WO2021022110A1 - Induction d'effets immunitaires à l'aide de bactéries du genre bifidobacterium - Google Patents

Induction d'effets immunitaires à l'aide de bactéries du genre bifidobacterium Download PDF

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WO2021022110A1
WO2021022110A1 PCT/US2020/044402 US2020044402W WO2021022110A1 WO 2021022110 A1 WO2021022110 A1 WO 2021022110A1 US 2020044402 W US2020044402 W US 2020044402W WO 2021022110 A1 WO2021022110 A1 WO 2021022110A1
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bifidobacterium
subject
increase
bacteria
cells
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PCT/US2020/044402
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Shannon ARGUETA
Mark BODMER
Loise FRANCISCO-ANDERSON
Humphrey Gardner
Michael F. GOLDBERG
Jason HUDAK
Shubhra KASHYAP
Pooja PARAMESWARAN
Holly PONICHTERA
Peter SANDY
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Evelo Biosciences, Inc.
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Publication of WO2021022110A1 publication Critical patent/WO2021022110A1/fr

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    • 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
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • 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 constitution of an individual’s gut microbiome can have a significant influence on their immunological health.
  • immune cells circulate via lymphatic vessels through the small intestine, where they become conditioned by exposure to antigens and immunomodulatory agents.
  • macrophages and dendritic cells can sample bacteria in the gut, and then these macrophages and dendritic cells can condition T cells in lymph nodes. Thereafter, the conditioned T cells can migrate to other parts of the body, such as the brain, the skin, the liver, and the joints.
  • provided herein are methods and compositions for inducing desired immune effects (e.g., an immune effect described herein) in a subject by administering to them bacteria of the genus Bifidobacterium.
  • desired immune effects e.g., an immune effect described herein
  • induction of such immune effects is useful in the treatment of cancer.
  • such immune effects can be used to identify subjects for whom administration of a pharmaceutical composition comprising Bifidobacterium will be an effective therapeutic strategy and/or for monitoring such therapies once they have been initiated.
  • methods of inducing an immune effect in a subject e.g. a subject in need thereof, such as a subject with cancer
  • a pharmaceutical composition that contains bacteria of the genus Bifidobacterium.
  • the immune effect in these and various other aspects can include one or more of the following: an increase in Ml polarization of macrophages in the subject; an increase in pro-inflammatory CD103 + dendritic cells within tumor-draining lymph nodes of the subject; an increase in expression of at least one co-stimulatory molecule (e.g., CD80, CD86, CD40, or a combination thereof) on CD1 lc + dendritic cells within mesenteric lymph nodes of the subject; an increase in production of at least one dendritic-cell-derived growth factor (e.g., GM-CSF) in the subject; an increase in expression of a pro- inflammatory cytokine (e.g., TNF-a, IL-I b, IL-12, or a combination thereof) by dendritic cells in the subject; an increase in production of a lymphocyte recruiting chemokine (e.g., CXCL9, CXCL10, or a combination thereof) in the subject; an increase in IFN
  • the antigen-presenting cells are monocytes.
  • the immune effect includes an increase in CD44 + effector lymphocytes in a tumor in the subject (e.g., natural killer cells, natural killer T cells, CD4 + cells, CD8 + cells).
  • the immune effect includes an increase in infiltration by activated XCR1 + conventional type 1 dendritic cells.
  • provided herein are methods of treating cancer in a subject that includes orally administering to the subject a pharmaceutical composition that has bacteria of the genus Bifidobacterium, in which the pharmaceutical composition induces an immune effect when administered to the subject.
  • the methods before administering the pharmaceutical composition, include selecting a subject having cancer in whom oral administration of a dose of a pharmaceutical composition comprising bacteria of the genus Bifidobacterium induces the immune effect.
  • provided herein are methods of treating a subject in need of induction of an immune effect that include orally administering to the subject a pharmaceutical composition having bacteria of the genus Bifidobacterium, in which the pharmaceutical composition induces the immune effect. In some aspects, before administering the
  • the methods include selecting a subject in need in whom oral administration of a dose of a pharmaceutical composition comprising bacteria of the genus Bifidobacterium induces the immune effect.
  • kits for screening a strain of bacteria of the genus Bifidobacterium as a cancer therapeutic that include orally administering a
  • composition comprising a strain of bacteria of the genus Bifidobacterium to a test subject (e.g., a non-human mammal, such as a mouse or a rat) or contacting a composition comprising a strain of bacteria of the genus Bifidobacterium with a test sample having dendritic cells, and determining at least one test level for an immune effect.
  • the modifier“test” in the term“test level” denotes that the level relates to a test subject or to a test sample (e.g., rather than to a control subject or to a control sample). In some embodiments, that determined level (i.e., test level) is compared to a control level or to a control threshold.
  • kits for selecting a subject with cancer for treatment with a pharmaceutical composition having bacteria of the genus Bifidobacterium that include orally administering to a subject a dose of a pharmaceutical composition having bacteria of the genus Bifidobacterium, determining at least one test level for an immune effect, and selecting the subject for treatment with a pharmaceutical composition having bacteria of the genus Bifidobacterium if the test level passes a control threshold.
  • methods of treating a subject for cancer that include orally administering to the subject an initial dose of a pharmaceutical composition having bacteria of the genus Bifidobacterium, determining at least one test level for an immune effect, and if the test level passes a control threshold, administering one or more additional doses of the pharmaceutical composition or if the test level fails to pass a control threshold, administering a higher dose of the pharmaceutical composition.
  • compositions that contain bacteria of the genus Bifidobacterium that induce an immune effect (e.g., an immune effect described herein) when orally administered to a subject.
  • an immune effect e.g., an immune effect described herein
  • pharmaceutical compositions having bacteria of the genus Bifidobacterium for use, via their oral administration to a subject, in inducement of an immune effect, in treatment of cancer, in screening of a strain of the bacteria, in selection of a subject with cancer for treatment with the bacteria, or in adjustment of a treatment regimen with the pharmaceutical compositions, e.g., in each case, as described herein.
  • compositions having bacteria of the genus Bifidobacterium for, via their oral administration to a subject, inducing an immune effect, treating cancer, screening a strain of the bacteria, selecting a subject with cancer for treatment with the bacteria, or adjusting a treatment regimen with the pharmaceutical compositions, e.g., in each case, as described herein.
  • the pharmaceutical composition contains at least 10 5 colony-forming units (CFU) of bacteria of the genus Bifidobacterium (e.g., at least 10 6 , 10 7 , or 10 8 CFU of the bacteria of the genus Bifidobacterium).
  • CFU colony-forming units
  • the pharmaceutical composition comprises no more than 10 10 CFUs of the bacteria of the genus Bifidobacterium. In some embodiments, the pharmaceutical composition comprises about 7.5 x 10 10 CFUs of the bacteria of the genus Bifidobacterium. In some embodiments, the pharmaceutical composition comprises about 15 x 10 10 CFUs of the bacteria of the genus Bifidobacterium. In some embodiments, the pharmaceutical composition comprises about 22.5 x 10 10 CFUs of the bacteria of the genus Bifidobacterium. In some embodiments, the
  • the pharmaceutical composition comprises about 30 x 10 10 CFUs of the bacteria of the genus Bifidobacterium. In some embodiments, the pharmaceutical composition is administered once a day. In some embodiments, the pharmaceutical composition is administered twice a day.
  • the pharmaceutical composition comprises about 7.5 x
  • 10 10 CFUs e.g., one capsule (e.g., enteric coated capsule) of the bacteria of the genus
  • the pharmaceutical composition comprises about 15 x 10 10 CFUs (e.g., two capsules (e.g., two enteric coated capsules)) of the bacteria of the genus Bifidobacterium. In some embodiments, the pharmaceutical composition comprises about 22.5 x 10 10 CFUs (e.g., three capsules (e.g., three enteric coated capsules)) of the bacteria of the genus Bifidobacterium. In some embodiments, the pharmaceutical composition comprises about 30 x 10 10 CFUs (e.g., four capsules (e.g., four enteric coated capsules)) of the bacteria of the genus Bifidobacterium.
  • the pharmaceutical composition is administered once a day. In some embodiments, the pharmaceutical composition is administered twice a day.
  • the cancer can be any cancer. In certain embodiments, the cancer is a cancer that can be treated by the therapeutic effect induced by the bacteria of the genus Bifidobacterium. In some embodiments, the cancer can be prostate cancer, lung cancer, colon cancer, colorectal cancer, melanoma, breast cancer, pancreatic cancer, hepatocellular carcinoma, or lymphoma.
  • the bacteria of the genus Bifidobacterium can include
  • Bifidobacterium adolescentis Bifidobacterium angulatum, Bifidobacterium animalis,
  • the bacteria of the genus Bifidobacterium are Bifidobacterium breve.
  • the Bifidobacterium breve is the Bifidobacterium breve strain deposited under accession number NCIMB 42380, also referred to as“MRx004” and“MRx4DP0004”.
  • the bacteria of the genus Bifidobacterium are Bifidobacterium animalis.
  • the bacteria of the genus Bifidobacterium are Bifidobacterium animalis ssp. lactis.
  • At least 90% (e.g ., at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%) of the bacteria in the pharmaceutical composition are from a single species of Bifidobacterium. In some embodiments, essentially all of the bacteria in the pharmaceutical composition are from a single species of Bifidobacterium. In some embodiments, all of the bacteria in the pharmaceutical composition are from a single species of Bifidobacterium. In certain embodiments, the bacteria in the pharmaceutical composition can be live and/or attenuated.
  • the bacteria of the genus Bifidobacterium include a strain of Bifidobacterium animalis ssp. lactis having at least 99% (e.g., at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%) genomic nucleotide sequence identity with Bifidobacterium animalis ssp. lactis Strain A (ATCC Deposit No. PTA-125097).
  • the bacteria of the genus Bifidobacterium include Bifidobacterium animalis ssp.
  • lactis Strain A ATCC Deposit No. PTA- 125097.
  • at least 90% e.g at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%
  • compositions are from a single strain of Bifidobacterium.
  • essentially all of the bacteria in the pharmaceutical composition are from a single strain of Bifidobacterium. In some embodiments, all of the bacteria in the pharmaceutical composition are from a single strain of Bifidobacterium.
  • the methods also include administering a prebiotic to the subject.
  • the methods also include administering a second therapy
  • Cancer therapies can include chemotherapy, radiotherapy, or immunotherapy (e.g., with a checkpoint inhibitor, such as atezolizumab, avelumab, durvalumab, ipilimumab, nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, BGB-A317, STI- A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0010718C, AUR-012, or STI- A1010).
  • the cancer therapy comprises a checkpoint inhibitor.
  • the immune checkpoint inhibitor is a CTLA-4 inhibitor.
  • the immune checkpoint inhibitor is ipilimumab. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the immune checkpoint inhibitor is pembrolizumab. In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor.
  • the immune checkpoint inhibitor is avelumab. In some embodiments, the immune checkpoint inhibitor is an antibody.
  • a control threshold for various aspects can be determined from one or more subjects without cancer, from a test subject before administering a dose of a pharmaceutical composition, or from a standard level.
  • the disclosure provides bacteria of the genus Bifidobacterium
  • cancer e.g., a bacteria described herein
  • a cancer described herein for use in treating cancer, e.g., a cancer described herein.
  • the disclosure provides the use of bacteria of the genus
  • Bifidobacterium e.g., a bacteria described herein
  • a medicament for treating cancer e.g., a cancer described herein.
  • the disclosure provides bacteria of the genus Bifidobacterium
  • the disclosure provides the use of bacteria of the genus
  • Bifidobacterium e.g., a bacteria described herein
  • a medicament for inducing an immune effect e.g., as described herein.
  • Fig. 1 A shows that stimulation of human monocyte-derived macrophages with
  • Bifidobacterium animalis ssp. lactis Strain A (ATCC Deposit No. PTA- 125097) (shown in second bar from left) in vitro leads to the strong induction of CXCL10, as compared to stimulation of human monocyte-derived macrophages with other strains of Bifidobacterium. (Ml polarized macrophages shown in black and unstimulated control shown in white).
  • Fig. IB shows that stimulation of human PBMCs with Bifidobacterium animalis ssp. lactis Strain A (ATCC Deposit No. PTA-125097) induces significant IL-12p70, as compared to stimulation of human monocyte-derived macrophages with other Bifidobacterium strains.
  • Fig.lC shows t at Bifidobacterium animalis ssp. lactis Strain A (ATCC Deposit
  • Fig. 2A shows results obtained after purified human monocytes were pulsed with
  • Bifidobacterium animalis ssp. lactis Strain A ATCC Deposit No. PTA-125097
  • an anti inflammatory microbe at an MOI of 1 for 18 hours and then cultured with autologous purified NK cells. NK cells were then transferred to wells containing CFSE-labeled K-562 cells. % cytotoxicity was measured by flow cytometry.
  • Fig. 2B shows results obtained after NK cells were cultured with microbe-treated monocyte-conditioned media and subsequently transferred to plates containing CFSE-labeled K- 562 cells to assess cytotoxic activity.
  • Fig. 2C shows results obtained after NK cells alone were incubated with
  • Bifidobacterium animalis ssp. lactis Strain A (ATCC Deposit No. PTA-125097) or an anti inflammatory microbe prior to culture with CFSE-labeled K-562 cells for 4 hours. % cytotoxicity was measured by flow cytometry. ⁇ * p > 0.05, ** p > 0.01, *** p > 0.001 ; Two-Way ANOVA).
  • Fig. 3 shows results obtained following overnight culture of mesenteric lymph node cells with Bifidobacterium animalis ssp. lactis Strain A (ATCC Deposit No. PTA-125097) ex vivo. The frequency (left panel) and number (right panel) of CD44+CD25 + IFN-y + effector cells was determined by flow cytometry.
  • Fig. 4A shows results obtained after syngeneic mice were inoculated with MC-38 tumor cells at 10 5 subcutaneously. Tumors were established for 10 days prior to initiation of treatment. Mice were treated with either sucrose vehicle or Bifidobacterium animalis ssp. lactis Strain A (ATCC Deposit No. PTA- 125097) via oral gavage daily or treated with anti -PD- 1 (Fig. 4A, Fig. 4C) or anti-PD-Ll (Fig. 4B) every four days I.P. for the duration of the study.
  • Fig. 4B shows results obtained after syngeneic mice were inoculated with B 16-
  • Fig. 4C shows results obtained after syngeneic mice were inoculated with CT-26 tumor cells at 10 5 subcutaneously. Tumors were established for 10 days prior to initiation of treatment. Mice were treated with either sucrose vehicle or Bifidobacterium animalis ssp. lactis Strain A (ATCC Deposit No. PTA- 125097) via oral gavage daily or treated with anti -PD- 1 (Fig. 4A, Fig. 4C) or anti-PD-Ll (Fig. 4B) every four days I.P. for the duration of the study.
  • Fig. 5 A shows that the RT-PCR analysis of mRNA derived from CT-26 tumor lysate indicates an increase in the relative expression of the pro-inflammatory chemokine CXCL10 following treatment with Bifidobacterium animalis ssp. lactis Strain A (ATCC Deposit No. PTA-125097).
  • Fig. 5B shows that ex vivo analysis of purified CD45 + cells from CT-26 tumors reveals strong induction of chemokines consistent with immunogenic remodeling of the TME following treatment with Bifidobacterium animalis ssp. lactis Strain A (ATCC Deposit No. PTA- 125097).
  • Fig.5C shows that RT-PCR analysis of mRNA derived from CT-26 tumor lysate indicates an increase in the relative expression of the antigen presenting molecule, b2M following treatment with Bifidobacterium animalis ssp. lactis Strain A (ATCC Deposit No. PTA- 125097).
  • Fig. 5D shows that CT-26 tumors from Bifidobacterium animalis ssp. lactis Strain
  • mice are infiltrated with greater numbers of activated XCR1 + cDCl than control mice.
  • Fig. 5E shows that the histological examination of CT26 tumors reveals increase
  • Fig. 5F shows the quantification of various TIL populations following
  • FIG. 6A shows that treatment with Bifidobacterium animalis ssp. lactis Strain A
  • FIG. 6B shows that treatment with Bifidobacterium animalis ssp. lactis Strain A
  • Fig. 6C shows that similar to anti-PD-1 , Bifidobacterium animalis ssp. lactis
  • Fig. 7A shows that Bifidobacterium animalis ssp. lactis Strain A (ATCC Deposit
  • Fig. 7B shows that depletion of NK cells with anti-asialoGMl reveals an NK- mediated anti-tumor response induced by Bifidobacterium animalis ssp. lactis Strain A (ATCC Deposit No. PTA-125097).
  • Fig. 7C shows the loss of efficacy of Bifidobacterium animalis ssp. lactis Strain
  • Fig. 7D shows that CD8 + T cells are required for Bifidobacterium animalis ssp. lactis Strain A (ATCC Deposit No. PTA-125097) efficacy as mice depleted of CD8 + T cells are unable to mount a productive anti-tumor response upon treatment with Bifidobacterium animalis ssp. lactis Strain A (ATCC Deposit No. PTA-125097).
