cancer (CRC).
  • the compositions and methods disclosed herein may also be useful for preventing one or more of the above diseases or conditions, when administered as vaccine compositions.
  • the bacterial strains provided herein are viable.
  • the bacterial strains are capable of at least partially or totally colonizing the gastrointestinal tract, e.g, the intestine.
  • the bacterial strains of the invention are viable and capable of at least partially or totally colonizing the gastrointestinal tract, e.g, the intestine.
  • the bacterial strains of the invention may be killed, inactivated or attenuated.
  • the compositions may comprise a vaccine adjuvant.
  • the compositions are for administration via injection, such as via subcutaneous injection.
  • compositions described herein can be used alone or in combination with other therapeutic agents and/or modalities.
  • administered “in combination,” as used herein, is understood to mean that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time.
  • the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.”
  • the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration.
  • the second treatment is more effective, e.g. , an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment.
  • delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other.
  • the effect of the two treatments can be partially additive, wholly additive, or greater than additive.
  • the delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.
  • a side effect of a first and/or second treatment is reduced because of combined administration.
  • a pharmaceutical composition or unit may include, or be administered in combination with, chemotherapy, e.g ., a cytotoxic agent.
  • cytotoxic agents include, for example, antimicrotubule agents, topoisomerase inhibitors, antimetabolites, protein synthesis and degradation inhibitors, mitotic inhibitors, alkylating agents, platinating agents, inhibitors of nucleic acid synthesis, histone deacetylase inhibitors (HD AC inhibitors, e.g., vorinostat (SAHA, MK0683), entinostat (MS-275), panobinostat (LBH589), trichostatin A (TSA), mocetinostat (MGCD0103), belinostat (PXD101), romidepsin (FK228, depsipeptide)), DNA methyltransferase inhibitors, nitrogen mustards, nitrosoureas, ethylenimines, alkyl sulfonates, triazene
  • HD AC inhibitors e
  • the cytotoxic agent is a platinum-based agent (such as cisplatin), cyclophosphamide, dacarbazine, methotrexate, fluorouracil, gemcitabine, capecitabine, hydroxyurea, topotecan, irinotecan, azacytidine, vorinostat, ixabepilone, bortezomib, taxanes (e.g, paclitaxel or docetaxel), cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, vinorelbine, colchicin, anthracyclines (e.g, doxorubicin or epirubicin) daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, adria
  • a pharmaceutical composition or unit may include, or be administered in combination with, a targeted therapy, e.g. a tyrosine kinase inhibitor, a proteasome inhibitor, or a protease inhibitor.
  • a targeted therapy e.g. a tyrosine kinase inhibitor, a proteasome inhibitor, or a protease inhibitor.
  • a pharmaceutical composition or unit may include, or be administered in combination with, an anti inflammatory, anti -angiogenic, anti-fibrotic, or anti -proliferative compound, e.g, a steroid, a biologic immunomodulator, a monoclonal antibody, an antibody fragment, an aptamer, an siRNA, an antisense molecule, a fusion protein, a cytokine, a cytokine receptor, a bronchodialator, a statin, an anti-inflammatory agent (e.g. methotrexate), or an NSAID.
  • an anti-inflammatory agent e.g. methotrexate
  • NSAID an anti-inflammatory agent
  • a pharmaceutical composition or unit may include, or be administered in combination with, a checkpoint inhibitor.
  • the checkpoint inhibitor may, for example, be selected from a PD-1 antagonist, PD-L1 antagonist, CTLA-4 antagonist, adenosine A2A receptor antagonist, B7-H3 antagonist, B7-H4 antagonist, BTLA antagonist, KIR antagonist, LAG3 antagonist, TIM-3 antagonist, VISTA antagonist or TIGIT antagonist.
  • the checkpoint inhibitor is a PD-1 or PD-L1 inhibitor.
  • Exemplary PD-1/PD-L1 based immune checkpoint inhibitors include antibody based therapeutics.
