WO2019169138A1 - Compositions et méthodes de traitement du cancer à l'aide de paraclostridium benzoelyticum - Google Patents

Compositions et méthodes de traitement du cancer à l'aide de paraclostridium benzoelyticum Download PDF

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WO2019169138A1
WO2019169138A1 PCT/US2019/020061 US2019020061W WO2019169138A1 WO 2019169138 A1 WO2019169138 A1 WO 2019169138A1 US 2019020061 W US2019020061 W US 2019020061W WO 2019169138 A1 WO2019169138 A1 WO 2019169138A1
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carcinoma
cancer
cell
protein
bacteria
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PCT/US2019/020061
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English (en)
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Brian Goodman
Maria Sizova
Holly PONICHTERA
Peter SANDY
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Evelo Biosciences, Inc.
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Publication of WO2019169138A1 publication Critical patent/WO2019169138A1/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
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/54—Pancreas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/55—Lung
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/56—Kidney
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • compositions and Methods for Treating Cancer Using Paraclostridium benzoelyticum Compositions and Methods for Treating Cancer Using Paraclostridium benzoelyticum
  • a subject e.g ., a human subject
  • administering a bacterial composition comprising Paraclostridium benzoelyticum, and/or a derivative of such bacteria (e.g., extracellular vesicles (EVs) and/or pharmacologically active biomasses (PhABs)), as well as methods of making and/or identifying such bacteria suitable for use in the pharmaceutical compostions disclosed herein.
  • a bacterial composition comprising Paraclostridium benzoelyticum, and/or a derivative of such bacteria (e.g., extracellular vesicles (EVs) and/or pharmacologically active biomasses (PhABs)
  • EVs extracellular vesicles
  • PhABs pharmacologically active biomasses
  • Paraclostridium benzoelyticum Strain A is provided as SEQ ID NO: 1.
  • EVs and PhABs are derived from or produced by Paraclostridium benzoelyticum.
  • the Paraclostridium benzoelyticum is a strain comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity) to the nucleotide sequence of (e.g., genomic sequence, 16S sequence,
  • the Paraclostridium benzoelyticum is a strain comprising a 16S sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 1.
  • the administration of the bacterial composition induces an immune response against a tumor in the subject.
  • the administration of the bacterial composition treats the cancer in the subject.
  • the administration augments a tumor microenvironment in the subject.
  • the cancer is a colorectal carcinoma.
  • compositions comprising Paraclostridium benzoelyticum provided herein (e.g., a killed bacterium, a live bacterium and/or an attenuated bacterium) and/or a product of such bacteria (e.g., extracellular vesicles (EVs) and/or pharmacologically active biomasses (PhABs)).
  • Paraclostridium benzoelyticum bacteria is Paraclostridium benzoelyticum Strain A.
  • benzoelyticum is a strain comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the nucleotide sequence of (e.g., genomic sequence, 16S sequence, CRISPR sequence) of
  • Paraclostridium benzoelyticum Strain A the Paraclostridium
  • benzoelyticum is a strain comprising a 16S sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 1. In some embodiments, at least 50%, 60%, 70%, 80%, 85%, 90%,
  • the bacteria in the bacterial composition are of the Paraclostridium benzoelyticum strain.
  • all or substantially all of the bacteria in the bacterial formulation are of the Paraclostridium
  • the bacterial formulation comprises at least 1 x 10 5 ,
  • the bacterial and/or pharmaceutical composition comprises EVs, and/or PhABs (e.g., whole cells, fractions of
  • EVs extracellular vesicles
  • the bacterial compositions comprise both Paraclostridium benzoelyticum EVs and whole Paraclostridium benzoelyticum (e.g., live bacteria, killed bacteria, attenuated bacteria).
  • bacterial and/or pharmaceutical compositions comprising Paraclostridium benzoelyticum bacteria in the absence (or substantially in the absence) of Paraclostridium benzoelyticum EVs.
  • the bacterial compositions comprise both Paraclostridium benzoelyticum EVs and whole Paraclostridium benzoelyticum (e.g., live bacteria, killed bacteria, attenuated bacteria).
  • bacterial and/or pharmaceutical compositions comprising Paraclostridium benzoelyticum bacteria in the absence (or substantially in the absence) of Paraclostridium benzoelyticum EVs.
  • the bacterial and/or pharmaceutical compositions comprising Paraclostridium benzoelyticum bacteria in the absence (or substantially in the absence) of Paraclostridium benzoelytic
  • compositions comprise Paraclostridium benzoelyticum EVs in the absence (or substantially in the absence) of Paraclostridium benzoelyticum bacteria.
  • PhABs made from and/or comprising an Paraclostridium benzoelyticum strain provided herein.
  • the PhABs comprise whole cells, fractions of cells, supernatant from fermentation, fractions of supernatant and/or extracellular vesicles derived from bacteria described herein.
  • the bacterial compositions provided herein comprise a PhAB derived from a Paraclostridium benzoelyticum strain PhAB provided herein.