  • Fig. 8B shows CXCL10 production from CD 1 lb- enriched primary human monocytes measured by ELISA from three individual healthy donors stimulated with a 10: 1 MOI of different microbes from the genus Bifidobacterium relative to unstimulated controls.
  • Fig. 8D shows cytokine production from human PMBC after stimulation with a
  • Fig. 8F shows the effect of Bifido Strain A on tumor growth following transplantation of B16-F10 melanoma cells into syngeneic mice.
  • MC-38 colon carcinoma cells were transplanted into syngeneic C57BL/6 mice.
  • mice were treated with Bifido Strain A and anti-PD-1 (clone RMPl-14; CT-26, MC-38) or anti-PD-Ll (clone 10F.9G2; B16-F10), representative of at least three independent experiments.
  • Fig. 9A shows the results of an experiment in which a single dose of Bifido Strain
  • Fig. 9B shows representative epi-fluorescent image of a thin (7 pm) section of fixed small intestinal tissue from a mouse treated with Alexa Fluor-647 labeled Bifido Strain A (red), mucin-2 (green), and DAPI (blue), to show the restriction of Bifido Strain A to the luminal surface of the small intestine and association with the mucus layer.
  • Fig. 9C shows representative flow cytometry analysis of different population of cells from Peyer’s patches from mice treated with unlabeled Bifido Strain A (top row) and Alexa Fluor-647 labeled Bifido Strain A (bottom row) one hour following oral treatment to indicate the percentage of each population containing labeled bacteria.
  • Fig. 10B shows representative FACS plots from ex vivo intracellular staining of tumor infiltrating MDSCs for CXCL9 and TNFa following treatment with vehicle, Bifido Strain A or anti-PD-1.
  • Fig. 10D shows the number of CD1 lc + I-A/I-E + XCR1 + DC1 (left) and SIRPa +
  • Fig. 10E shows the geometric mean fluorescence intensity (gMFI) of CD86 on
  • CD1 lc + I-A/I-E + XCR1 + DC1 (left) and SIRPa + DC2 (right) following treatment with vehicle, Bifido Strain A or anti-PD-1. (n.s. not significant, **** p > 0.0001, ***p > 0.001, **p > 0.01, *p > 0.05)
  • Fig. 10F shows ex vivo intracellular staining of TNFa by SIRPa + DC2 in CT-26 tumors following treatment with vehicle, Bifido Strain A or anti-PD-1 (top three panels).
  • Fig. 10G shows staining of XCR1 + DC1 for IF-12p40 expression by flow cytometry following treatment with vehicle, Bifido Strain A or anti-PD-1 (top three panels). Frequencies of XCR1 + DC1 expressing IF-12p40 in the tumors is shown in the bottom panel.
  • FIG. 11 A shows the frequency of each TIL population positive for intracellular IFN-g after ex vivo stimulation with phorbol 12-myristate 13 -acetate (PMA) and ionomycin. Data was analyzed using Welch’s ANOVA with Dunnet’s T3 multiple comparisons test.
  • Fig. 11B shows the number of total IFN-g- producing CTL per gram of CT-26 tumor following treatment with vehicle, Bifido Strain A or anti-PD-1; geometric means ⁇ geometric S.D. Data was analyzed using Welch’s ANOVA with Dunnet’s T3 multiple comparisons test.
  • Fig. llC shows representative staining of CD4 + FoxP3 + tumor-infiltrating ( ⁇ )
  • Tregs for antigen-sensing and proliferation PD-1 vs. Ki67 (y-axis) following treatment with vehicle, Bifido Strain A or anti-PD-1.
  • Fig. 11D shows the quantitation of the results presented in Fig. 11C, displaying frequency of activated ⁇ -Tregs; means ⁇ SEM. Data was analyzed using Welch’s ANOVA with Dunnet’s T3 multiple comparisons test.
  • Fig. HE shows the ratio of the cells per (g) of tumor of IFN-y + CD8 + CD44 + CTL divided by activated Ki67 + PD-1 + CD4 + FoxP3 + Tregs of CD45 + TIL from tumors of vehicle, Bifido Strain A, and anti-PD-1 treated mice; geometric means ⁇ geometric S.D., Values displayed on a logarithmic scale were transformed. Data was analyzed using Welch’s ANOVA with Dunnet’s T3 multiple comparisons test.
  • Fig. 11F shows representative gp70p:H-2L d tetramer staining (y-axis) of tumor infiltrating lymphocytes in CT-26 tumors versus CD8a (x-axis), gated on total CD90.2 + T cells from the tumor 20 days after tumor implantation, and 10 days after the start of treatment.
  • Fig. 11G shows representative staining of tumor infiltrating CD8a + gp70p:H-2L d tetramer+ cells for surface-expressed CX3CRI and intracellular IFN-g following ex vivo PMA and ionomycin stimulation following treatment with vehicle, Bifido Strain A or anti-PD-1.
  • Fig. 11H shows the frequency of IFN-y + CX3CR1 + of tumor infiltrating gp70p:H-
  • Fig. 11 J shows the frequency of IFN-y + CXA’RP of tumor infiltrating gp70p:H-
  • Fig. 12A shows flow cytometry analysis of splenic DC infected ex vivo overnight with Bifido Strain A, for the expression levels of MHC class I (left) and CD80 (middle) and amounts of IL-12p70 in the supernatants of the cells analyzed by FACS (right). Comparisons from were done using two-tailed student’s t-test.
  • Fig. 12B shows the results of an experiment in which Bifido Strain A-treated and untreated DC cultures were pulsed with increasing amounts of SIINFEKL peptide and combined with purified CD8 + T cells from OT-I mice at a T celkDC ratio of 10: 1, IL-2 production was measured in the supernatant (left) and T cell expansion (right) was measured after 72 hours,
  • 12D shows FACS plots representative of four individual mice, showing the expression of CD25 and intracellular IFN-g in mesenteric lymph node suspensions, unstimulated (left column) or stimulated ex vivo with Bifido Strain A for 18 hours (right column), of NKp46 + NK cells (top row), or CD44 + CD8a + memory T cells (bottom row).
  • Fig. 13A shows monocytes from healthy donors were infected with Bifido Strain
  • Fig 14A shows the results of an experiment in which mesenteric lymph node suspensions from individual mice were infected ex vivo with Bifido Strain A similar to Fig. 12D- Fig. 12F and incubated for 18 hours.
  • Fig 14B shows the results of an experiment in which mesenteric lymph node suspensions from individual mice were infected ex vivo with Bifido Strain A similar to Fig. 12D- Fig. 12F and incubated for 18 hours.
  • Neutralizing antibodies against the indicated cytokines known to stimulate bystander activation of innate and adaptive lymphocytes were added to the cultures at a final concentration of 5 pg/ml, and the percent of CD25 + IFN-y + among CD8 + T cells are indicated by mean ⁇ SEM (left panel).
  • Fig. 14E shows representative staining of CD44 + gp70p:H-2L d+
  • compositions related to the induction of certain immune effects by bacteria of the genus Bifidobacterium include an increase in Ml polarization of macrophages in the subject; an increase in pro-inflammatory CD103 + dendritic cells within tumor-draining lymph nodes of the subject; an increase in expression of at least one co-stimulatory molecule (e.g., CD80, CD86, CD40, or a combination thereof) on CD1 lc + dendritic cells within mesenteric lymph nodes of the subject; an increase in production of at least one dendritic-cell-derived growth factor (e.g., GM-CSF) in the subject; an increase in expression of a pro- inflammatory cytokine (e.g., TNF-a, IL-I b, IL-12, or a combination thereof) by dendritic cells in the subject; an increase in production of a pro- inflammatory cytokine (e.g., TNF-a, IL-I b, IL-12,
  • the antigen-presenting cells are monocytes.
  • the immune effect includes an increase in CD44 + effector lymphocytes in a tumor in the subject (e.g., natural killer cells, natural killer T cells, CD4 + cells, CD8 + cells).
  • the immune effect includes an increase in infiltration by activated XCR1 + conventional type 1 dendritic cells.
  • provided herein are methods that treat cancer in a subject, induce an immune effect, screen strains of bacteria of the genus Bifidobacterium as a cancer therapeutic, select a subject with cancer for treatment, or adjust the treatment regimen with the disclosed pharmaceutical compositions by relying on one or more of the disclosed immune effects induced by the pharmaceutical compositions.
  • adjuvant or“Adjuvant therapy” broadly refers to an agent that affects an immunological or physiological response in a patient or subject.
  • an adjuvant might increase the presence of an antigen over time or to an area of interest like a tumor, help absorb an antigen presenting cell antigen, activate macrophages and lymphocytes, and support the production of cytokines.
  • an adjuvant might permit a smaller dose of an immune interacting agent to increase the effectiveness or safety of a particular dose of the immune interacting agent.
  • an adjuvant might prevent T cell exhaustion and thus increase the effectiveness or safety of a particular immune interacting agent.
  • the term“antibody” may refer to both an intact antibody and an antigen-binding fragment thereof.
  • Intact antibodies are glycoproteins that include at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the term“antibody” includes, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies ( e.g ., bispecific antibodies), single-chain antibodies, and antigen-binding antibody fragments.
  • antibody binding fragment and“antigen-binding portion” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen.
  • binding fragments encompassed within the term "antigen binding fragment” of an antibody include Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chain antibodies, NANOBODIES®, isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of an intact antibody.
  • These antibody fragments can be obtained using conventional recombinant and/or enzymatic techniques and can be screened for antigen binding in the same manner as intact antibodies.
  • “Cancer” broadly refers to an uncontrolled, abnormal growth of a host’s own cells leading to invasion of surrounding tissue and potentially tissue distal to the initial site of abnormal cell growth in the host.
  • Major classes include carcinomas which are cancers of the epithelial tissue (e.g., skin, squamous cells); sarcomas which are cancers of the connective tissue (e.g., bone, cartilage, fat, muscle, blood vessels, etc.); leukemias which are cancers of blood forming tissue (e.g., bone marrow tissue); lymphomas and myelomas which are cancers of immune cells; and central nervous system cancers which include cancers from brain and spinal tissue.“Cancer(s),”“neoplasm(s),” and“tumor(s)” are used herein interchangeably.
  • cancer refers to all types of cancer or neoplasm or malignant tumors including leukemias, carcinomas and sarcomas, whether new or recurring. Specific examples of cancers are: carcinomas, sarcomas, myelomas, leukemias, lymphomas and mixed type tumors. Non- limiting examples of cancers are new or recurring cancers of the brain, melanoma, bladder, breast, cervix, colon, head and neck, kidney, lung, non-small cell lung, mesothelioma, ovary, prostate, sarcoma, stomach, uterus and medulloblastoma.
  • Pediatric and adult tumors include, but not limited to, those of bladder, brain, breast, bone, cervix, colon, connective tissue, fat, head and neck, kidney, liver, lung, mesothelium, melanocytes (melanoma), muscle, ovary, pancreas, prostate, stomach, small intestine, and uterus
  • the term“decrease” or“deplete” means a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000 or undetectable after treatment when compared to a pre-treatment state.
  • Properties that may be decreased include the number of immune cells, bacterial cells, stromal cells, myeloid derived suppressor cells, fibroblasts, metabolites; the level of a cytokine; or another physical parameter (such as ear thickness (e.g., in a DTH animal model) or tumor size (e.g., in an animal tumor model)).
  • the term“ecological consortium” is a group of bacteria that trades metabolites and positively co-regulates one another, in contrast to two bacteria that induce host synergy through activating complementary host pathways for improved efficacy.
  • epitope means a protein determinant capable of specific binding to an antibody.
  • Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. Certain epitopes can be defined by a particular sequence of amino acids to which an antibody is capable of binding.
  • the term“gene” is used broadly to refer to any nucleic acid associated with a biological function.
  • the term“gene” applies to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence.
  • “Identity” as between nucleic acid sequences of two nucleic acid molecules can be determined as a percentage of identity using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, T, et al. , Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., et al, J Molec Biol 215:403 (1990); Guide to Huge Computers, Mrtin J.
  • Immunotherapy is treatment that uses a subject’s immune system to treat cancer and includes, for example, checkpoint inhibitors, cancer vaccines, cytokines, cell therapy, CAR- T cells, and dendritic cell therapy.
  • the term“increase” means a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 4-fold, 10- fold, 100-fold, 10 L 3 fold, 10 L 4 fold, 10 L 5 fold, 10 L 6 fold, and/or 10 L 7 fold greater after treatment when compared to a pre-treatment state.
  • Properties that may be increased include the number of immune cells, bacterial cells, stromal cells, myeloid derived suppressor cells, fibroblasts, metabolites; the level of a cytokine; or another physical parameter (such as ear thickness (e.g., in a DTH animal model) or tumor size (e.g., in an animal tumor model).
  • “Innate immune agonists” or“immuno-adjuvants” are small molecules, proteins, or other agents that specifically target innate immune receptors including Toll-Like Receptors, NOD receptors, STING Pathway components.
  • LPS is a TLR-4 agonist that is bacterially derived or synthesized and aluminum can be used as an immune stimulating adjuvant.
  • Immuno-adjuvants are a specific class of broader adjuvant or adjuvant therapy.
  • isolated or“enriched” encompasses a microbe, bacteria or other entity or substance that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by the hand of man. Isolated microbes may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated.
  • isolated microbes are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure, e.g., substantially free of other components.
  • the terms“purify,”“purifying” and“purified” refer to a microbe or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production.
  • a microbe or a microbial population may be considered purified if it is isolated at or after production, such as from a material or environment containing the microbe or microbial population, and a purified microbe or microbial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “isolated.”
  • purified microbes or microbial population are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • the one or more microbial types present in the composition can be independently purified from one or more other microbes produced and/or present in the material or environment containing the microbial type.
  • Microbial compositions and the microbial components thereof are generally purified from residual habitat products.
  • a gene is“overexpressed” in bacteria if it is expressed at a higher level in engineered bacteria under at least some conditions than it is expressed by wild-type bacteria of the same species under the same conditions.
  • a gene is“underexpressed” in bacteria if it is expressed at a lower level in engineered bacteria under at least some conditions than it is expressed by wild-type bacteria of the same species under the same conditions.
  • polynucleotide and“nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • loci locus
  • polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide may be further modified, such as by conjugation with a labeling component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.
  • “Operational taxonomic units” or“OTU(s)” refers to a terminal leaf in a phylogenetic tree, and is defined by a nucleic acid sequence (e.g., the entire genome, or a specific genetic sequence) and all sequences that share sequence identity to this nucleic acid sequence at the level of species.
  • the specific genetic sequence may be the 16S sequence or a portion of the 16S sequence.
  • the entire genomes of two entities are sequenced and compared.
  • select regions such as multilocus sequence tags (MLST), specific genes, or sets of genes may be genetically compared.
  • OTUs that share > 97% average nucleotide identity across the entire 16S or some variable region of the 16S are considered the same OTU. See e.g. Claesson MJ, Wang Q, O’Sullivan O, Greene- Diniz R, Cole JR, Ross RP, and O’Toole PW. 2010. Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Res 38: e200. Konstantinidis KT, Ramette A, and Tiedje JM. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361 : 1929-1940.
  • OTUs For complete genomes, MLSTs, specific genes, other than 16S, or sets of genes OTUs that share > 95% average nucleotide identity are considered the same OTU. See e.g., Achtman M, and Wagner M. 2008. Microbial diversity and the genetic nature of microbial species. Nat. Rev. Microbiol. 6: 431-440. Konstantinidis KT, Ramette A, and Tiedje JM. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361 : 1929-1940. OTUs are frequently defined by comparing sequences between organisms. Generally, sequences with less than 95% sequence identity are not considered to form part of the same OTU.
  • OTUs may also be characterized by any combination of nucleotide markers or genes, in particular highly conserved genes (e.g.,“house-keeping” genes), or a combination thereof.
  • Operational Taxonomic Units (OTUs) with taxonomic assignments made to, e.g., genus, species, and phylogenetic clade are provided herein.
  • “specific binding” refers to the ability of an antibody to bind to a predetermined antigen or the ability of a polypeptide to bind to its predetermined binding partner.
  • an antibody or polypeptide specifically binds to its predetermined antigen or binding partner with an affinity corresponding to a KD of about 10 7 M or less, and binds to the predetermined antigen/binding partner with an affinity (as expressed by KD) that is at least 10 fold less, at least 100 fold less or at least 1000 fold less than its affinity for binding to a non specific and unrelated antigen/binding partner (e.g., BSA, casein).
  • a non specific and unrelated antigen/binding partner e.g., BSA, casein
  • specific binding applies more broadly to a two-component system where one component is a protein, lipid, or carbohydrate, or combination thereof, which engages with the second component that is a protein, lipid, carbohydrate, or combination thereof in a specific way.
  • the terms“subject” or“patient” refers to any animal.
  • a subject or a patient described as“in need thereof’ refers to one in need of a treatment for a disease.
  • Mammals i.e., mammalian animals
  • mammals include humans, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows, sheep, goats, pigs), and household pets (e.g., dogs, cats, rodents).