  • Exemplary treatment methods that employ PD-1/PD-L1 based immune checkpoint inhibition are described in U.S. Patent Nos. 8,728,474 and 9,073,994, and EP Patent No. 1537878B1, and, for example, include the use of anti-PD-1 antibodies.
  • Exemplary anti-PD-1 antibodies are described, for example, in U.S. Patent Nos. 8,952,136,
  • Exemplary anti-PD-1 antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.), pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), and pidilizumab (CT- 011, Cure Tech).
  • Exemplary anti-PD-Ll antibodies are described, for example, in U.S.
  • Exemplary anti-PD-Ll antibodies include, for example, atezolizumab (Tecentriq®, Genentech), durvalumab (AstraZeneca), MEDI4736, avelumab, and BMS 936559 (Bristol Myers Squibb Co.).
  • the checkpoint inhibitor is a CTLA-4 inhibitor.
  • CTLA-4 based immune checkpoint inhibition methods are described in U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227.
  • Exemplary anti-CTLA-4 antibodies are described in U.S. Patent Nos. 6,984,720, 6,682,736, 7,311,910; 7,307,064, 7,109,003, 7,132,281, 6,207,156,
  • CTLA-4 antibodies include ipilimumab or tremelimumab.
  • a pharmaceutical composition or unit may include, or be administered in combination with, an anti -bacterial agent, e.g ., an antibiotic.
  • a disclosed method may comprise pretreatment with an antibiotic, e.g, administration of an antibiotic to a subject prior to administration of a disclosed pharmaceutical composition or unit.
  • antibiotics for use in combination therapy include vancomycin, metronidazole, gentamicin, colistin, fidaxomicin, telavancin, oritavancin, dalbavancin, daptomycin, cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, ceftobiprole, cipro, Levaquin, floxin, tequin, avelox, norflox, tetracycline, minocycline, oxytetracycline, doxycycline, amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, methicillin, ertapenem, doripenem, imipenem/cilastatin, meropenem, amikacin, kanamycin, neomycin, netilmicin, to
  • a pharmaceutical composition or unit may include, or be administered in combination with, an anti-fungal or anti-viral agents.
  • anti-viral agents include abacavir, acyclovir, adefovir, amprenavir, atazanavir, cidofovir, darunavir, delavirdine, didanosine, docosanol, efavirenz, elvitegravir, emtricitabine, enfuvirtide, etravirine, famciclovir, foscarnet, fomivirsen, ganciclovir, indinavir, idoxuridine, lamivudine, lopinavir, maraviroc, MK-2048, nelfmavir, nevirapine, penciclovir, raltegravir, rilpivirine, ritonavir, saquinavir, stavudine,
  • anti-fungal agents include natamycin, rimocidin, filipin, nystatin, amphotericin B, candicin, and hamycin, miconazole, ketoconazole, clotrimazole, econazole, omoconazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole, and albaconazole, abafungin, terbinafme, naftifme, butenafme, anidulafungin, caspofungin, micafungin, polygodial, benzoic acid, ciclopirox, tolnaftate, undecylenic acid, flucytos
  • compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps.
  • Isolate P121-D5a was isolated from the stool sample of a healthy human donor. The donor underwent comprehensive clinical and laboratory testing to confirm healthy status including screening for infectious agents to minimize risk of transmissible infection. Serology screening included HIV-l/HIV-2 (IgG and EIA), HTLV-I and HTLV-II (Ab), Hepatitis A virus (IgM), Hepatitis B virus (HBSAg, anti-HBc IgG and IgM), Hepatitis C virus (anti-HCV IgG), Treponema pallidum (EIA, or RPR if EIA is positive),
  • Example 2 Taxonomic Characterization of Isolate P121-D5a [0113] 2.1 16S Sequencing and Phylogenetic Analysis.
  • a taxonomic characterization of purified isolate P121-D5a was performed using full length 16S rRNA gene sequencing data. Homology searches were performed against existing publicly available strains present in the National Center for Biotechnology Information (NCBI) taxonomy database.