  • the bacterial and/or pharmaceutical composition is administered orally, intravenously, intratumorally, or subcutaneously.
  • the bacterial composition is administered in 2 or more doses (e.g., 3 or more, 4 or more or 5 or more doses).
  • the administration to the subject of the two or more doses are separated by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days,
  • a second bacterial strain is administered as part of an ecological consortium.
  • the bacterial and/or pharmaceutical composition comprises a specific ratio of Paraclostridium benzoelyticum bacteria to Paraclostridium benzoelyticum EV particles.
  • the pharmaceutical composition comprises a specific ratio of Paraclostridium benzoelyticum bacteria to Paraclostridium benzoelyticum EV particles.
  • composition comprises at least 1 Paraclostridium benzoelyticum bacterium for every 1, 1.1, 1.2,
  • the bacterial and/or pharmaceutical composition comprises about 1 Paraclostridium benzoelyticum bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,
  • the bacterial and/or pharmaceutical composition comprises no more than 1 Paraclostridium benzoelyticum bacteriafor every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3,
  • the bacterial and/or pharmaceutical composition comprises at least 1
  • Paraclostridium benzoelyticum EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2,
  • benzoelyticum EV particle for every 1, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3,
  • the bacterial and/or pharmaceutical composition comprises no more than 1 Paraclostridium benzoelyticum EV particle for every 1, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3,
  • provided herein are methods of treating a subject who has cancer, comprising administering to the subject a bacterial and/or pharmaceutical composition comprising Paraclostridium benzoelyticum provided herein (e.g., a killed bacterium, a live bacterium and/or an attenuated bacterium) and/or a derivative of such bacteria (e.g., extracellular vesicles (EVs) and/or pharmacologically active biomasses (PhABs)).
  • the Paraclostridium benzoelyticum bacteria is Paraclostridium benzoelyticum Strain A.
  • the Paraclostridium benzoelyticum is a strain comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., 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 (e.g., genomic sequence, 16S sequence, CRISPR sequence) of Paraclostridium benzoelyticum Strain A.
  • sequence identity e.g., 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
  • the Paraclostridium benzoelyticum is a strain comprising a 16S sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., 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 SEQ ID NO: 1.
  • sequence identity e.g., 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%, 60%, 70%, 80%, 85%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bacteria in the bacterial composition are of the Paraclostridium benzoelyticum strain. In some embodiments, all or substantially all of the bacteria in the bacterial formulation are of the Paraclostridium benzoelyticum strain.
  • the bacterial formulation comprises at least 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 2 x 10 6 , 3 x 10 6 , 4 x 10 6 , 5 x 10 6 , 6 x 10 6 , 7 x 10 6 , 8 x 10 6 , 9 x 10 6 , 1 x 10 7 , 2 x 10 7 , 3 x 10 7 , 4 x 10 7 , 5 x 10 7 , 6 x 10 7 , 7 x 10 7 , 8 x 10 7 , 9 x 10 7 , 1 x 10 8 , 2 x 10 8 , 3 x 10 8 , 4 x 10 8 , 5 x 10 8 , 6 x 10 8 , 7 x 10 8 , 8 x 10 8 , 9 x l0 8 or 1 x 10 9 colony forming units of the Paraclostridium benzoelyticum strain.
  • the method further comprises administering to the subject an antibiotic.
  • the method further comprises administering to the subject one or more other cancer therapies.
  • the other cancer therapy is the surgical removal of a tumor, the administration of a chemotherapeutic agent, the administration of radiation therapy, the administration of an antibiotic, the administration of a cancer immunotherapy (e.g ., an immune checkpoint inhibitor, a cancer-specific antibody, a cancer vaccine, a primed antigen presenting cell, a cancer-specific T cell, a cancer-specific chimeric antigen receptor (CAR) T cell, an immune activating protein, an adjuvant), and/or the administration of another therapeutic bacterial strain.
  • a cancer immunotherapy e.g ., an immune checkpoint inhibitor, a cancer-specific antibody, a cancer vaccine, a primed antigen presenting cell, a cancer-specific T cell, a cancer-specific chimeric antigen receptor (CAR) T cell, an immune activating protein, an adjuvant
  • the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal (e.g., 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).
  • a non-human mammal e.g., 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.
  • Figure 1 shows the efficacy of an exemplary Paraclostridium benzoelyticum strain (Paraclostridium benzoelyticum Strain A) compared to that of intraperitoneally (i.p.) administered anti-PD-l or vehicle in a mouse colorectal carcinoma model.
  • Figure 2 shows the efficacy of an exemplary Paraclostridium benzoelyticum strain (Paraclostridium benzoelyticum Strain A) compared to that of intraperitoneally (i.p.) administered anti-PD-l or vehicle in a mouse colorectal carcinoma model at day 7.
  • Figure 3 shows the efficacy of an exemplary Paraclostridium benzoelyticum strain (Paraclostridium benzoelyticum Strain A) compared to that of intraperitoneally (i.p.) administered anti-PD-l or vehicle in a mouse colorectal carcinoma model at day 9.