  • the subject may be a non- human mammal including but not limited to of a dog, a cat, a cow, a horse, a pig, a donkey, a goat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, a gorilla or a chimpanzee.
  • the subject or patient may be healthy or may be suffering from a neoplasm at any developmental stage, wherein any of the stages are either caused by or opportunistically supported of a cancer associated or causative pathogen, or may be at risk of developing a neoplasm, or transmitting to others a cancer associated or cancer causative pathogen.
  • patients have lung cancer, bladder cancer, prostate cancer, ovarian cancer, and/or melanoma.
  • the patients may have tumors that show enhanced
  • patients suffer from other cancers.
  • the subject has undergone a cancer therapy.
  • strain refers to a member of a bacterial species with a genetic signature such that it may be differentiated from closely related members of the same bacterial species.
  • the genetic signature may be the absence of all or part of at least one gene, the absence of all or part of at least on regulatory region (e.g ., a promoter, a terminator, a riboswitch, a ribosome binding site), the absence (“curing”) of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one foreign gene (a gene derived from another species), the presence at least one mutated regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof.
  • regulatory region e.g ., a promoter, a terminator
  • strains may be identified by PCR amplification optionally followed by DNA sequencing of the genomic region(s) of interest or of the whole genome.
  • strains may be differentiated by selection or counter-selection using an antibiotic or nutrient/metabolite, respectively.
  • the term“treating” a disease in a subject or“treating” a subject having or suspected of having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of one or more agents, such that at least one symptom of the disease is decreased or prevented from worsening.
  • “treating” refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof.
  • a cancer is treated if the subject experiences a reduction in tumor size, a reduced number of tumors, a reduction in tumor growth, a reduction in cancer metastasis and/or a reduced number of total cancer cells following treatment than would be expected in the absence of treatment.
  • bypasses a control threshold indicates that, for a test level determined for a particular immune effect, the test level is consistent with the presence of or with an increase in the immune effect. If the method used to determine the immune effect results in higher values when the immune effect is present or increased, then the test level passes a control threshold when it is higher than the control threshold. Conversely, if the method used to determine the immune effect results in lower values when the immune effect is present or increased, then the test levels passes the control threshold when it is lower than the control threshold.
  • test level is lower than or equal to the control threshold when the degree of the immune effect positively correlates with the determined level values, the test level is higher than or equal to the control threshold when the degree of the immune effect negatively correlates with the determined level values
  • the control level can be determined from a control sample, but need not directly equal to a measured level from that control sample.
  • the control threshold can be set as half (or another fraction or multiple, such as 0.3, 0.4, 0.5 (“half’), 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, or 1.4) of the measured value from the control sample.
  • test values that pass the control threshold would be higher than 50% of the control level measurement (e.g., 50.1%, 51%, 60%, 100%, 200%, 400%, 1000%).
  • compositions comprising bacteria of the genus Bifidobacterium.
  • the bacteria of the genus Bifidobacterium can include Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium gallicum, Bifidobacterium infantis, Bifidobacterium kashiwanohense, Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum, Bifidobacterium scardovii, Bifidobacterium sp. HM2,
  • the bacteria of the genus Bifidobacterium are Bifidobacterium breve.
  • the Bifidobacterium breve is the Bifidobacterium breve strain deposited under accession number NCIMB 42380, also referred to as“MRx004” and “MRx4DP0004”.
  • the bacteria of the genus Bifidobacterium are Bifidobacterium sp. TM_7, Bifidobacterium thermophilum, Bifidobacterium urinalis, or a combination thereof.
  • the bacteria of the genus Bifidobacterium are Bifidobacterium breve.
  • the Bifidobacterium breve is the Bifidobacterium breve strain deposited under accession number NCIMB 42380, also referred to as“MRx004” and “MRx4DP0004”.
  • the bacteria of the genus Bifidobacterium are
  • the bacteria of the genus Bifidobacterium are Bifidobacterium animalis ssp. lactis. In some embodiments, at least 90% (e.g., at least 95%,
  • the bacteria in the pharmaceutical composition are from a single species of Bifidobacterium. In some embodiments, essentially all of the bacteria in the pharmaceutical composition are from a single species of Bifidobacterium. In some embodiments, all of the bacteria in the pharmaceutical composition are from a single species of Bifidobacterium. In some embodiments, all of the bacteria in the
  • compositions are from a single species of Bifidobacterium.
  • the bacteria in the pharmaceutical composition can be live and/or attenuated.
  • compositions comprising Bifidobacterium animalis ssp. lactis (e.g., an effective amount of Bifidobacterium animalis ssp. lactis) and methods of using such pharmaceutical compositions.
  • the Bifidobacterium animalis ssp. lactis is Bifidobacterium animalis ssp. lactis Strain A (ATCC Deposit Number PTA-125097).
  • lactis is a strain comprising at least 90% sequence identity (e.g., at least 92% sequence identity, at least 94% sequence identity, at least 96% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence of the Bifidobacterium animalis ssp. lactis Strain A.
  • sequence identity e.g., at least 92% sequence identity, at least 94% sequence identity, at least 96% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity
  • At least 90% (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%) of the bacteria in the pharmaceutical composition are from a single strain of Bifidobacterium. In some embodiments, essentially all of the bacteria in the pharmaceutical composition are from a single strain of Bifidobacterium. In some embodiments, all of the bacteria in the pharmaceutical composition are from a single strain of Bifidobacterium .
  • Applicant represents that the ATCC is a depository affording permanence of the deposit and ready accessibility thereto by the public if a patent is granted. All restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent. The material will be available during the pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR ⁇ 1.14 and 35 U.S.C. ⁇ 122.
  • the deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited plasmid, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of the patent, whichever period is longer. Applicant acknowledges its duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit.
  • the bacteria described herein are modified to improve colonization and/or engraftment in the mammalian gastrointestinal tract (e.g., modified metabolism, such as improved mucin degradation, enhanced competition profile, increased motility, increased adhesion to gut epithelial cells, modified chemotaxis).
  • the bacteria described herein are modified to enhance their immunomodulatory and/or therapeutic effect (e.g., either alone or in combination with another therapeutic agent).
  • the bacteria described herein are modified to enhance immune activation (e.g., through modified production of polysaccharides, pili, fimbriae, adhesins, outer membrane vesicles).
  • the bacteria described herein are modified to improve bacterial manufacturing (e.g., higher oxygen tolerance, improved freeze-thaw tolerance, shorter generation times).
  • the Bifidobacterium animalis ssp. lactis is grown in
  • RCM medium Reinforced Clostridium medium
  • LMRS medium Lactobacilli MRS medium
  • Bifidobactrium animalis ssp. lactis is grown using RCM medium ATCC 1053 and/or Trypticase soy agar/broth with defibrimated sheep blood ATCC 260 per the manufacturer’s instructions.
  • compositions comprising bacteria of the genus Bifidobacterium.
  • the pharmaceutical formulation comprises a bacterium and/or a combination of bacteria described herein and a pharmaceutically acceptable carrier.
  • the bacteria of the genus Bifidobacterium are
  • the bacteria of the genus Bifidobacterium are Bifidobacterium animalis ssp. Lactis.
  • the Bifidobacterium animalis ssp. lactis is Bifidobacterium animalis ssp. lactis Strain A (ATCC Deposit Number PTA-125097).
  • lactis is a strain comprising at least 90% sequence identity (e.g ., at least 92% sequence identity, at least 94% sequence identity, at least 96% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence of the Bifidobacterium animalis ssp. lactis Strain A.
  • sequence identity e.g ., at least 92% sequence identity, at least 94% sequence identity, at least 96% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity
  • At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the bacteria in the pharmaceutical composition are Bifidobacterium animalis ssp. lactis (e.g., a strain of Bifidobacterium animalis ssp. lactis disclosed herein). In certain embodiments, substantially all of the bacteria in the pharmaceutical composition are Bifidobacterium animalis ssp. lactis (e.g., a strain of
  • Bifidobacterium animalis ssp. lactis disclosed herein 100% of the bacteria in the pharmaceutical composition are Bifidobacterium animalis ssp. lactis (e.g., a strain of Bifidobacterium animalis ssp. lactis disclosed herein).
  • the pharmaceutical composition comprises at least 1 x 10 3 colony forming units (CFUs), 1 x 10 4 colony forming units (CFUs), 1 x 10 5 colony forming units (CFUs), 5 x 10 5 colony forming units (CFUs), 1 x 10 6 colony forming units (CFUs), 2 x 10 6 colony forming units (CFUs), 3 x 10 6 colony forming units (CFUs), 4 x 10 6 colony forming units (CFUs), 5 x 10 6 colony forming units (CFUs), 6 x 10 6 colony forming units (CFUs), 7 x 10 6 colony forming units (CFUs), 8 x 10 6 colony forming units (CFUs), 9 x 10 6 colony forming units (CFUs), 1 x 10 7 colony forming units (CFUs), 2 x 10 7 colony forming units (CFUs), 3 x 10 7 colony forming units (CFUs),
  • the pharmaceutical composition comprises about 7.5 x 10 10 CFUs of the bacteria of the genus Bifidobacterium. In some embodiments, the
  • composition comprises about 15 x 10 10 CFUs of the bacteria of the genus Bifidobacterium. In some embodiments, the pharmaceutical composition comprises about 22.5 x 10 10 CFUs of the bacteria of the genus Bifidobacterium. In some embodiments, the
  • the pharmaceutical composition comprises about 30 x 10 10 CFUs of the bacteria of the genus Bifidobacterium. In some embodiments, the pharmaceutical composition is administered once a day. In some embodiments, the pharmaceutical composition is administered twice a day.
  • formulations containing the bacteria of the genus are provided.
  • Bifidobacterium are provided as encapsulated, enteric coated, or powder forms, with doses ranging up to 5 xlO 11 CFU (e.g., orup to 3 x 10 11 CFU).
  • the composition comprises 5 x 10 11 CFU of Bifidobacterium animalis ssp. lactis in a capsule.
  • the composition comprises 7.5 x 10 10 CFU of Bifidobacterium animalis ssp. lactis in a capsule.
  • 1, 2, 3, or 4 capsules are administered, e.g., once or twice daily.
  • the capsule is enteric coated, e.g., for duodenal release at pH 5.5.
  • the composition comprises a powder of freeze-dried Bifidobacterium animalis ssp. lactis that is deemed“Qualified Presumption of Safety” (QPS) status.
  • the composition is stable at frozen or refrigerated temperature.
  • Methods for producing pharmaceutical compositions may include these three main processing steps: organism banking, organism production, and preservation.
  • a sample that contains an abundance of Bifidobacterium animalis ssp. lactis may be cultured by avoiding an isolation step.
  • the strains included in the microbial composition may be (1) isolated directly from a specimen or taken from a banked stock, (2) optionally cultured on a nutrient agar or broth that supports growth to generate viable biomass, and (3) the biomass optionally preserved in multiple aliquots in long-term storage.
  • the agar or broth may contain nutrients that provide essential elements and specific factors that enable growth.
  • An example would be a medium composed of 20 g/L glucose, 10 g/L yeast extract, 10 g/L soy peptone, 2 g/L citric acid, 1.5 g/L sodium phosphate monobasic, 100 mg/L ferric ammonium citrate, 80 mg/L magnesium sulfate, 10 mg/L hemin chloride, 2 mg/L calcium chloride, 1 mg/L menadione.
  • Another example would be a medium composed of 10 g/L beef extract, 10 g/L peptone, 5 g/L sodium chloride, 5 g/L dextrose, 3 g/L yeast extract, 3 g/L sodium acetate, 1 g/L soluble starch, and 0.5 g/L L- cysteine HC1, at pH 6.8.
  • a variety of microbiological media and variations are well known in the art (e.g., R.M. Atlas, Handbook of Microbiological Media (2010) CRC Press). Culture media can be added to the culture at the start, may be added during the culture, or may be
  • the strains in the pharmaceutical composition may be cultivated alone, as a subset of the microbial composition, or as an entire collection comprising the microbial composition.
  • a first strain may be cultivated together with a second strain in a mixed continuous culture, at a dilution rate lower than the maximum growth rate of either cell to prevent the culture from washing out of the cultivation.
  • the inoculated culture is incubated under favorable conditions for a time sufficient to build biomass.
  • microbial compositions for human use this is often at 37°C temperature, pH, and other parameter with values similar to the normal human niche.
  • the environment may be actively controlled, passively controlled ( e.g ., via buffers), or allowed to drift.
  • an anoxic/reducing environment may be employed for anaerobic pharmaceutical compositions. This can be accomplished by addition of reducing agents such as cysteine to the broth, and/or stripping it of oxygen.
  • a culture of a pharmaceutical composition may be grown at 37°C, pH 7, in the medium above, pre-reduced with 1 g/L cysteine-HCl.
  • the culture When the culture has generated sufficient biomass, it may be preserved for banking.
  • the organisms may be placed into a chemical milieu that protects from freezing (adding‘cryoprotectants’), drying (Tyoprotectants’), and/or osmotic shock (‘osmoprotectants’), dispensing into multiple (optionally identical) containers to create a uniform bank, and then treating the culture for preservation.
  • Containers are generally impermeable and have closures that assure isolation from the environment. Cryopreservation treatment is accomplished by freezing a liquid at ultra-low temperatures (e.g., at or below -80°C).
  • Dried preservation removes water from the culture by evaporation (in the case of spray drying or‘cool drying’) or by sublimation (e.g., for freeze-drying, spray freeze-drying). Removal of water improves long-term microbial composition storage stability at temperatures elevated above cryogenic conditions. If the microbial composition comprises, for example, spore forming species and results in the production of spores, the final composition may be purified by additional means such as density gradient centrifugation and preserved using various techniques. Microbial composition banking may be done by culturing and preserving the strains individually, or by mixing the strains together to create a combined bank.
  • a microbial composition culture may be harvested by centrifugation to pellet the cells from the culture medium, the supernatant decanted and replaced with fresh culture broth containing 15% glycerol. The culture can then be aliquoted into 1 mL cryotubes, sealed, and placed at -80°C for long-term viability retention. This procedure achieves acceptable viability upon recovery from frozen storage.
  • Microbial production may be conducted using similar culture steps to banking, including medium composition and culture conditions described herein. It may be conducted at larger scales of operation, especially for clinical development or commercial production. At larger scales, there may be several sub-cultivations of the microbial composition prior to the final cultivation. At the end of cultivation, the culture is harvested to enable further formulation into a dosage form for administration. This can involve concentration, removal of undesirable medium components, and/or introduction into a chemical milieu that preserves the microbial composition and renders it acceptable for administration via the chosen route.
  • a microbial composition may be cultivated to a concentration of 10 10 CFU/mL, then concentrated 20-fold by tangential flow microfiltration; the spent medium may be exchanged by diafiltering with a preservative medium consisting of 2% gelatin, 100 mM trehalose, and 10 mM sodium phosphate buffer. The suspension can then be freeze-dried to a powder and titrated.
  • the powder may be blended to an appropriate potency, and mixed with other cultures and/or a filler such as microcrystalline cellulose for consistency and ease of handling, and the pharmaceutical composition formulated as provided herein.
  • compositions for administration subjects are combined with additional active and/or inactive materials in order to produce a final product, which may be in single dosage unit or in a multi-dose format.
  • the composition comprises at least one carbohydrate.
  • a “carbohydrate” refers to a sugar or polymer of sugars. The terms“saccharide,”“polysaccharide,” “carbohydrate,” and“oligosaccharide” may be used interchangeably. Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one on each carbon atom of the molecule. Carbohydrates generally have the molecular formula CnFhnOn. A carbohydrate may be a monosaccharide, a disaccharide, trisaccharide, oligosaccharide, or polysaccharide.
  • the most basic carbohydrate is a monosaccharide, such as glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, and fructose.
  • Disaccharides are two joined monosaccharides. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose.
  • an oligosaccharide includes between three and six monosaccharide units ( e.g ., raffinose, stachyose), and
  • polysaccharides include six or more monosaccharide units.
  • Exemplary polysaccharides include starch, glycogen, and cellulose.
  • Carbohydrates may contain modified saccharide units such as 2’- deoxyribose wherein a hydroxyl group is removed, 2’-fluororibose wherein a hydroxyl group is replaced with a fluorine, or N-acetylglucosamine, a nitrogen- containing form of glucose (e.g., T- fluororibose, deoxyribose, and hexose).
  • Carbohydrates may exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.
  • the composition comprises at least one lipid.
  • a“lipid” includes fats, oils, triglycerides, cholesterol, phospholipids, and fatty acids in any form including free fatty acids. Fats, oils and fatty acids can be saturated, unsaturated (cis or trans) or partially unsaturated (cis or trans).
  • the lipid comprises at least one fatty acid selected from lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16: 1), margaric acid (17:0), heptadecenoic acid (17: 1), stearic acid (18:0), oleic acid (18: 1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20: 1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoic acid (22: 1),
  • the composition comprises at least one modified lipid, for example a lipid that has been modified by cooking.