  • NCBI National Center for Biotechnology Information
  • DNA sequences were filtered for low quality (Q-Score ⁇ 20) and length ( ⁇ 50), and adapter sequences were trimmed using Cutadapt v.1.15 (Martin, EMBnet Journal, [S.I.], v. 17, n. 1, p. pp. 10-12, (2011)).
  • Taxonomic identities were made using appropriate score cut offs on average nucleotide identity and alignment fraction scores. These scores were calculated using Joint Genome Institute’s Microbial Specie Identifier (MiSi) Version 0.3 (Varghese et al., Nucleic Acids Research, 43(14):6761-6771 (2015)); and FastANI (Jain et al, Nat Commun. 9(1):5114 (2018)) using reference genome databases curated by Assembly Biosciences.
  • PI primary isolate
  • Mb megabase pairs
  • CDS coding sequence
  • tRNA transfer ribonucleic acid
  • the P121-D5a PI whole genome sequence was compared against other strains and species of Collinsella by assessing average nucleotide identity (ANI) and alignment fraction (AF) to these reference organisms, in order to determine the extent of genomic relatedness to previously identified species of Collinsella.
  • ANI average nucleotide identity
  • AF alignment fraction
  • Table 3 The results, determined using two independent calculators of ANI and AF (FastANI, which uses alignment-free approximate sequence mapping to assess genomic relatedness; and Mi SI, an alignment-based method), are summarized in Table 3 below.
  • Reference (R) Collinsella organisms.
  • FastANI ANI determined using the method of Jain et al. (2016).
  • fa AF AF determined using the method of Jain et al. (2018).
  • MiSi ANI ANI determined using the method of Varghese etal. (2015).
  • MiSi AF AF determined using the method of Varghese etal. (2015).
  • ANI values for the most closely related Collinsella strain to Collinsella P121-D5a were in the range of 95.76-95.90%, with corresponding AF values of 0.85-0.88.
  • This strain is annotated as a member of Collinsella aerofaciens (RefSeq accession no. GCF 003856815.1).
  • other strains annotated as Collinsella aerofaciens had ANI values below 95%, for example, the type strain Collinsella aerofaciens ATCC 25986 (ANI range: 93.33 - 93.52; AF range: 0.77 - 0.79), which otherwise shared 98.57% identity at the 16S rRNA level (see Table 1).
  • Collinsella P121-D5a cells are obligate anaerobes, non-motile, non-spore forming, and appear as rods in short chains.
  • P152-H6d was evaluated for its ability to utilize 190 different carbon sources and 95 nitrogen sources, using Phenotypic Microarrays (Biolog, Hayward, CA).
  • the carbon sources utilized by P121-D5a include N-Acetyl-D-glucosamine, D-Galactose, L-Proline, D-Mannose, D-Fructose, a-D-Glucose, a-D-Lactose, Lactulose, b- Methyl-D-Glucoside, Maltotriose, D-Psicose, a-Cyclodextrin, Amygdalin, Arbutin, Lactitol, b-Methyl-D-Galactoside, D-Raffmose, Stachyose, and D-Glucosamine.
  • Nitrogen sources utilized by P121-D5a include, Nitrite, Nitrate, Urea, Biuret, L- Alanine, L- Arginine, L- Asparagine, L-Cysteine, N-Acetyl-D-Glucosamine, and Thymidine.
  • Freshly cultured bacteria from overnight cultures of the Collinsella ASMB P121- D5a strain were prepared in anaerobic conditions. Bacteria were centrifuged at 4300 x g for four minutes. Bacteria were washed once with pre-reduced anaerobic PBS (Gibco). Working stock solutions were prepared by resuspending washed bacteria with anaerobic PBS to the total surface area of - lx 10 L 10 pm 2 . Total surface area was calculated by determining the number of particles (bacterial cells) in solution, then multiplying the total number of particles by tlm average surface area (pm 2 ) of each particle, as measured by a particle counter (Beckman Coulter Counter). 10-fold serial dilutions were made using anaerobic PBS for specific assays.