  • the composition comprises at least one supplemental mineral or mineral source.
  • supplemental mineral or mineral source examples include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium.
  • Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.
  • the composition comprises at least one supplemental vitamin.
  • the at least one vitamin can be fat-soluble or water-soluble vitamins.
  • Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin.
  • Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.
  • the composition comprises an excipient.
  • suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.
  • the excipient is a buffering agent.
  • suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.
  • the excipient comprises a preservative.
  • suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.
  • the composition comprises a binder as an excipient.
  • suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof.
  • the composition comprises a lubricant as an excipient.
  • suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc,
  • polyethyleneglycol sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil.
  • the composition comprises a dispersion enhancer as an excipient.
  • suitable dispersants include starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose as high HLB emulsifier surfactants.
  • the composition comprises a disintegrant as an excipient.
  • the disintegrant is a non-effervescent disintegrant.
  • suitable non-effervescent disintegrants include starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro- crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pectin, and tragacanth.
  • the disintegrant is an effervescent disintegrant.
  • Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.
  • the bacterial formulation comprises an enteric coating or micro encapsulation.
  • the enteric coating or micro encapsulation improves targeting to a desired region of the gastrointestinal tract.
  • the pharmaceutical composition comprises an enteric coating and/or
  • microcapsules that dissolves at a pH associated with a particular region of the gastrointestinal tract.
  • the enteric coating and/or microcapsules dissolve at a pH of about 5.5-6.2 to release in the duodenum, at a pH value of about 7.2-7.5 to release in the ileum, and/or at a pH value of about 5.6-6.2 to release in the colon.
  • Exemplary enteric coatings and microcapsules are described, for example, in U.S. Pat. Publ’n No. 2016/0022592, which is hereby incorporated by reference in its entirety.
  • the composition is a food product (e.g ., a food or beverage) such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.
  • a food product e.g ., a food or beverage
  • the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, and Chinese soups; soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products, including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, carb
  • the bacteria disclosed herein are administered in conjunction with a prebiotic to the subject.
  • Prebiotics are carbohydrates that are generally indigestible by a host animal and are selectively fermented or metabolized by bacteria.
  • Prebiotics may be short-chain carbohydrates (e.g., oligosaccharides) and/or simple sugars (e.g., mono- and di-saccharides) and/or mucins (heavily glycosylated proteins) that alter the composition or metabolism of a microbiome in the host.
  • the short chain carbohydrates are also referred to as oligosaccharides, and usually contain from 2 or 3 and up to 8, 9, 10, 15 or more sugar moieties.
  • a prebiotic composition can selectively stimulate the growth and/or activity of one of a limited number of bacteria in a host.
  • Prebiotics include oligosaccharides such as fructooligosaccharides (FOS) (including inulin),
  • GOS galactooligosaccharides
  • XOS xylooligosaccharides
  • COS chitooligosaccharides
  • soy oligosaccharides e.g., stachyose and raffinose
  • Oligosaccharides are not necessarily single components, and can be mixtures containing oligosaccharides with different degrees of oligomerization, sometimes including the parent disaccharide and the monomeric sugars.
  • oligosaccharides are found as natural components in many common foods, including fruits, vegetables, milk, and honey.
  • Specific examples of oligosaccharides are lactulose, lactosucrose, palatinose, glycosyl sucrose, guar gum, gum Arabic, tagalose, amylose, amylopectin, pectin, xylan, and cyclodextrins.
  • Prebiotics may also be purified or chemically or enzymatically synthesized.
  • kits for delivering bacteria and/or a pharmaceutical composition described herein to a subject (e.g ., a subject in need of a immune effect induced by the bacteria of the genus Bifidobacterium provided herein).
  • the subject has cancer.
  • the bacteria are administered in conjunction with the administration of a cancer therapeutic.
  • the bacteria are co-formulated in a pharmaceutical composition with the cancer therapeutic.
  • the bacteria are co-administered with the cancer therapeutic.
  • the cancer therapeutic is administered to the subject before administration of the bacteria (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes before, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours before, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days before).
  • the cancer therapeutic is administered to the subject after administration of the bacteria (e.g ., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes after, about 1, 2, 3,
  • the same mode of delivery is used to deliver both the bacteria and the cancer therapeutic.
  • different modes of delivery are used to administer the bacteria and the cancer therapeutic.
  • the bacteria are administered orally while the cancer therapeutic is administered via injection (e.g., an intravenous, intramuscular, and/or intratumoral injection).
  • the pharmaceutical compositions, dosage forms, and kits described herein can be administered in conjunction with any other conventional anti-cancer treatment, such as, for example, radiation therapy, and surgical resection of the tumor. These treatments may be applied as necessary and/or as indicated and may occur before, concurrent with or after administration of the pharmaceutical compositions, dosage forms, and kits described herein.
  • the dosage regimen can be any of a variety of methods and amounts, and can be determined by one skilled in the art using methods provided herein.
  • the dose of the pharmaceutical composition can be determined based on the dose required to induce an immune effect disclosed herein.
  • the dose of the pharmaceutical compositions described herein may be appropriately set or adjusted in accordance with the dosage form, the route of administration, the degree or stage of a target disease, and the like.
  • the general effective dose of the agents may range between 0.01 mg/kg body weight/day and 1000 mg/kg body weight/day, between 0.1 mg/kg body weight/day and 1000 mg/kg body weight/day, 0.5 mg/kg body weight/day and 500 mg/kg body weight/day, 1 mg/kg body weight/day and 100 mg/kg body weight/day, or between 5 mg/kg body weight/day and 50 mg/kg body weight/day.
  • the effective dose may be 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, or 1000 mg/kg body weight/day or more, but the dose is not limited thereto.
  • the dose administered to a subject is sufficient to induce an immune effect disclosed herein in the subject.
  • dosage will depend upon a variety of factors including the strength of the particular compound employed, as well as the age, species, condition, and body weight of the subject.
  • the size of the dose will also be determined by the route, timing, and frequency of administration as well as the existence, nature, and extent of any adverse side effects that might accompany the administration of a particular compound and the desired physiological effect.
  • Suitable doses and dosage regimens can be determined using the methods provided herein.
  • treatment is initiated with one or more smaller dosages and the subject is monitored for the desired immune effect. Thereafter, the dosage is increased by small increments as necessary until the immune effect is achieved.
  • Separate administrations can include any number of two or more administrations
  • the doses may be separated by at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days or 1, 2, 3, or 4 weeks. Accordingly, the methods provided herein include methods of providing to the subject one or more
  • administrations of a pharmaceutical composition where the number of administrations can be determined by monitoring the subject for the immune effects disclosed herein, and, based on the results of the monitoring, determining whether to provide one or more additional administrations.
  • the time between administrations can be any of a variety of times.
  • the time period between administrations can be a function of any of a variety of factors, including monitoring steps, as described in relation to the number of administrations, the time period for a subject to mount an immune response and/or the time period for a subject to clear the bacteria from normal tissue.
  • the time period can be a function of the time period for a subject to mount an immune effect disclosed herein; for example, the time period can be more than the time period for a subject to mount an immune effect, such as more than about one week, more than about ten days, more than about two weeks, or more than about a month; in another example, the time period can be less than the time period for a subject to mount an immune effect, such as less than about one week, less than about ten days, less than about two weeks, or less than about a month.
  • the time period can be a function of the time period for a subject to clear the bacteria from normal tissue; for example, the time period can be more than the time period for a subject to clear the bacteria from normal tissue, such as more than about a day, more than about two days, more than about three days, more than about five days, or more than about a week.
  • the administration of the pharmaceutical composition induces one or more immune effects in the subject.
  • the administration of the pharmaceutical composition treats the cancer.
  • the pharmaceutical composition induces an anti -tumor immune response in the subject.
  • compositions and/or methods provided herein induce one or more immune effects in a subject.
  • the compositions and/or methods induce at least 1, 2, 3, 4, 5, 6, or 7 of the immune effects disclosed herein.
  • the immune effect includes an increase in Ml polarization of macrophages in the subject and/or an increase in pro-inflammatory CD103 + dendritic cells within tumor-draining lymph nodes of the subject.
  • the number and/or percentage of pro-inflammatory CD103 + dendritic cells within tumor-draining lymph nodes of the subject is/are increased by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400% or at least 500%.
  • the number and/or percentage of pro- inflammatory CD103 + dendritic cells within tumor-draining lymph nodes of the subject can be determined using methods available in the art, e.g., by obtaining a lymph node biopsy from the subject and then detecting in pro-inflammatory CD103 + dendritic cells using flow cytometry and/or microscopy.
  • the immune effect includes an increase in expression of at least one co-stimulatory molecule on CD1 lc + dendritic cells within mesenteric lymph nodes of the subject.
  • the co-stimulatory molecule is CD80, CD86, or CD40.
  • the expression of at least one co-stimulatory molecule on CD1 lc + dendritic cells is increased by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400% or at least 500%.
  • the expression of at least one co- stimulatory molecule on CD1 lc + dendritic cells can be determined using methods available in the art, e.g., using flow cytometry and/or microscopy.
  • the immune effect includes an increase in production of at least one dendritic-cell-derived growth factor in the subject.
  • the dendritic- cell-derived growth factor is GM-CSF.
  • the production of at least one dendritic-cell-derived growth factor is increased by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400% or at least 500%.
  • production of at least one dendritic-cell-derived growth factor can be determined using methods available in the art, e.g., using an ELISA assay, flow cytometry and/or microscopy.
  • the immune effect includes an increase in expression of a pro-inflammatory cytokine by dendritic cells in the subject.
  • the pro- inflammatory cytokine is TNF-a, IL-I b, IL-12, or a combination thereof.
  • the expression of a pro-inflammatory cytokine by dendritic cells in the subject is increased by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400% or at least 500%.
  • the expression of a pro-inflammatory cytokine by dendritic cells in the subject can be determined using methods available in the art, e.g., using an ELISA assay, flow cytometry and/or microscopy.
  • the immune effect includes an increase in production of a lymphocyte-recruiting chemokine in the subject.
  • the lymphocyte recruiting chemokine is CXCL9, CXCL10, or a combination thereof.
  • the production of a lymphocyte-recruiting chemokine is increased by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400% or at least 500%.
  • the e production of a lymphocyte-recruiting chemokine can be determined using methods available in the art, e.g., using an ELISA assay, flow cytometry and/or microscopy.
  • the immune effect includes an increase in IFN-y-producing by CD8 + T cells in a tumor in the subject.
  • the number and/or percentage of IFN-y-producing by CD8 + T cells in a tumor in the subject are increased by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400% or at least 500%.
  • the number and/or percentage IFN-g- producing by CD8 + T cells in a tumor in the subject can be determined using methods available in the art, e.g., obtaining a biopsy of the tumor and then using flow cytometry and/or microscopy to detect the IFN-y-producing by CD8 + T cells in the biopsy.
  • the immune effect includes an increase in Ki67+ natural killer cells in a tumor in the subject.
  • the number and/or percentage of Ki67+ natural killer cells in a tumor in the subject are increased by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400% or at least 500%.
  • the number and/or percentage of Ki67+ natural killer cells in a tumor in the subject can be determined using methods available in the art, e.g., obtaining a biopsy of the tumor and then using flow cytometry and/or microscopy to detect the Ki67+ natural killer cells in the biopsy.
  • the immune effect is activation of one or more pattern- recognition receptors (e.g., TLR2) in the subject.
  • activation of one or more pattern-recognition results in stimulation of cytolytic activity of natural killer cells in the subject.
  • activation of one or more pattern-recognition results in antigen- presenting cells acquiring a pro-inflammatory phenotype in the subject (e.g., expression of IL12p70, TNFa, IL-Ib, CD80, CD86 and/or CD40).
  • a pro-inflammatory phenotype e.g., expression of IL12p70, TNFa, IL-Ib, CD80, CD86 and/or CD40.
  • Various methods such as immunoblotting, RNASeq, flow cytometry, immunofluorescence, and other suitable methods can be used to measure or otherwise detect many of the immune effects.
  • the immune effect is stimulation of systemic anti-tumor immunity via activation of antigen-presenting cells and natural killer cells in the subject .
  • the immune effect includes production of high levels of IL-12p70 from peripheral blood mononuclear cells (PBMCs) or purified human monocytes, for example as determined by an assay provided herein (e.g., in Example 3).
  • PBMCs peripheral blood mononuclear cells
  • human monocytes for example as determined by an assay provided herein (e.g., in Example 3).
  • the immune effect includes production of high levels of CXCL10 from peripheral blood mononuclear cells (PBMCs) or purified human monocytes, for example as determined by an assay provided herein (e.g., in Example 3).
  • PBMCs peripheral blood mononuclear cells
  • the immune effect includes a decrease in the activity of myeloid-derived suppressor cells and regulatory T cells and/or an increase in the activation of immunostimulatory dendritic cells and/or in the production of IFN-g by tumor infiltrating lymphocytes, for example as determined by an assay provided herein (e.g., in Example 3).
  • the immune effect includes differentiation and
  • the immune effect includes activation of innate immunity, for example as determined by an assay provided herein (e.g., in Example 3).
  • the immune effect includes engagement of TLR2, for example as determined by an assay provided herein (e.g., in Example 3).
  • the immune effect includes engagement of TLR4, for example as determined by an assay provided herein (e.g., in Example 3).
  • the immune effect includes a greater increase in the production of TNFa, IL-1 b, and IFN-g as compared to an increase from an individual TLR agonist, for example as determined by an assay provided herein (e.g., in Example 3).
  • the immune effect includes an increase in the production of IL-12p70, for example as determined by an assay provided herein (e.g., in Example 3).
  • the immune effect includes a reduction in expression of TNFa in tumor infiltrating myeloid-derived suppressor cells (MDSCs) with no effect (or no significant effect) on CXCL9, for example as determined by an assay provided herein (e.g., in Example 3).
  • MDSCs tumor infiltrating myeloid-derived suppressor cells
  • the immune effect includes a significant reduction in MDSC TNFa expression relative to a reduction from a vehicle and/or from an anti-PD-1 agent, for example as determined by an assay provided herein (e.g., in Example 3).
  • the immune effect includes an increase in tumor- infiltrating XCR1 + DC1 and/or SIRPa + DC2, and/or upregulation of co- stimulatory CD86, for example as determined by an assay provided herein (e.g., in Example 3).
  • the immune effect includes a reduction in production of TNFa by SIRPa + tumor DC2 (e.g., compared to production from vehicle-treated and/or anti-PD- 1 agent-treated controls), for example as determined by an assay provided herein (e.g., in Example 3).
  • the immune effect includes expression of significantly more IL-12p40 in situ by XCR1 + DC1 (e.g., in Bifido Strain A treated tumors relative to vehicle- treated and/or anti-PD-1 agent-treated controls, thereby enhancing immunostimulatory activity), for example as determined by an assay provided herein (e.g., in Example 3).
  • the immune effect includes a significant reduction in the activation and proliferation of the Tregs (e.g., in Bifido Strain A treatment compared to vehicle, mirroring the effects of anti-PD-1 treatment), for example as determined by an assay provided herein.
  • the immune effect includes a significant increase in the ratio of IFN-g producing CTL to activated PD-1 + Ki67 + Treg in responsive tumors (e.g., similarly to anti-PD-1 treated controls), for example as determined by an assay provided herein (e.g., in Example 3).
  • the immune effect includes activation of IFN-y-producing lymphocytes, for example as determined by an assay provided herein (e.g., in Example 3).
  • the immune effect includes differentiation of tumor antigen-specific CTL into functional cytotoxic effectors, for example as determined by an assay provided herein (e.g., in Example 3).
  • the immune effect includes the stimulation of dendritic cell maturation supporting antigen-independent bystander activation of effector lymphocytes, for example as determined by an assay provided herein (e.g., in Example 3).
  • the immune effect includes lymphocyte activation, for example as determined by an assay provided herein (e.g., in Example 3).
  • the immune effect includes IL-12p70-dependent activation of tumor antigen-specific effector cells in the spleen, for example as determined by an assay provided herein (e.g., in Example 3).
  • Methods of treating cancer in a subject (who may be a subject having cancer in whom oral administration of a dose of a pharmaceutical composition having bacteria of the genus Bifidobacterium induces an immune effect), methods of treating a subject in need of induction of an immune effect, as well as methods of inducing an immune effect all rely on the disclosed pharmaceutical compositions that when orally administered to the subject induce one or more of the disclosed immune effects.
  • the methods include orally administering a composition having a strain of bacteria of the genus Bifidobacterium to a test subject and/or contacting bacteria of the genus Bifidobacterium to one or more test cells, and then determining at least one test level for one or more of the disclosed immune effects.
  • a finding that the test level passes a control threshold can indicate that the screened strain is one that is an effective or promising cancer therapeutic, whereas a finding that the test level fails to pass a control threshold can indicate that the screened strain is one that is not an effective or promising cancer therapeutic.
  • the control threshold can be obtained in numerous ways. For example, it can be determined from one or more subjects without cancer, from the subject before administering the dose of the pharmaceutical composition, or from a standard level.