  • Cryopreserved PBMC were thawed in a 37 °C water bath, diluted in warm RPMI 1640 supplemented with 10% heat-inactivated FBS and L-glutamine, and centrifuged (515 x g; four minutes). Cells were resuspended in PBS buffer containing 0.5 % bovine serum albumin (BSA) and 2 mM EDTA and CD14+ monocyte cells were isolated by selection using Miltenyi CD 14 Microbeads according to manufacturer’s directions.
  • BSA bovine serum albumin
  • CD14+ monocyte cells were isolated by selection using Miltenyi CD 14 Microbeads according to manufacturer’s directions.
  • Isolated CD 14+ monocytes were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, L- glutamine, penicillin/streptomycin antibiotic, 50 ng/mL recombinant human IL-4 (R&D Systems), and 100 ng/mL recombinant human GM-CSF (Biolegend). Media was replenished on days 3 and 6. On day 7 after isolation, cells were diluted to 5xl0 5 cells/mL in RPMI 1640 containing L-glutamine (Corning) supplemented with 10% heat-inactivated FBS (Tissue Culture Biologicals) and 0.292 mg/mL L-glutamine (Corning). A 100 pL aliquot of the 5xl0 5 cells/mL cell suspension was added to each well within a flat-well 96 well plate and cultured for 24 hours at 37 °C and 5% CO2 before addition of test articles.
  • P121-D5a test articles were prepared to a total surface area of lx 10 8 pm 2 and lx
  • test articles, vehicle (PBS) control and moDCs were co-incubated for 3 hours in 37 °C and 5% CO2.
  • the plates were then centrifuged (515 x g; four minutes), media removed, and replaced with RPMI 1640 supplemented with 10% heat-inactivated FBS, L-glutamine, and penicillin/streptomycin antibiotic. Culture plates were then incubated for an additional 15 h at 37 °C and 5% CO2.
  • the plates were centrifuged (515 x g; four minutes), and supernatant was collected and analyzed by a custom U-plex multiplex kit from Meso Scale Discovery according to manufacturer’s instructions. Results were averaged from 4 human donors with two experimental replicates from each donor.
  • Trima residual blood product containing concentrated blood mononuclear cells was obtained from anonymous donors through Blood Centers of the Pacific (San Francisco, CA) and processed within 24 hours of collection. Blood samples were tested negative for HIV, HBV, HCV, HTLV, Syphilis, West Nile Virus and Zika Virus. PBMC were isolated using a ficoll gradient as described previously (Sim et ak, J. Vis. Exp. (112), e541282016). Briefly, 50 mL of Trima residual was diluted with 50 mL of sterile PBS (Gibco) and 25 mL was overlaid on 15 mL Ficoll-Paque Plus (GE Healthcare) in 50 mL conical tubes.
  • sterile PBS Gibco
  • Ficoll-Paque Plus GE Healthcare
  • the samples were centrifuged at 450 x g for 30 min at room temperature and allowed to stop without brake.
  • the PBMC interphase was collected, washed with PBS and resuspended in RPMI 1640 containing 2.05 mM L-glutamine (Coming) supplemented with 10% heat-inactivated FBS (Tissue Culture Biologicals) and 0.292 mg/mL L-glutamine (Corning).
  • the cells were maintained by incubation in 37 °C and 5% CO2 and used for assay evaluation within 24 h or frozen for later use.
  • Cells were cryopreserved in RPMI 1640 supplemented with 50% FBS and 10% DMSO (Sigma Aldrich) at a concentration of 5 x 10 7 cells/mL and stored in liquid nitrogen until ready for use.
  • Human PBMCs used immediately after isolation or thawed from cryo-storage, were diluted to 5xl0 6 cells/mL in RPMI 1640 containing L-glutamine (Coming) supplemented with 10% heat-inactivated FBS (Tissue Culture Biologicals) and 0.292 mg/mL L-glutamine (Corning). A 100 pL aliquot of the 5xl0 6 cells/mL cell suspension was added to each well within a round-bottom 96 well plate and cultured for 24 hours at 37 °C and 5% CO2 before addition of test articles.