  • test subject which can be a non-human mammal, such as a mouse or a rat.
  • the tested pharmaceutical compositions can be contacted with a test sample having dendritic cells for determining a test level for one of the applicable immune effects (e.g., an increase in expression of CD103 + by dendritic cells, an increase in expression of at least one co-stimulatory molecule on CD1 lc + dendritic cells, an increase in production of at least one growth factor by dendritic cells, an increase in expression of a pro-inflammatory cytokine by dendritic cells, an increase in production of a lymphocyte-recruiting chemokine by dendritic cells).
  • a test level for one of the applicable immune effects e.g., an increase in expression of CD103 + by dendritic cells, an increase in expression of at least one co-stimulatory molecule on CD1 lc + dendritic cells, an increase in production of at least one growth factor by dendritic cells, an increase in expression of a pro-inflammatory cytokine by dendritic cells, an increase in production of a lymph
  • kits for selecting a subject with cancer for treatment or methods of treating a subject for cancer wherein the methods include orally administering to the subject a dose of a pharmaceutical composition comprising bacteria of the genus Bifidobacterium, and then determining at least one test level for one of the disclosed immune effects.
  • The“test level,” as used herein, is merely the level that is determined for the subject (or sample) of interest.
  • the modifier“test” is used to differentiate the level (i.e., test level) from another level, such as a“control” level or a control threshold.
  • the test level passes a control threshold
  • the subject is then selected for treatment with a pharmaceutical composition comprising bacteria of the genus Bifidobacterium.
  • a pharmaceutical composition comprising bacteria of the genus Bifidobacterium.
  • one or more additional doses of the pharmaceutical composition comprising bacteria of the genus Bifidobacterium can be administered if the test level passes a control threshold, or a higher dose of the pharmaceutical composition comprising bacteria of the genus Bifidobacterium can be administered if the test level fails to pass the control threshold.
  • the control threshold can be obtained in numerous ways. For example, it can be determined from one or more subjects without cancer, from the subject before administering the dose of the pharmaceutical composition, or from a standard level.
  • the control threshold can be chosen to be different for different methods. For instance, it can be set as an easier to pass value for screening method when a high sensitivity of screening is desired, or it can be set as a harder to pass value for screening methods when a high selectivity of screening is desired.
  • the methods provided herein include the administration to a subject of a pharmaceutical composition described herein (e.g., a Bifidobacterium animalis ssp. /.ac rs-containing pharmaceutical composition) either alone or in combination with a second therapy (e.g., cancer therapy).
  • a second therapy e.g., cancer therapy
  • the other cancer therapeutic may include e.g., surgical resection, radiotherapy, or a cancer therapeutic agent.
  • the pharmaceutical composition and the other cancer therapy can be administered to the subject in any order.
  • the pharmaceutical composition and the other cancer therapy are administered conjointly.
  • the pharmaceutical composition is administered to the subject before the cancer therapeutic is administered (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days before).
  • the pharmaceutical composition is administered to the subject after the cancer therapeutic is administered (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours after or at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days after).
  • the pharmaceutical composition and the cancer therapeutic are administered to the subject simultaneously or nearly simultaneously (e.g ., administrations occur within an hour of each other).
  • the pharmaceutical composition is administered daily (e.g., once daily or twice daily) and the cancer therapeutic is administered once every 1, 2, 3, or 4 weeks.
  • the pharmaceutical composition is administered daily (e.g., once daily or twice daily) and the cancer therapeutic is administered once every 3 weeks.
  • the subject is administered an antibiotic before the pharmaceutical composition is administered to the subject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
  • the subject is administered an antibiotic after the pharmaceutical composition is administered to the subject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days before).
  • the subject is administered an antibiotic after the pharmaceutical composition is administered to the subject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
  • the pharmaceutical composition and the antibiotic are administered to the subject simultaneously or nearly simultaneously (e.g., administrations occur within an hour of each other).
  • the subject may undergo surgery.
  • Types of surgery include but are not limited to preventative, diagnostic or staging, curative and palliative surgery.
  • Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.
  • Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed.
  • Tumor resection refers to physical removal of at least part of a tumor.
  • treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). Upon excision of part of all of cancerous cells, tissue, or tumor, a cavity may be formed in the body.
  • the subject may undergo radiation therapy.
  • Radiation therapy includes the administration or application of a radiotherapeutic agents and factors including but not limited to trays, UV-irradiation, microwaves, electronic emissions, and radioisotopes.
  • the localized tumor site may be irradiated, including by one or more forms of radiations described herein. All of these factors may affect a broad range of damage DNA, on the precursors of DNA, the replication and repair of DNA, and the assembly and maintenance of chromosomes.
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
  • the methods provided herein further comprise administering another cancer therapeutic to the subject.
  • the cancer therapeutic is a chemotherapeutic agent.
  • chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin;
  • alkylating agents such as thiotepa and cyclosphosphamide
  • alkyl sulfonates such as busulfan, improsulfan and piposulfan
  • aziridines such as
  • callystatin including its adozelesin, carzelesin and bizelesin synthetic analogues
  • cryptophycins particularly cryptophycin 1 and cryptophycin 8
  • dolastatin duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1)
  • eleutherobin pancratistatin
  • a sarcodictyin spongistatin
  • nitrogen mustards such as chlorambucil, chlornaphazine
  • cholophosphamide estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g ., calicheamicin, especially calicheamicin gammall and calicheamicin omegall; dynemicin, including dynemicin A;
  • bisphosphonates such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, no
  • demecolcine diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;
  • losoxantrone podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;
  • pipobroman gacytosine; arabinoside ("Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine;
  • methotrexate platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine
  • the cancer therapeutic is a cancer immunotherapy agent.
  • Immunotherapy refers to a treatment that uses a subject’s immune system to treat cancer, e.g., checkpoint inhibitors, cancer vaccines, cytokines, cell therapy, CAR-T cells, and dendritic cell therapy.
  • checkpoint inhibitors include
  • Nivolumab (BMS, anti-PD-1), Pembrolizumab (Merck, anti-PD-1), Ipilimumab (BMS, anti- CTLA-4), MEDI4736 (AstraZeneca, anti-PD-Ll), and MPDL3280A (Roche, anti-PD-Ll).
  • Other immunotherapies may be tumor vaccines, such as Gardail, Cervarix, BCG, sipulencel-T, Gpl00:209-217, AGS-003, DCVax-L, Algenpantucel-L, Tergenpantucel-L, TG4010, ProstAtak, Prostvac-V/R-TRICOM, Rindopepimul, E75 peptide acetate, IMA901, POL-103 A,
  • tumor vaccines such as Gardail, Cervarix, BCG, sipulencel-T, Gpl00:209-217, AGS-003, DCVax-L, Algenpantucel-L, Tergenpantucel-L, TG4010, ProstAtak, Prostvac-V/R-TRICOM, Rindopepimul, E75 peptide acetate, IMA901, POL-103 A,
  • Immunotherapy may be administered via injection (e.g ., intravenously, intratumorally, subcutaneously, or into lymph nodes), but may also be administered orally, topically, or via aerosol.
  • Immunotherapies may comprise adjuvants such as cytokines.
  • the immunotherapy agent is an immune checkpoint inhibitor.
  • Immune checkpoint inhibition broadly refers to inhibiting the checkpoints that cancer cells can produce to prevent or downregulate an immune response.
  • immune checkpoint proteins include, but are not limited to, CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7- H3, B7-H4, BTLA, KIR, LAG3, TIM-3, or VISTA.
  • Immune checkpoint inhibitors can be antibodies or antigen binding fragments thereof that bind to and inhibit an immune checkpoint protein.
  • the immune checkpoint inhibitor is an antibody.
  • immune checkpoint inhibitors include, but are not limited to, nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0010718C (avelumab), AUR-012, and STT-AIOIO.
  • the cancer immunotherapy includes administering an additional immune checkpoint inhibitor (e.g., administering 2, 3, 4, or 5, immune checkpoint inhibitors) to the subject.
  • the immune checkpoint inhibitor is a CTLA-4 inhibitor.
  • the immune checkpoint inhibitor is ipilimumab.
  • the immune checkpoint inhibitor is a PD-1 inhibitor.
  • the immune checkpoint inhibitor is pembrolizumab.
  • the immune checkpoint inhibitor is a PD-L1 inhibitor.
  • the immune checkpoint inhibitor is avelumab. In some embodiments, the immune checkpoint inhibitor is an antibody. In some embodiments, the pharmaceutical composition is administered daily (e.g., once daily or twice daily) and the immune checkpoint inhibitor is administered once every 1, 2, 3 or 4 weeks.
  • the pharmaceutical composition is administered daily (e.g., once daily or twice daily) and the immune checkpoint inhibitor is pembrolizumab.
  • the pharmaceutical composition is administered daily (e.g., once daily or twice daily) and pembrolizumab is administered once every 3 weeks, e.g., at a dose of 200 mg (e.g., intravenously).
  • immune checkpoint inhibitors can be an inhibitory nucleic acid molecule (e.g., an siRNA molecule, an shRNA molecule or an antisense RNA molecule) that inhibits expression of an immune checkpoint protein that inhibits expression of an immune checkpoint protein.
  • an inhibitory nucleic acid molecule e.g., an siRNA molecule, an shRNA molecule or an antisense RNA molecule
  • the immune checkpoint inhibitor is a siRNA molecule.
  • siRNA molecules should include a region of sufficient homology to the target region, and be of sufficient length in terms of nucleotides, such that the siRNA molecule down-regulate target RNA (e.g., RNA of an immune checkpoint protein).
  • target RNA e.g., RNA of an immune checkpoint protein.
  • ribonucleotide or nucleotide can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions.
  • the sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double-strand character of the molecule.
  • an siRNA molecule may be modified or include nucleoside surrogates.
  • Single stranded regions of an siRNA molecule may be modified or include nucleoside surrogates, e.g., the unpaired region or regions of a hairpin structure, e.g., a region which links two complementary regions, can have modifications or nucleoside surrogates.
  • Modification to stabilize one or more 3'- or 5'-terminus of an siRNA molecule, e.g., against exonucleases, or to favor the antisense siRNA agent to enter into RISC are also useful.
  • Modifications can include C3 (or C6, C7, Cl 2) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, Cl 2, abasic, tri ethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that come as phosphoramidites and that have another DMT- protected hydroxyl group, allowing multiple couplings during RNA synthesis.
  • Each strand of an siRNA molecule can be equal to or less than 35, 30, 25, 24, 23, 22, 21, or 20 nucleotides in length. In some embodiments, the strand is at least 19 nucleotides in length. For example, each strand can be between 21 and 25 nucleotides in length. In some embodiments, siRNA agents have a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more overhangs, such as one or two 3' overhangs, of 2-3 nucleotides.
  • the immune checkpoint inhibitor is a shRNA molecule.
  • a “small hairpin RNA” or“short hairpin RNA” or“shRNA” includes a short RNA sequence that makes a tight hairpin turn that can be used to silence gene expression via RNA interference.
  • the shRNAs provided herein may be chemically synthesized or transcribed from a transcriptional cassette in a DNA plasmid. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • shRNAs are about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, or are about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g ., each complementary sequence of the double-stranded shRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, or about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded shRNA is about 15- 60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, or about 18-22, 19-20, or 19-21 base pairs in length).
  • shRNA duplexes may comprise 3’ overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides on the antisense strand and/or 5’-phosphate termini on the sense strand.
  • the shRNA comprises a sense strand and/or antisense strand sequence of from about 15 to about 60 nucleotides in length (e.g., about 15-60, 15-55, 15- 50, 15-45, 15-40, 15-35, 15-30, or 15-25 nucleotides in length), or from about 19 to about 40 nucleotides in length (e.g., about 19-40, 19-35, 19-30, or 19-25 nucleotides in length), or from about 19 to about 23 nucleotides in length (e.g., 19, 20, 21, 22, or 23 nucleotides in length).
  • Non-limiting examples of shRNA include a double-stranded polynucleotide molecule assembled from a single-stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; and a double-stranded polynucleotide molecule with a hairpin secondary structure having self-complementary sense and antisense regions.
  • the sense and antisense strands of the shRNA are linked by a loop structure comprising from about 1 to about 25 nucleotides, from about 2 to about 20 nucleotides, from about 4 to about 15 nucleotides, from about 5 to about 12 nucleotides, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides.
  • a loop structure comprising from about 1 to about 25 nucleotides, from about 2 to about 20 nucleotides, from about 4 to about 15 nucleotides, from about 5 to about 12 nucleotides, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides.
  • the immune checkpoint inhibitor is an antisense oligonucleotide compounds that inhibits expression of an immune checkpoint protein.
  • the degree of complementarity between the target sequence and antisense targeting sequence is sufficient to form a stable duplex.
  • the region of complementarity of the antisense oligonucleotides with the target RNA sequence may be as short as 8-11 bases, but can be 12-15 bases or more, e.g., 10-40 bases, 12-30 bases, 12-25 bases, 15-25 bases, 12-20 bases, or 15-20 bases, including all integers in between these ranges.
  • An antisense oligonucleotide of about 14-15 bases is generally long enough to have a unique complementary sequence.
  • antisense oligonucleotides may be 100% complementary to the target sequence, or may include mismatches, e.g., to improve selective targeting of allele containing the disease-associated mutation, as long as a heteroduplex formed between the oligonucleotide and target sequence is sufficiently stable to withstand the action of cellular nucleases and other modes of degradation which may occur in vivo.
  • mismatches e.g., to improve selective targeting of allele containing the disease-associated mutation
  • oligonucleotides may have about or at least about 70% sequence complementarity, e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity, between the oligonucleotide and the target sequence.
  • Oligonucleotide backbones that are less susceptible to cleavage by nucleases are discussed herein.
  • Mismatches are typically less destabilizing toward the end regions of the hybrid duplex than in the middle.
  • the number of mismatches allowed will depend on the length of the oligonucleotide, the percentage of G:C base pairs in the duplex, and the position of the mismatch(es) in the duplex, according to well understood principles of duplex stability.
  • the inhibitory nucleic acid molecule can be prepared, for example, by chemical synthesis, in vitro transcription, or digestion of long dsRNA by Rnase III or Dicer. These can be introduced into cells by transfection, electroporation, or other methods known in the art. See Hannon, GJ, 2002, RNA Interference, Nature 418: 244-251; Bernstein E et al., 2002, The rest is silence. RNA 7: 1509-1521 ; Hutvagner G et al., RNAi: Nature abhors a double-strand. Curr. Opin.
  • Short hairpin RNAs induce sequence-specific silencing in mammalian cells. Genes & Dev. 16:948-958; Paul CP, Good PD, Winer I, and Engelke DR. (2002). Effective expression of small interfering RNA in human cells. Nature Biotechnol. 20:505-508; Sui G, Soohoo C, Affar E-B, Gay F, Shi Y, Forrester WC, and Shi Y. (2002). A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl.
  • RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells Proc. Natl. Acad. Sci. USA 99(9): 6047-6052.
  • the inhibitory nucleic acid molecule can be administered to the subject, for example, as naked nucleic acid, in combination with a delivery reagent, and/or as a nucleic acid comprising sequences that express an interfering nucleic acid molecule.
  • the nucleic acid comprising sequences that express the interfering nucleic acid molecules are delivered within vectors, e.g. plasmid, viral and bacterial vectors. Any nucleic acid delivery method known in the art can be used in the methods described herein. Suitable delivery reagents include, but are not limited to, e.g., the Mirus Transit TKO lipophilic reagent;
  • atelocollagen as a delivery vehicle for nucleic acid molecules is described in Minakuchi et al. Nucleic Acids Res., 32(13):el09 (2004); Hanai et al. Ann NY Acad Sci., 1082:9-17 (2006); and Kawata et al. Mol Cancer Ther., 7(9):2904-12 (2008); each of which is incorporated herein in their entirety.
  • Exemplary interfering nucleic acid delivery systems are provided m U.S. Patent Nos. 8,283,461, 8,313,772, 8,501,930. 8,426,554, 8,268,798 and
  • the immunotherapy agent is an antibody or antigen binding fragment thereof that, for example, binds to a cancer-associated antigen.
  • cancer-associated antigens include, but are not limited to, adipophilin, AIM-2, ALDHIAI, alpha- actinm-4, alpha-fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (“CEA”), CASP-5, C ASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin Dl, Cyclin-Al, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM,
  • the immunotherapy agent is a cancer vaccine and/or a component of a cancer vaccine (e.g ., an antigenic peptide and/or protein).
  • the cancer vaccine can be a protein vaccine, a nucleic acid vaccine, or a combination thereof.
  • the cancer vaccine comprises a polypeptide comprising an epitope of a cancer- associated antigen.
  • the cancer vaccine comprises a nucleic acid (e.g., DNA or RNA, such as mRNA) that encodes an epitope of a cancer-associated antigen.
  • the nucleic acid is a vector (e.g., a bacterial vector, viral vector).
  • bacterial vectors include, but are not limited to, Mycobacterium bovis (BCG), Salmonella Typhimurium ssp., Salmonella Typhi ssp., Clostridium sp. spores, Escherichia cob Nissle 1917, Escherichia coli K-12/LLO, Listeria monocytogenes, and Shigella flexneri.
  • viral vectors include, but are not limited to, vaccinia, adenovirus, RNA viruses, and replication- defective avipox, replication-defective fowlpox, replication-defective canarypox, replication- defective MVA and replication-defective adenovirus.
  • the cancer immunotherapy comprises administration of an antigen presenting cell (APC) primed with a cancer-specific antigen.
  • APC antigen presenting cell
  • the APC is a dendritic cell, a macrophage, or a B cell.
  • cancer-associated antigens include, but are not limited to, adipophilin, AIM-2, ALDHIAI, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTCl, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenm, BING-4, CA-125, CALC A, carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cychn Dl, Cyclm-Al, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen (“ETA”), ETV6-AML1 fusion protein, EZH
  • Kallikrein 4 KIF20A, KK-LC-1, KKLCl, KM-HN-1, KMHNl also known as CCDC110, LAGE-1, LDLR-fucosyltransferaseAS fusion protein, Lengsin, M-CSF, MAGE-Al, MAGE- A10, MAGE-Al 2, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-Cl, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, MEl, Melan- A/MART - 1 , Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY
  • the CAR binds specifically to a cancer-associated antigen.
  • the cancer immunotherapy comprises administration of a cancer-specific T cell to the subject.
  • the T cell is a CD4 + T cell.
  • the CD4 + T cell is a THI T cell, a TH2 T cell, or a TH17 T cell.
  • the T cell expresses a T cell receptor specific for a cancer-associated antigen.
  • the cancer vaccine is administered with an adjuvant.
  • adjuvants include, but are not limited to, an immune modulatory protein, Adjuvant 65, a-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, b-Glucan Peptide, CpG DNA, GPI-0100, lipid A, lipopolysaccharide, Lipovant, Montanide, N-acetyl-muramyl-L- alanyl-D-isoglutamine, Pam3CSK4, quil A, and trehalose dimycolate.
  • the immunotherapy agent is an immune modulating protein to the subject.
  • the immune modulatory protein is a cytokine.
  • immune modulating proteins include, but are not limited to, B lymphocyte chemoattractant ("BLC"), C-C motif chemokine 11 (“Eotaxin-1”), Eosinophil chemotactic protein 2 (“Eotaxin-2”), Granulocyte colony-stimulating factor (“G-CSF”), Granulocyte macrophage colony-stimulating factor (“GM-CSF”), 1-309, Intercellular Adhesion Molecule 1 (“ICAM-1”), Interferon gamma ("IFN-gamma”), Interlukin-1 alpha (“IL-1 alpha”), Interlukin-1 beta (“IL-1 beta”), Interleukin 1 receptor antagonist (“IL-1 ra”), Interleukin-2 (“IL-2”),
  • BLC B lymphocyte chemoattractant
  • Eotaxin-2 Eosinophil chemotactic protein 2
  • Interleukin-4 ("IL-4"), Interleukin-5 (“IL-5"), Interleukin-6 (“IL-6”), Interleukin-6 soluble receptor (“IL-6 sR”), Interleukin-7 (“IL-7”), Interleukin-8 (“IL-8”), Interleukin- 10 (“IL-10”), Interleukin- 11 (“IL-11”), Subunit beta of Interleukin- 12 (“IL-12 p40” or “IL-12 p70"), Interleukin- 13 (“IL-13”), Interleukin- 15 (“IL-15”), Interleukin- 16 (“IL-16”), Interleukin- 17 (“IL- 17”), Chemokine (C-C motif) Ligand 2 (“MCP-1 "), Macrophage colony-stimulating factor (“M- CSF”), Monokine induced by gamma interferon (“MIG”), Chemokine (C-C motif) ligand 2 (“MIP-1 alpha”), Chemokine (C-C motif) ligand 4 ("MIP-1
  • PIGF Phosphatidylinositol-glycan biosynthesis
  • Skp Skp
  • SCF Stem cell factor receptor
  • SCF R Stem cell factor receptor
  • TGFalpha Transforming growth factor alpha
  • TGF beta 1 Transforming growth factor beta-1
  • TGF beta-3 TGF beta 3
  • VEGF Vascular endothelial growth factor
  • VEGFR2 Vascular endothelial growth factor receptor 2
  • VGFR3 Vascular endothelial growth factor receptor 3
  • VEGF-D 6Ckine Tyrosine-protein kinase receptor UFO (“Axl)
  • Betacellulin BTC
  • Mucosae- associated epithelial chemokine CL28
  • C-C motif Chemokine (C-C motif) ligand 27
  • CXCL16 Chemokine (C-X-C motif) ligand 16
  • CXCL16 C-X-C motif chem
  • ErbB3 Endothelial-leukocyte adhesion molecule 1
  • E-Selectin Endothelial-leukocyte adhesion molecule 1
  • Fas Fms-like tyrosine kinase 3
  • GITR Tumor necrosis factor receptor superfamily member 1
  • HVEM Tumor necrosis factor receptor superfamily member 14
  • ICM-3 Intercellular adhesion molecule 3
  • IL-1 R4 IL-1 RI, IL-10 Rbeta, IL-17R, IL- 2Rgamma, IL-21R
  • Lysosome membrane protein 2 ("LIMPII"
  • Neutrophil gelatinase-associated lipocalin (“Lipocalin-2")
  • CD62L L-Selectin
  • LYVE-l Lymphatic endothelium
  • MHC class I polypeptide-related sequence A MICA
  • MHC class I polypeptide-related sequence B MHC class I polypeptide-related sequence B
  • TGF beta 2 Transforming growth factor-beta 2
  • Tie-2 Tie-2
  • TPO Tumor necrosis factor receptor superfamily member 10D
  • TME Tumor necrosis factor receptor superfamily member 10D
  • TME-1 Tumor necrosis factor receptor superfamily member 1
  • VEGF-C Vascular endothelial growth factor C
  • VEGFRIAdiponectin Adipsin ("AND), Alpha-fetoprotein ("AFP”), Angiopoietin-like 4 (“ANGPTL4"), Beta-2-microglobulin (“B2M”), Basal cell adhesion molecule (“BCAM”), Carbohydrate antigen 125 (“CA125”), Cancer Antigen 15-3 (“CA15-3"), Carcinoembryonic antigen (“CEA”), cAMP receptor protein (“CRP”), Human Epidermal Growth Factor Receptor 2 (“ErbB2”), Follistatin, Follicle-stimulating hormone (“FSH”), Chemokine (C-X-C motif) ligand 1 ("GRO alpha"), human chorionic gonadotropin (“beta HCG”), Insulin-like growth factor 1 receptor (“IGF-1 sR”), IL-1 sRII, IL-3, IL-18 Rb, IL-21, Leptin, Matrix metalloproteinase- 1 (“MMP-1 "), Matr
  • Interleukin 24 Interleukin 24
  • Interleukin 33 Interleukin 33
  • Kallikrein 14 Asparaginyl endopeptidase
  • Legumain Oxidized low-density lipoprotein receptor 1
  • MBL Mannose-binding lectin
  • NEP Neprilysin
  • Notch- 1 Notch homolog 1, translocation-associated (Drosophila)
  • NOV Nephroblastoma overexpressed
  • Osteoactivin Programmed cell death protein 1
  • PGRP-5" N-acetylmuramoyl-L-alanine amidase
  • Serpin A4 Secreted frizzled related protein 3
  • sFRP-3 Thrombomodulin
  • TLR2 Tumor necrosis factor receptor superfamily member 10A
  • TRF Tumor necrosis factor receptor superfamily member 10A
  • TRF Tumor necrosis factor receptor superfamily member 10A
  • TRF Tumor necrosis factor receptor superfamily member 10A
  • TRF Transfer
  • FLR1 Furin
  • GASP-1 GPCR-associated sorting protein 1
  • GASP-2 GPCR-associated sorting protein 2
  • GCSF R Granulocyte colony- stimulating factor receptor
  • HAI-2 Serine protease hepsin
  • IL-17B R Interleukin 17B Receptor
  • IL-27 Interleukin 27
  • LAG-3 Lymphocyte-activation gene 3
  • LDL R Apolipoprotein A-V
  • Pepsinogen I Pepsinogen I
  • Retinol binding protein 4 (“RBP4"), SOST, Heparan sulfate proteoglycan (“Syndecan-1”),
  • Tumor necrosis factor receptor superfamily member 13B (“TACI”), Tissue factor pathway inhibitor (“TFPI”), TSP-1, Tumor necrosis factor receptor superfamily, member 10b (“TRAIL R2”), TRANCE, Troponin I, Urokinase Plasminogen Activator (“uPA”), Cadherin 5, type 2 or VE-cadherin (vascular endothelial) also known as CD144 (“VE-Cadherin”), WNTl-inducible- signabng pathway protein 1 (“WISP-1 "), and Receptor Activator of Nuclear Factor k B
  • the cancer therapeutic is a radioactive moiety that comprises a radionuclide.
  • radionuclides include, but are not limited to Cr-51, Cs-131, Ce-134, Se-75, Ru-97, 1-125, Eu-149, Os- 189m, Sb-119, 1-123, Ho- 161, Sb-117, Ce-139, In-111, Rh-103m, Ga-67, Tl-201, Pd-103, Au-195, Hg-197, Sr-87m, Pt-191, P-33, Er-169, Ru-103, Yb- 169, Au-199, Sn-121, Tm-167, Yb-175, In-113m, Sn-113, Lu-177, Rh-105, Sn-117m, Cu-67, Sc- 47, Pt-195m, Ce-141, 1-131, Tb-161, As-77, Pt-197, Sm-153, Gd-159, Tm-173, Pr-143
  • the cancer therapeutic is an angiogenesis inhibitor to the subject.
  • angiogenesis inhibitors include, but are not limited to Bevacizumab (Avastin®), Ziv-aflibercept (Zaltrap®), Sorafenib (Nexavar®), Sunitinib (Sutent®), Pazopanib (Votrient®), Regorafenib (Stivarga®), and Cabozantinib (CometriqTM).
  • the cancer therapeutic is an antibiotic.
  • antibiotics can be administered to eliminate the cancer-associated bacteria from the subject.
  • Antibiotics broadly refers to compounds capable of inhibiting or preventing a bacterial infection. Antibiotics can be classified in a number of ways, including their use for specific infections, their mechanism of action, their
  • antibiotics can be used to selectively target bacteria of a specific niche.
  • antibiotics known to treat a particular infection that includes a cancer niche may be used to target cancer-associated microbes, including cancer-associated bacteria in that niche.
  • antibiotics are administered after the bacterial treatment.
  • antibiotics are administered after the bacterial treatment to remove the engraftment.
  • antibiotics can be selected based on their bactericidal or bacteriostatic properties.
  • Bactericidal antibiotics include mechanisms of action that disrupt the cell wall (e.g., b-lactams), the cell membrane (e.g., daptomycin), or bacterial DNA (e.g., fluoroquinolones).
  • Bacteriostatic agents inhibit bacterial replication and include sulfonamides, tetracyclines, and macrolides, and act by inhibiting protein synthesis.
  • some drugs can be bactericidal in certain organisms and bacteriostatic in others, knowing the target organism allows one skilled in the art to select an antibiotic with the appropriate properties.
  • bacteriostatic antibiotics inhibit the activity of bactericidal antibiotics.
  • bactericidal and bacteriostatic antibiotics are not combined.
  • Antibiotics include, but are not limited to aminoglycosides, ansamycins, carbacephems, carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptide antibiotics, quinolones, fluoroquinolone, sulfonamides, tetracyclines, and anti-mycobacterial compounds, and combinations thereof.
  • Aminoglycosides include, but are not limited to Amikacin, Gentamicin,
  • Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, and Spectinomycin are examples of the compounds listed in the following paragraphs.
  • Aminoglycosides are effective, e.g., against Gram-negative bacteria, such as Escherichia coli, Klebsiella, Pseudomonas aeruginosa, and Francisella tularensis, and against certain aerobic bacteria but less effective against obligate/facultative anaerobes. Aminoglycosides are believed to bind to the bacterial 30S or 50S ribosomal subunit thereby inhibiting bacterial protein synthesis.
  • Ansamycins include, but are not limited to, Geldanamycin, Herbimycin, Rifamycin, and Streptovaricin.
  • Geldanamycin and Herbimycin are believed to inhibit or alter the function of Heat Shock Protein 90.
  • Carbacephems include, but are not limited to, Loracarbef. Carbacephems are believed to inhibit bacterial cell wall synthesis.
  • Carbapenems include, but are not limited to, Ertapenem, Doripenem,
  • Carbapenems are bactericidal for both Gram-positive and Gram-negative bacteria as broad-spectrum antibiotics. Carbapenems are believed to inhibit bacterial cell wall synthesis.
  • Cephalosporins include, but are not limited to, Cefadroxil, Cefazolin, Cefalotin, Cefalothin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil, and Ceftobiprole. Selected
  • Cephalosporins are effective, e.g., against Gram-negative bacteria and against Gram-positive bacteria, including Pseudomonas, certain Cephalosporins are effective against methicillin- resistant Staphylococcus aureus (MRSA). Cephalosporins are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Glycopeptides include, but are not limited to, Teicoplanin, Vancomycin, and Telavancin. Glycopeptides are effective, e.g, against aerobic and anaerobic Gram-positive bacteria including MRSA and Clostridium difficile. Glycopeptides are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Lincosamides include, but are not limited to, Clindamycin and Lincomycin. Lincosamides are effective, e.g., against anaerobic bacteria, as well as Staphylococcus, and Streptococcus. Lincosamides are believed to bind to the bacterial 50S ribosomal subunit thereby inhibiting bacterial protein synthesis.
  • Lipopeptides include, but are not limited to, Daptomycin. Lipopeptides are effective, e.g., against Gram-positive bacteria. Lipopeptides are believed to bind to the bacterial membrane and cause rapid depolarization.
  • Macrolides include, but are not limited to, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, and Spiramycin. Macrolides are effective, e.g., against Streptococcus and Mycoplasma. Macrolides are believed to bind to the bacterial or 50S ribosomal subunit, thereby inhibiting bacterial protein synthesis.
  • Monobactams include, but are not limited to, Aztreonam. Monobactams are effective, e.g, against Gram-negative bacteria. Monobactams are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Nitrofurans include, but are not limited to, Furazolidone and Nitrofurantoin.
  • Oxazolidonones include, but are not limited to, Linezolid, Posizolid, Radezolid, and Torezolid. Oxazolidonones are believed to be protein synthesis inhibitors.
  • Penicillins include, but are not limited to, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Temocillin and Ticarcillin. Penicillins are effective, e.g, against Gram-positive bacteria, facultative anaerobes, e.g., Streptococcus,
  • Penicillins are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Penicillin combinations include, but are not limited to, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam, and Ticarcillin/clavulanate.
  • Polypeptide antibiotics include, but are not limited to, Bacitracin, Colistin, and Polymyxin B and E.
  • Polypeptide Antibiotics are effective, e.g, against Gram-negative bacteria. Certain polypeptide antibiotics are believed to inhibit isoprenyl pyrophosphate involved in synthesis of the peptidoglycan layer of bacterial cell walls, while others destabilize the bacterial outer membrane by displacing bacterial counter-ions.
  • Quinolones and Fluoroquinolone include, but are not limited to, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Fevofloxacin, Fomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, and Temafloxacin.
  • Quinolones/Fluoroquinolone are effective, e.g. , against Streptococcus and Neisseria.
  • Quinolones/Fluoroquinolone are believed to inhibit the bacterial DNA gyrase or topoisomerase IV, thereby inhibiting DNA replication and transcription.
  • Sulfonamides include, but are not limited to, Mafenide, Sulfacetamide,
  • Sulfadiazine Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole,
  • Sulfanilimide Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole (Co-trimoxazole), and Sulfonamidochrysoidine.
  • Sulfonamides are believed to inhibit folate synthesis by competitive inhibition of dihydropteroate synthetase, thereby inhibiting nucleic acid synthesis.
  • Tetracyclines include, but are not limited to, Demeclocycline, Doxycycline, Minocycline, Oxytetracy cline, and Tetracycline. Tetracyclines are effective, e.g., against Gram negative bacteria. Tetracyclines are believed to bind to the bacterial 30S ribosomal subunit thereby inhibiting bacterial protein synthesis.
  • Anti-mycobacterial compounds include, but are not limited to, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, and Streptomycin.
  • Suitable antibiotics also include arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin/dalfopristin, tigecycline, tinidazole, trimethoprim amoxicillin/clavulanate, ampicillin/sulbactam, amphomycin ristocetin, azithromycin, bacitracin, buforin II, carbomycin, cecropin PI, clarithromycin, erythromycins, furazolidone, fusidic acid, Na fusidate, gramicidin, imipenem, indolicidin, josamycin, magainan II, metronidazole, nitroimidazoles, mikamycin, mutacin B-Ny266, mutacin B-JH1 140, mutacin J-T8, nisin, nisin A, novobiocin, o
  • the cancer therapy comprises administering a therapeutic bacteria and/or a therapeutic combination of bacteria to the subject so a healthy microbiome can be reconstituted in the subject.