  • Test articles were prepared and added to the PBMCs as described above for the moDC assay. After 3 hours of incubation in 37 °C with 5% CO2, the plates containing cocultures were centrifuged (515 x g; four minutes), media removed, and replaced with RPMI 1640 supplemented with 10% heat-inactivated FBS, L-glutamine, and penicillin/streptomycin antibiotic. Culture plates were then incubated for an additional 15 h at 37 °C and 5% CO2. The plates were centrifuged (515 x g; four minutes) and supernatant was collected and analyzed by a custom U-plex multiplex kit from Meso Scale Discovery according to manufacturer’s instructions. Results were averaged from 4 human donors with two experimental replicates from each donor.
  • the THP-1 human monocyte cell line (ATCC cat# TIB-202) was cultured in 37 °C and 5% CO2 using RPMI 1640 containing 2.05mM L-glutamine (Corning) supplemented with 10% heat-inactivated FBS (Corning), 100 I.U/mL Penicillin, 100 pg/mL Streptomycin and 0.292 mg/mL L-glutamine (Coming). Passage number was restricted to 8 passages. The THP-1 human monocyte cell line was grown until 70-80% confluent. Cells were counted and resuspended in culture media. 100,000 cells were plated per well onto 96 well plates.
  • THP-1 human M2 macrophages were made by culturing the THP-1 human monocyte cells with 10 ng/mL phorbol 12-myristate 13-acetate (PMA) (InvivoGen) for 24 hours followed by 20ng/mL IL-4 (R&D Systems) and 20 ng/mL IL-13 (R&D Systems) for 48 hours in 37 °C and 5% CCh as described previously (Genin etal ., BMC Cancer 15:577 (2015)). One day before the experiment, cells were washed and resuspended in RPMI culture media without antibiotics containing 20 ng/mL IL-4 and 20 ng/mL IL-13.
  • PMA phorbol 12-myristate 13-acetate
  • P121-D5a test articles were prepared to a total surface area of lx 10 8 pm 2 , lx 10 7 pm 2 , lx 10 6 pm 2 and respectively.
  • the test articles and vehicle (PBS) control were added onto THP-1 macrophages at 10% v/v and centrifuged down onto the THP-1 cells at 515 x g for four minutes.
  • the test articles, control and THP-1 macrophages were co-incubated for 3 hours in 37 °C and 5% CO2. The co-incubation media was then replaced with fresh RPMI culture media supplemented with antibiotics to limit excess bacteria growth.
  • THP-1 cells were incubated after culture media replacement for 15 hours in 37 °C and 5% CO2 .
  • THP-1 cell supernatants were collected and analyzed by ELISA. Levels of selected cytokines in culture supernatants were quantified by using commercial enzyme-linked immunosorbent assay (ELISA) kits from Biolegend or R&D Systems with TMB detection according to manufacturer’s specifications.
  • ELISA enzyme-linked immunosorbent assay
  • Collinsella ASMB P121-D5a was tested for efficacy in two different well-validated mouse tumor models: (1) a subcutaneous B16F10 syngeneic melanoma model and (2) a subcutaneous CT26 syngeneic colon carcinoma model.
  • B16F10 cells were thawed from low passage number stock, expanded using standard tissue culture technique, and maintained in DMEM with 10% fetal bovine plasma (FBS) in a 37°C incubator with 5% CO2. Cells growing in logarithmic phase (50-60% confluency) were harvested, and 1 x 10 5 cells per animal were implanted subcutaneously into female C57BL/6 mice (Taconic) in the right lower flank. Upon tumors reaching an average size of 100 mm 3 in volume, animals were stratified into positive control, experimental, or negative control groups. The positive control group received anti-PD-Ll antibody (200 pg, via intraperitoneal route) every 4 days for the duration of the study.
  • FBS fetal bovine plasma
  • Experimental groups received either oral administrations of Collinsella ASMB P121-D5a once daily for the duration of the study, or the combination of Collinsella ASMB P121-D5a once daily and anti- PD-Ll antibody (200 pg, via intraperitoneal route) every 4 days for the duration of the study.
  • the negative control group received vehicle only once daily for the duration of the study. Tumor volume and body weights were assessed 3 times a week, and tumor weights were assessed at the end of the study.
  • CT-26 cells were thawed from low passage number stock, expanded using standard tissue culture technique, and maintained in DMEM with 10% fetal bovine plasma (FBS) in a 37°C incubator with 5% CO2. Cells growing in logarithmic phase (50-60% confluency) were harvested, and 1 x 10 5 cells per animal were implanted subcutaneously into female BALB/c mice (Taconic) in the right lower flank. Upon tumors reaching an average size of 100 mm 3 in volume, animals were stratified into experimental, negative control or positive control groups.
  • FBS fetal bovine plasma
  • Animals in groups receiving bacterial test articles were administered once daily bacteria as single strains (mixed in equal volumes with vehicle), bacteria as 2-strain combinations ( Collinsella ASMB P121-D5a strain and Butyricimonas faecihominis P40-F2a in equal amounts), or bacteria as 2-strain combinations ⁇ Collinsella ASMB P121-D5a strain and Butyricimonas faecihominis P40-F2a in equal amounts) plus anti-PD-1 antibody.
  • Anti-PD-1 antibody was administered (alone as positive control or with a 2-strain combination) in an amount of 10 mg/kg, in 100 m ⁇ , via intraperitoneal route every 3 days for the duration of the study.
  • tumor reduction when compared to administration of vehicle only, was observed in groups administered: Collinsella ASMB P121-D5a alone (Fig. 10A); Butyricimonas faecihominis P40-F2a alone (Fig. 10B); anti-PD-1 antibody alone (Fig. IOC); the combination of Collinsella ASMB P121-D5a plus Butyricimonas faecihominis P40- F2a (Fig. 10D) (tumor volume versus vehicle only displayed in mm 3 in the left graph and as fold-change in the right graph).
  • FIG. 11 provides representative immunohistochemistry staining for CD8+ T cells in formalin-fixed paraffin-embedded (FFPE) tumor sections following treatment with (Fig. 11A) Collinsella ASMB P121-D5a + Butyricimonas faecihominis P40-F2a (Ca + Bf); (Fig. 11A) Collinsella ASMB P121-D5a + Butyricimonas faecihominis P40-F2a (Ca + Bf); (Fig.
  • each of anti-PD-1 antibody alone (a-PD-1), the combination of Collinsella ASMB P121-D5a and Butyricimonas faecihominis P40-F2a (Ca + Bf) strains, and the combination of Collinsella ASMB P121-D5a and Butyricimonas faecihominis P40-F2a strains anti-PD-1 antibody (Ca + Bf + a-PD-1) increased tumor-infiltrating T cells relative to administration of vehicle only, with the highest T cell score observed for the combination of Ca + Bf+ a-PD-1.
  • IFN-g concentrations were also assessed in tumor samples by Meso Scale Discovery (MSD) analysis of tumor tissue homogenate and normalized to homogenate protein concentration.
  • MSD Meso Scale Discovery
  • each of anti-PD-1 antibody alone (a-PD-1)
  • the combination of Collinsella ASMB P121-D5a and Butyricimonas faecihominis P40-F2a (Ca + Bf) strains
  • the combination of Collinsella ASMB P121-D5a and Butyricimonas faecihominis P40-F2a strains anti -PD- 1 antibody (Ca + Bf + a-PD-1) increased IFN-g concentrations in tumors relative to administration of vehicle alone.

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Abstract

L'invention concerne de manière générale des souches bactériennes du genre Collinsella , par exemple, des souches bactériennes de Collinsella ASMB, et des compositions, par exemple des compositions pharmaceutiques, comprenant de telles souches. L'invention concerne en outre des procédés d'utilisation de telles souches et compositions pour prévenir ou traiter un trouble, par exemple un cancer, lorsqu'elles sont administrées à un sujet en ayant besoin.
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