  • the therapeutic bacteria is a non-cancer- associated bacteria.
  • the therapeutic bacteria is a probiotic bacteria.
  • the methods and compositions described herein relate to the treatment of cancer.
  • cancers that may treated by methods described herein include, but are not limited to, hematological malignancy, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia
  • immunoblastic sarcoma of B cells lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, rhabdosarcoma, serocystic sarcoma, synovial sarcoma, telangiectaltic sarcoma, Hodgkin's Disease, Non-Hodgkin's
  • Lymphoma multiple myeloma, neuroblastoma, bladder cancer, breast cancer, ovarian cancer, lung cancer, colorectal cancer, rhabdomyosarcoma, primary thrombocytosis, primary
  • macroglobulinemia small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.
  • the cancer comprises renal cell carcinoma.
  • the cancer comprises breast cancer (e.g., triple negative breast cancer).
  • the cancer comprises colorectal cancer (e.g., microsatellite stable (MSS) colorectal cancer).
  • MSS microsatellite stable
  • the cancer comprises renal cell carcinoma.
  • the cancer comprises lung cancer (e.g., non small cell lung cancer).
  • the cancer comprises bladder cancer.
  • the cancer comprises gastroesophageal cancer.
  • the methods and compositions provided herein relate to the treatment of a leukemia.
  • leukemia is meant broadly progressive, malignant diseases of the hematopoietic organs/systems and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow.
  • Non-limiting examples of leukemia diseases include, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leuk
  • carcinoma refers to a malignant growth made up of epithelial cells tending to infiltrate the surrounding tissues, and/or resist physiological and non- physiological cell death signals and gives rise to metastases.
  • carcinomas include, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma,
  • basosquamous cell carcinoma bronchioalveolar carcinoma
  • bronchiolar carcinoma basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma,
  • bronchogenic carcinoma cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, signet ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinoma villosum, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-
  • the methods and compositions provided herein relate to the treatment of a sarcoma.
  • sarcoma generally refers to a tumor that is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar, heterogeneous, or homogeneous substance.
  • Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing' s sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic s
  • Additional exemplary neoplasias that can be treated using the methods and compositions described herein include Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenal cortical cancer.
  • the cancer treated is a melanoma.
  • melanoma is taken to mean a tumor arising from the melanocytic system of the skin and other organs.
  • melanomas are Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.
  • the cancer comprises a solid tumor.
  • compositions described herein include lymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, colorectal cancer, pancreatic cancer, cancer of the thyroid, head and neck cancer, cancer of the central nervous system, cancer of the peripheral nervous system, skin cancer, kidney cancer, as well as metastases of all the above.
  • tumors include hepatocellular carcinoma, hepatoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, invasive ductal carcinoma, papillary
  • adenocarcinoma melanoma
  • pulmonary squamous cell carcinoma basal cell carcinoma
  • adenocarcinoma well differentiated, moderately differentiated, poorly differentiated or undifferentiated
  • bronchioloalveolar carcinoma renal cell carcinoma, hypernephroma, hypernephroid adenocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor
  • lung carcinoma including small cell, non-small and large cell lung carcinoma, bladder carcinoma, glioma, astrocyoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, retinoblastoma, neuroblastoma, colon carcinoma, rectal carcinoma, hematopoietic malignancies including all types of leukemia and lymphoma including: acute myelogenous leukemia, acute myelocy
  • Cancers treated in certain embodiments also include precancerous lesions, e.g., actinic keratosis (solar keratosis), moles (dysplastic nevi), acitinic chelitis (farmer's lip), cutaneous horns, Barrett's esophagus, atrophic gastritis, dyskeratosis congenita, sideropenic dysphagia, lichen planus, oral submucous fibrosis, actinic (solar) elastosis and cervical dysplasia.
  • precancerous lesions e.g., actinic keratosis (solar keratosis), moles (dysplastic nevi), acitinic chelitis (farmer's lip), cutaneous horns, Barrett's esophagus, atrophic gastritis, dyskeratosis congenita, sideropenic dysphagia, lichen
  • Cancers treated in some embodiments include non-cancerous or benign tumors, e.g., of endodermal, ectodermal or mesenchymal origin, including, but not limited to
  • cholangioma cholangioma, colonic polyp, adenoma, papilloma, cystadenoma, liver cell adenoma,
  • hydatidiform mole renal tubular adenoma, squamous cell papilloma, gastric polyp, hemangioma, osteoma, chondroma, lipoma, fibroma, lymphangioma, leiomyoma, rhabdomyoma, astrocytoma, nevus, meningioma, and ganglioneuroma.
  • Systemic immunity is regulated by interactions of commensal bacteria with immune cells in the gut. Enrichment of specific intestinal microbes has been shown to enhance the anti -tumor response to PD-1 blockade in both murine models and cancer patients.
  • Oral administration of a monoclonal microbial that includes Bifidobacterium animalis ssp. lactis Strain A (ATCC Deposit No. PTA-125097) (“ Bifido Strain A”) induces systemic anti-tumor immunity via the direct activation of antigen presenting cells (APCs) and natural killer (NK) cells.
  • APCs antigen presenting cells
  • NK natural killer
  • Bifido Strain A increases expression of costimulatory molecules on CD1 lc + dendritic cells (DCs) within the mesenteric LNs with an accompanying increase in
  • Bifido Strain A stimulates direct stimulation of DCs with Bifido Strain A results in the upregulation of the proinflammatory cytokines TNF-a, IL-Ib, and IL-12.
  • Bifido Strain A amplifies both myeloid and lymphocyte responses via production of DC-derived growth factors, Ml polarization of macrophages, and production of the lymphocyte-recruiting chemokines, CXCL9 and CXCL10.
  • Bifido Strain A triggers specific pattern recognition receptors, resulting in a pro-inflammatory signature from APCs and enhanced cytolytic activity by NK cells.
  • Example 2 Monoclonal microbial Bifidobacterium animalis ssp. lactis Strain A induces antitumor responses via gut-mediated activation of both innate and adaptive immunity
  • Bifido Strain A (ATCC Deposit No. PTA-125097) (“ Bifido Strain A” or“Strain A”) has potent anti-tumor activity.
  • Treatment with Bifido Strain A in vitro promotes the production of type-I effector molecules by cells of the innate immune system, including the transactivation of human NK cells.
  • Oral delivery of Bifido Strain A monotherapy leads to tumor control comparable to checkpoint inhibition in multiple subcutaneous syngeneic tumor models. This treatment promotes the immunogenic remodeling of the tumor microenvironment to favor the infiltration of protective effector cells.
  • NK cells are insufficient to mediate protection in the absence of CD8 + cytotoxic T lymphocytes (CTL).
  • CTL cytotoxic T lymphocytes
  • PBMCs were stimulated with Bifido Strain A biomass (shown in second bar from left) or other strains of Bifidobacterium (grey) in vitro for 24 hours in 1% O2 (Fig. IB). Supernatants were harvested and analyzed by MSD for IL-12p70. PBMCs showed significant IL-12p70 induction when stimulated with Bifido Strain A lyophilized powder (second bar from left) (Fig. IB). To further assess the cytokine signature induced by Bifido Strain A and differentiate this signature from TLR2 or TLR9 agonists, PBMCs were stimulated with Bifido Strain A biomass for 24 hours in 1 %Ch (Fig. 1C).
  • NK cells are critical effector cells involved in the anti-tumor response.
  • human monocytes were pulsed with Bifido Strain A or an anti-inflammatory microbe at an MOI of 1 for 18 hours and then cultured with purified autologous NK cells.
  • NK cells were then transferred to wells containing CFSE-labeled K-562 tumor target cells for 4 hours and % cytotoxicity was measured by flow cytometry ⁇ Bifido Strain A is shown in shading; an anti-inflammatory control microbe is shown in white) (Fig. 2A).
  • Bifido Strain A significantly induces monocyte activation of NK cell killing of K562 target cells as read by flow cytometry.
  • NK cells were cultured with microbe-treated monocyte-conditioned media only.
  • NK cells were then subsequently transferred to plates containing CFSE-labeled K- 562 cells to assess cytotoxic activity (Fig. 2B).
  • Bifido Strain A-treated monocyte conditioned media alone can stimulate NK cell killing significantly.
  • To control for non-specific activation and also assess for direct activation of NK cells by Bifido Strain A NK cells alone were incubated with Bifido Strain A or an anti-inflammatory microbe prior to culture with CFSE- labeled K-562 cells ( Figure 2C). (* p > 0.05, ** p > 0.01, *** p > 0.001 ; Two-Way ANOVA).
  • RT-PCR analysis was performed from mRNA derived from CT-26 tumor lysate from mice orally treated with Bifido Strain A or vehicle for 11 days. Treatment was begun 10- days post tumor implant. CXCL10 (IP10) and B2M was assessed by RT-PCR and an increase in the relative expression of the pro- inflammatory chemokine and the antigen presenting molecule was increased with Bifido Strain A (Fig. 5A and Fig. 5C).
  • Tumors were homogenized following oral Bifido Strain A treatment and cells were analyzed by flow cytometry (Fig. 5F).
  • Balbc mice were inoculated with CT26 tumors for 10 days and orally treated with Bifido Strain A or sucrose vehicle for 5 days. Tumors were harvested and single cells suspensions were isolated and analyzed by flow cytometry.
  • Bifido Strain A treatment of CT26 bearing mice have increased NKp46 + NKG2A/C/E + NK cells with the TME comparable to anti- PD-1 treatment (quantified in Fig. 6A).
  • Bifido Strain A results in elevated IFNg production by CD44 + CD8 CTF within the TME (Fig.6B). Similar to anti-PD-1, Bifido Strain A treatment results in decreased Treg to CTF ratio within the TME as assessed by flow cytometry (Fig.6C). Tumors were analyzed following 5 days of Bifido Strain A treatment.
  • NK cells and CD8a + CTLs cooperate for optimal control of tumor growth
  • NK and CD8 cells were depleted of NK cells with anti-asialoGMl antibody or isotype control prior to treatment with Bifido Strain A (4mg) (Fig. 7B). Mice were challenged with CT26 tumor cells (10 5 ) 10 days prior to treatment with either Bifido Strain A or vehicle.
  • mice were challenged with CT26 tumor cells (10 5 ) 10 days prior to treatment with either Bifido Strain A or vehicle.
  • CD8 + T cells are required for Bifido Strain A efficacy as mice depleted of CD8 + T cells are unable to mount a productive anti-tumor response upon treatment with Bifido Strain A.
  • Bifido Strain A is effective in multiple tumor models (CT26, B16-F10, and MC38); (2) Bifido Strain A induces a proinflammatory gene signature (CXCL10, IL12p70, IFNg, IL-lb, TNFa and IL-6); (3) Bifido Strain A-treated Monocyte-conditioned sups induces NK cell cytolytic activity; (4) CD44 + effector lymphocyte populations (NK, NKT, CD4, CD8) upregulate CD25 and IFNg upon stimulation with Bifido Strain A; (5) EDP induces a proinflammatory chemotactic signature within the TME; (6) NK and CD8 TILs display an activated phenotype post -Bifido Strain A treatment; (7) both NK and CD8 T cells are required for a productive anti-tumor response induced by Bifido Strain A; and (8) NK
  • Example 3 Oral delivery of a single microbial strain mediates therapeutic anti-tumor immunity though IL-12p70-dependent activation of multiple IFN-y-prodiicing lymphocyte populations
  • a library of commensal bacterial strains including 33 Bifidobacteria, was screened for their ability to induce inflammatory responses in primary human immune cells in vitro.
  • Bifidobacterium animalis ssp. lactis Strain A induced high levels of both IL-12p70 and CXCL10 from peripheral blood mononuclear cells (PBMCs) or purified human monocytes, respectively (Figs. 8A and 8B).
  • Bifidobacterium animalis ssp. lactis Strain A possesses known and predicted PRR ligands. A panel of embryonic alkaline phosphatase-secreting HEK-293 cell lines expressing individual PRRs was tested. At low multiplicities of co-culture Bifidobacterium animalis ssp. lactis Strain A engaged human TLR2, and at higher multiplicities TLR9 and cGAS-STING, to levels similar to PRR agonist controls (Fig. 8C). Bifidobacterium animalis ssp. lactis Strain A also triggered TLR4, consistent with reports that purified exopolysaccarides from
  • PBMCs from five healthy donors were stimulated with Bifidobacterium animalis ssp. lactis Strain A or saturating concentrations of specific agonists toTLR2/6, ParmCSKI, and to TLR9, CpG-ODN 2008.
  • Bifidobacterium animalis ssp. lactis Strain A induced greater production of TNFa, IL-1 b, and IFN-g than the individual TLR agonists. Consistent with Fig.
  • Oral Bifidobacterium animalis ssp. lactis Strain A has in vivo systemic anti-tumor efficacy
  • lactis Strain A also reduced tumor growth in the C57BL/6 B16-F10 melanoma and the MC-38 colon carcinoma models (Figs. 8F & 8G).
  • C57BL/6 mice were treated with Bifidobacterium animalis ssp. lactis Strain A one day prior to intravenous challenge with B16-F10 cells.
  • Bifidobacterium animalis ssp. lactis Strain A significantly reduced tumor foci in the lungs 17 days later relative to vehicle control and comparably to checkpoint inhibition (Figs. 8H & 81).
  • mice were orally administered fluorescently-labeled Bifidobacterium animalis ssp. lactis Strain A. Labeled bacteria were restricted to the intestinal mucosa and not detected beyond the epithelium (Fig. 9B). Flow cytometry was then used to quantitate the interactions of CD45 + cells in the SI with Alexa Flour 647-label ed Bifidobacterium animalis ssp.
  • lactis Strain A Bifido Strain A AF64" ⁇ A
  • lactis Strain A caused a reduction in expression of TNFa in tumor infiltrating myeloid-derived suppressor cells (MDSCs) with no effect on CXCL9 (Fig. 10B).
  • MDSCs tumor infiltrating myeloid-derived suppressor cells
  • Fig. 10B Bifidobacterium animalis ssp. lactis Strain A significantly reduced MDSC TNFa expression relative to vehicle and anti-PD-1 (Fig. IOC).
  • Responses from other myeloid subsets were investigated to determine if a concomitant immunostimulatory response accompanied the reduction in MDSC activation.
  • Tumor-infiltrating XCR1 + DC1 and SIRPa + DC2 were significantly increased in
  • Bifidobacterium animalis ssp. lactis Strain A treated mice relative to vehicle-treated controls (Fig. 10D), accompanied by upregulation of co- stimulatory CD86 (Fig. 10E).
  • Bifido Strain A treatment reduced TNFa production by SIRPa + tumor DC2 compared to vehicle and anti-PD-1 (Fig. 10F).
  • the differential activation profile of tumor DC1 and DC2 indicated a signature of oral Bifidobacterium animalis ssp. lactis Strain A treatment.
  • the ratio of IL-12p40 + DC1 over TNFa + DC2 illustrated the magnitude of this signature (Fig. 10H).
  • lactis Strain A increases effector lymphocyte activation and IFN-g production
  • TME tumor infiltrating lymphocytes
  • Ratios of CTL to Treg in a tumor is a prognostic indicator that correlates with patient response to cancer immunotherapy and survival.
  • lactis Strain A-responsive tumors there was a significant increase in terminally-differentiated effector CX3CR1 + CTL expressing IFN-g and increased granzyme-B (Fig. 11G & 11H), suggesting enhanced cellular cytotoxicity.
  • the increases in the total numbers of CTLs per gram of tumor compared to vehicle were not significant (Fig. 111).
  • these cells were significantly more frequent in tumor draining lymph nodes of Bifidobacterium animalis ssp.
  • lactis Strain A-treated mice (Fig. 11 J) suggesting antigen presentation in the local lymphatics.
  • Oral Bifidobacterium animalis ssp. lactis Strain A treatment results in activation of IFN-g- producing lymphocytes and differentiation of tumor antigen- specific CTL into functional cytotoxic effectors.
  • Bifidobacterium animalis ssp. lactis Strain A stimulates dendritic cell maturation supporting antigen-independent bystander activation of effector lymphocytes
  • Magnetically-enriched DCs were co-cultured with Bifidobacterium animalis ssp. lactis Strain A anaerobically in vitro. MHC class I, CD80, and IL-12p70 were increased in Bifidobacterium animalis ssp. lactis Strain A-treated DC, elevating signals 1 , 2, and 3 required for naive T cell priming (Fig. 12A). DCs were then pulsed with SIINFEKL, an H-2K d -restricted chicken ovalbumin peptide, and co-cultured with spleen CD8 + T cells from OT-I mice.
  • Bifidobacterium animalis ssp. lactis Strain A stimulated greater antigen-dependent response than vehicle, both of IL-2 production and OT-I T cell number, after four days (Fig. 12B).
  • Bifidobacterium animalis ssp. lactis Strain A-treated DCs supported T cell expansion and IFN-g production (Fig. 12C).
  • Cells from mLN of naive SPF mice were cultured with Bifidobacterium animalis ssp. lactis Strain A and treated with brefeldin-A to trap cytokines intracellularly.
  • NK and NKT cells were activated by Bifidobacterium animalis ssp. lactis Strain A with the greatest percentage. The largest number of activated cells were the memory CD8 + T cell compartment (Fig. 12F).
  • Lymphocyte activation is required for the anti-tumor effect of Bifidobacterium animalis ssp. lactis Strain A
  • Bifidobacterium animalis ssp. lactis Strain A triggers IL-12p70-dependent activation of tumor antigen-specific effector cells in the spleen
  • lactis Strain A mLN suspensions from naive mice with Bifidobacterium animalis ssp. lactis Strain A were co-cultured overnight, similar to Figs. 12D-12F. Cells were incubated with neutralizing antibodies against cytokines involved in bystander activation of effector lymphocytes. IL-15, IL-18, and IL-23pl9 blockade had no effect on Bifidobacterium animalis ssp. lactis Strain A NK cell activation. IL-2 and IL-12p40 neutralization significantly inhibited the response (Fig. 14A).
  • the CD8 + T cell profile was similar (Fig. 14B).
  • IL-2 blockade reduced expression of IFN-g, but not of CD25 and the early activation marker CD69 (Fig. 14B), revealing a potential synergy between IL-2 and IFN-g.
  • the gp70p:H2-L D tetramer showed that while most tumor antigen-specific memory CD8 + T cells are in the TME, there are substantial numbers these cells that are less-differentiated in secondary lymphoid organs, which do not exhibit the effects of chronic antigen stimulation, or exhaustion. These cells display higher levels of T cell receptor and lower levels of co-inhibitory receptors indicating a large reservoir of effector cells poised to respond to an appropriate stimulus.
  • mice were treated with Bifidobacterium animalis ssp. lactis Strain A for 48-hours to evaluate short-term effects.
  • Gp70p:H-2L d+ CTLs were significantly increased in the spleens of Bifidobacterium animalis ssp. lactis Strain A-treated mice (Fig. 14C) but not in the tumor draining lymph node (Fig. 14D).
  • IL- 12p70 promotes differentiation of memory T cells, changing adhesion molecule and chemokine receptor expression to modify positioning and function. Bifidobacterium animalis ssp.
  • lactis Strain A treatment shifted gp70p:H-2L d+ CTL in the spleen from CD62L-expressing central memory to CX3CRI -expressing cytotoxic effectors (Fig. 14E). These results were consistent with data in Fig. 10A-10H, indicating that the anti-tumor effects of Bifidobacterium animalis ssp. lactis Strain A are mediated by the IL-12p70-driven differentiation of tumor antigen-specific central memory cells outside of the tumor.
  • Bifidobacterium animalis ssp. lactis Strain A was isolated from commercial probiotics powders and maintained in Evelo Biosciences bacterial strain library. All strains were grown in Reinforced Clostridial Broth (RCB) under anaerobic conditions at 37°C for 12-18 hours. Total bacterial cell count (TCC) was measured by using Coulter Counter Multisizer4e (Beckman Coulter). Bacterial cultures were mixed with 40% anaerobic glycerol at 1 : 1 ratio and distributed into multiple repetitive 0.5ml matrix test tubes under anaerobic conditions and stored at -80°C. The glycerol stocks TCC concentration varied from le+8/ml to le+9/ml.
  • Bacterial strain identity was confirmed by 16S rDNA sequencing.
  • bacterial stocks were diluted in antibiotic-free cell culture media to a particular multiplicity of infection (MOI), and combined with cells under low oxygen conditions.
  • MOI multiplicity of infection
  • a lyophilized preparation of Bifidobacterium animalis ssp. lactis Strain A was used. Briefly, the powder was resuspended in anaerobic yeast extract-sucrose solution at room temperature. IOOmI of suspension was administered orally to each mouse daily for 11-13 days. The regular daily dose was calculated based on VCC: 10 mg of bacterial powder was
  • mice administered to mice and corresponded to a dose of 6.55 x 10 9 CFU.
  • Fresh, unpurified healthy donor human huffy coats were obtained from Research Blood Components, LLC. Peripheral blood mononuclear cells were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). Residual red blood cells were lysed using Ammonium Chloride lysis buffer (Qiagen) for 5 minutes on ice. PBMCs were re suspended in complete media without antibiotics and counted with viability dye to obtain total viable cell count for each donor. For some experiments, phagocytes were isolated from PBMC cells using anti-human/mouse CD1 lb microbeads (Miltenyi) and enriched via positive selection using MACS column-based magnetic enrichment. For other experiments, PBMC were isolated from leukopaks obtained from Stem Cell Technologies, and monocytes were purified using positive selection with the StemSep human CD14 + monocyte isolation kit. NK cells were isolated
  • mice For in vivo studies, six to eight- week-old female C57BL/6nTac, BALB/cnTac, and ICR-SCID mice were purchased from Taconic. C57BL/6-Tg (TcraTcrb - )1100Mjb /J (OT-I) and C57BL/6J controls were purchased from Jackson Laboratories. Mice were housed under specific pathogen free conditions and maintained under strict IACUC-approved guidelines.
  • CT-26 colon carcinoma and B16-F10 melanoma cell lines were from ATCC, MC-
  • CT-26 was grown in RPMI1640/Glutamax and B16-F10 and MC-38 were passaged in DMEM/Glutamax both supplemented with 10% FCS (ThermoFisher Scientific), 1% HEPES, and 100 U/ml penicillin/100 pg/ml streptomycin. Low passage (>3) cells were used for tumor implantation experiments. Cells were split upon reaching 80% confluency (2-3 days), and detached from culture flasks with Trypsin-EDTA, washed several times with PBS and resuspended in 50% phenol red free growth factor reduced (PRF- GFR) matrigel (corning) prior to implantation. 10 5 cells in 100 m ⁇ were implanted
  • mice were treated daily with vehicle +/- Bifido Strain A alone via oral gavage or in combination or as a monotherapy with purified anti PD-1 intraperitoneally every 4 days. 5 days post onset of treatment mice were euthanized by CO2 asphyxiation and death confirmed by cervical dislocation. Tumors and tumor draining lymph nodes (tDLNs) (inguinal, axillary and cervical) on the corresponding flank were removed and placed on ice. Single cell suspensions were obtained via mashing of tDLNs through a lOOum cell strainer and resuspending with complete RPMI1640 (cRPMI) media.
  • tDLNs tumor draining lymph nodes
  • Tumors were digested using the gentleMACs tissue dissociator (Miltenyi) following the manufacturer’s instructions. Following digestion steps cells were washed and resuspended in 40% isotonic percoll (Millipore Sigma) and cells pelleted at 650xG for 10 minutes. After centrifugation the percoll was discarded and pellets were washed and resuspended in enriched cRPMI for further analysis.
  • permeabilized using the FoxP3 staining kit (eBioscience) following the manufacturer’s instructions. After two washes in permeabilization buffer containing saponin, cells were stained for intracellular transcription factors and trapped cytokines at 4°C overnight. Cells were washed twice with permeabilization buffer, resuspended in FACS buffer and acquired on a BD LSR Fortessa flow cytometer.
  • Human PBMCs were isolated from leukopaks (Stemcell Tech) by Ficoll density gradient centrifugation. NK cells and monocytes were purified from PBMCs using human NK cell negative isolation and CD14+ isolation kits (Stemcell Tech). 20,000, 300,000, or 900,000 NK cells per well were plated with or without donor matched CD14 + monocytes (4,000, 60,000, or 180,000 cells per well respectively) in 96 well plates. Cells were then incubated with microbe or vehicle at an MOI of 1 for 18 hours at 37°C and 1% O2.
  • purified human CD14 + monocytes were incubated at 10 6 cells per mL with microbe or vehicle at an MOI of 1 for 24 hours at 37°C and 1% O2. Following the incubation supernatant was harvested and filter sterilized through 0.4 um filters, then applied to donor matched human NK cells and incubated for 18 hours in 96-well plates. At the end of the incubation period, microbe was washed off by centrifugation, and 20,000 CFSE-labeled K-562 tumor cells were added per well to generate the given (E)ffector:(T)arget ratios and incubated for 4 hrs.
  • Cells were then labeled with 7-AAD viability dye and acquired by FACS to assess specific cell killing, measured as frequency of CFSE + , 7-AAD + double positive cells from co-incubated wells minus background cell death frequency of K-562 cells cultured alone. IFN-g in cell culture supernatants at the end of the K-562 incubation period was measured using MSD V-plex.
  • Anti-PD-1 (clone RMP1-14) or anti-PD-Ll (10F-9G2) was used in in vivo tumor studies as an immunotherapy standard of care. 200 pg of therapeutic antibody was administered at the For the depletion of NK or CD8 + T cells, 200 pg of either anti-Asialo-GMl or control polyclonal rabbit IgG (BioXcell), or anti-mouse CD8a (clone 2.43) or rat IgG2b isotype (clone LTF-2), were injected intraperitoneally the day before tumor implantation and subsequently every four days thereafter until the study was terminated.
  • MNN Mesenteric lymph nodes
  • digestion buffer RPMI1640 + 1% FCS, 1 mM HEPES, and 1 mM glutathione with 0.15 Wunsch units/ml Liberase grade TL and 20 pg/ml DNAse I (Roche)
  • single cell suspensions were then plated in 96- well plates at 2 x 10 6 cells/100 pi and infected with an MOI of 10 Bifidobacterium animalis ssp.
  • lactis Strain A for 1 hour, after which the cells were washed and media containing 20 pg/ml of gentamicin was added to complete DMEM and the cells were incubated overnight at 37°C, for indicated wells isotype control antibodies or neutralizing antibodies against different cytokines were added at 5 pg/ml prior to stimulation with Bifidobacterium animalis ssp. lactis Strain A. Brefeldin-A and monensin to trap
  • mice were treated orally with Alexa Flour-647 labeled and unlabeled Bifidobacterium animalis ssp. lactis Strain A. Briefly, 1 hour after dosing, small intestines were dissected, and mesenteric fat was aggressively removed with forceps followed by scraping with a dull scalpel, visible PP on the anti-mesenteric side of the SI were removed with sharp scissors, placed in digestion buffer and processed similar to MLN. After PP removal, SI was cut into three equivalent sections and cut longitudinally to fully expose the intestinal lumen.
  • LP lamina limbal
  • PP Peyer’s patch
  • the SI was rinsed in several changes of PBS and transferred to tubes containing epithelial stripping buffer (Earle’s balanced salt solution + 5% dithiothreitol, 5 mM EDTA and 1% FCS) for two-15-minute incubations at 37°C with shaking at 250 RPM to remove mucus and intestinal epithelial cells.
  • epithelial stripping buffer Esarle’s balanced salt solution + 5% dithiothreitol, 5 mM EDTA and 1% FCS
  • the SI fragments were washed twice in complete RPMI1640 to remove residual DTT and EDTA, and placed in tubes with digestion buffer, minced vigorously with scissors, and with shaken at 37°C/250 RPM for 30-45 minutes.
  • Bifidobacterium animalis ssp. lactis Strain A strain specific primers were designed by identifying regions of genomic DNA and single nucleotide polymorphisms (SNPs) that are unique to Bifido Strain A using whole genome sequencing and publicly available databases.
  • SNPs single nucleotide polymorphisms
  • LNA locked nucleic acid
  • a synthetically designed gBlock fragment identical to the PCR amplicon was used as a standard to enable the calculation of the bacterial numbers in each sample.
  • The“click-chemistry” reagents 3-azido-D-alanine and DIBAC-Cy5 were utilized for fluorescent labeling of the bacteria.
  • a saturated culture of bacterial cells in a volume of 100 pL was inoculated into 10 mL of TSB+ broth containing 0.8 mM 3-azido-D-alanine and cultured overnight for 16-24 hours in an anaerobic chamber (5% H2, 5% CO2, 90% N2) at 37 °C.
  • the cells were centrifuged and resuspended in 900 pL of 1% BSA in PBS followed by centrifugation at 5000 x g for 3 minutes at room temperature.
  • IHC immunohistochemistry
  • the tissue sections were blocked and permeabilized by incubation in 10% FBS, 1% BSA, and 0.1% Triton X-100 in PBS for 1 h, washed twice with PBST, and incubated with antibody to MUC2 (1 :200 in 2% BSA/PBS) for 16 h (protected from light) at 4 °C.
  • Secondary antibody to rabbit IgG (1 : 1 ,000 in 2% BSA/PBS) was applied for 1 h at room temperature for MUC2 staining.
  • Nuclear staining was performed with Hoechst 33342 (0.1 pg/mL) in PBST for 10 min. Confocal images were acquired with a Zeiss AxioObserver 710 laser scanning inverted confocal microscope equipped with 3 PMT detectors and 6 visible laser lines.
  • SEAP Secreted embryonic alkaline phosphatase
  • HEK-Blue cell lines overexpressing human TLRs (hTLR2, hTLR4, hTLR7 and hTLR9) and the monocyte THP-1 ISG (STING) reporter cell line were obtained from InvivoGen.
  • TLR agonist controls (Par CSKl for TLR2, ultrapure LPS for TLR4, R848 for TLR7/8, ODN2006 for TLR9, 3’3’-cGAMP for THP-1 ISG) were also obtained from InvivoGen.
  • HEK-Blue cell lines were routinely grown in DMEM, 4.5 g/1 glucose supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 100 mg/ml Normocin, 2 mM L-glutamine and selection antibiotics (HEK-Blue selection for TLR2 and TLR4, 10 pg/ml of blasticidin and 100 mg/ml of Zeocin for TLR7 and TLR9).
  • HEK-Blue cells were passaged at 1 : 10 dilution in DMEM media containing the respective selection antibiotics by detaching in pre warmed PBS and used up to passage 20 according to the manufacturer’s instructions.
  • THP-1 ISG cells were maintained in RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum (30 min at 56°C), 100 pg/ml Normocin, Pen-Strep (100 U/ml-100 pg/ml) routinely passaged at 1 : 10 dilution.
  • TLR agonist activity HEK-Blue cells were seeded in 96-well plates at 40,000 cells/well in selection media and incubated for 48hr at 37°C in a CO2.
  • THP-1 ISG cells were seeded at 100,000 cells/well in selection media then differentiated with 20 nM PMA for 72 hr.
  • the selection media of both cell types was replaced with media without antibiotics (DMEM + FBS for HEK-cells and RPMI + 10% FBS for THP-1 cells) then incubated with Bifido Strain A at multiplicity of infection (MOI) of 1, 10, and 100, or TLR ligand controls, for 24 hr at 37°C in CO2 incubator.
  • MOI multiplicity of infection
  • TLR or STING agonist activity was assessed by measuring reporter SEAP activity using QUANTI-blue.
  • Cell supernatants were diluted 1 : 10 in QUANTI-Blue, incubated for lhr at 37°C; the OD630nm was determine using a microplate reader.

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Abstract

L'invention concerne des procédés et des compositions associés à l'induction d'un ou plusieurs des effets immunitaires décrits par administration orale d'une composition pharmaceutique contenant des bactéries du genre Bifidobacterium.
PCT/US2020/044402 2019-08-01 2020-07-31 Induction d'effets immunitaires à l'aide de bactéries du genre bifidobacterium WO2021022110A1 (fr)

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EP4046643A1 (fr) * 2021-02-23 2022-08-24 University College Cork - National University of Ireland, Cork Procédé de traitement ou de prévention du cancer colorectal
WO2023036214A1 (fr) * 2021-09-10 2023-03-16 香港中文大学 Composition pour la prévention, le traitement ou le contrôle du risque de tumeurs colorectales
WO2023118868A1 (fr) * 2021-12-21 2023-06-29 Quadram Institute Bioscience Bifidobacterium et ses compositions pour le traitement du cancer du sein
WO2023141119A1 (fr) * 2022-01-18 2023-07-27 The Regents Of The University Of California Compositions bactériennes et méthodes de traitement du cancer et de maladies immunitaires

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EP4046643A1 (fr) * 2021-02-23 2022-08-24 University College Cork - National University of Ireland, Cork Procédé de traitement ou de prévention du cancer colorectal
WO2022180135A1 (fr) 2021-02-23 2022-09-01 University College Cork - National University Of Ireland, Cork Procédé de traitement ou de prévention du cancer colorectal
WO2023036214A1 (fr) * 2021-09-10 2023-03-16 香港中文大学 Composition pour la prévention, le traitement ou le contrôle du risque de tumeurs colorectales
WO2023118868A1 (fr) * 2021-12-21 2023-06-29 Quadram Institute Bioscience Bifidobacterium et ses compositions pour le traitement du cancer du sein
WO2023141119A1 (fr) * 2022-01-18 2023-07-27 The Regents Of The University Of California Compositions bactériennes et méthodes de traitement du cancer et de maladies immunitaires

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