  • Figure 4 shows the efficacy of an exemplary Paraclostridium benzoelyticum strain (Paraclostridium benzoelyticum Strain A) compared to that of intraperitoneally (i.p.) administered anti-PD-l or vehicle in a mouse colorectal carcinoma model at day 11.
  • Figure 5 shows the efficacy of the combination of an exemplary Paraclostridium benzoelyticum strain ⁇ Paraclostridium benzoelyticum Strain A) and anti -PD- 1 compared to that of intraperitoneally (i.p.) administered anti-PD-l alone or vehicle in a mouse colorectal carcinoma model.
  • Figure 6 shows the efficacy of the combination of an exemplary Paraclostridium benzoelyticum strain ⁇ Paraclostridium benzoelyticum Strain A) and anti-PD-l compared to that of intraperitoneally (i.p.) administered anti-PD-l alone or vehicle in a mouse colorectal carcinoma model at day 21.
  • compositions for treating cancer in a subject comprising administering to the subject a
  • Paraclostridium benzoelyticum e.g., Paraclostridium benzoelyticum Strain A
  • a derivative of such bacteria e.g., extracellular vesicles (EVs) and/or pharmacologically active biomasses (PhABs)
  • EVs extracellular vesicles
  • PhABs pharmacologically active biomasses
  • 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.
  • administering broadly refers to a route of administration of a composition to a subject.
  • routes of administration include oral administration, rectal administration, topical administration, inhalation (nasal) or injection.
  • Administration by injection includes intravenous (IV), intramuscular (IM), intratumoral (IT) and subcutaneous (SC) administration.
  • compositions described herein can be administered in any form by any effective route, including but not limited to intratumoral, oral, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal ( e.g ., using any standard patch), intradermal, ophthalmic, (intra)nasally, local, non-oral, such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, (trans)rectal, vaginal, intra-arterial, and intrathecal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), intravesical, intrapulmonary, intraduodenal, intragastrical, and intrabronchial.
  • transdermal e.g ., using any standard patch
  • intradermal e.g ., using any standard patch
  • intradermal e.g ., using any standard patch
  • compositions described herein are administered orally, rectally, intratumorally, topically, intravesically, by injection into or adjacent to a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously.
  • 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.
  • the terms“antigen binding fragment” and“antigen-binding portion” of an antibody 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.
  • 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,
  • Plasmacytoma colorectal cancer, rectal cancer, Merkel Cell carcinoma, salivary gland carcinoma, 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.
  • the term“ecological consortium” is a group of bacteria which trades metabolites and positively co-regulates one another, in contrast to two bacteria which 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, I, 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, lOO-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 immune cells, bacterial cells, stromal cells, myeloid derived suppressor cells, fibroblasts, metabolites, and cytokines.
  • ‘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.
  • a substance is“pure” if it is 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.
  • LPS mutant or bpopolysaccharide mutant broadly refers to selected bacteria that comprises loss of LPS. Loss of LPS might be due to mutations or disruption to genes involved in lipid A biosynthesis, such as IpxA, IpxC, and IpxD. Bacteria comprising LPS mutants can be resistant to aminoglycosides and polymyxins (polymyxin B and cobstin).
  • “Operational taxonomic units” and“OTU(s)” refer 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.
  • a gene is“overexpressed” in a bacteria if it is expressed at a higher level in an engineered bacteria under at least some conditions than it is expressed by a wild-type bacteria of the same species under the same conditions.
  • a gene is “underexpressed” in a bacteria if it is expressed at a lower level in an engineered bacteria under at least some conditions than it is expressed by a wild-type bacteria of the same species under the same conditions.
  • the terms“pharmacologically active biomass” or“PhABs” broadly refer to a composition containing pharmacologically active bacterial components, for example, derived from lysed or otherwise disrupted cells.
  • 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.
  • a substance is“pure” if it is substantially free of other components.
  • the terms“purify,”“purifying” and“purified” refer to a EV 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.
  • An EV may be considered purified if it is isolated at or after production, such as from one or more other bacterial components, 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“purified.”
  • purified EVs 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.
  • EV compositions and the microbial components thereof are, e.g., purified from residual habitat products.
  • the term“purified EV composition” or“EV composition” refer to a preparation that includes EVs that have been separated from at least one associated substance found in a source material ( e.g . separated from at least one other bacterial component) or any material associated with the EVs in any process used to produce the EV composition. It also refers to a composition that has been significantly enriched or concentrated. In some
  • the EVs are concentrated by 2 fold, 3-fold, 4-fold, 5-fold, 10-fold, lOO-fold, 1000- fold, 10, 000-fold or more than 10,000 fold.
  • “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).
  • specific binding applies more broadly to a two component system where one component is a protein, lipid, or carbohydrate or combination thereof and engages with the second component which is a protein, lipid, carbohydrate or combination thereof in a specific way.
  • 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.
  • 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 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, plasmacytoma, colorectal cancer, rectal cancer, Merkel Cell carcinoma, salivary gland carcinoma, ovarian cancer, and/or melanoma.
  • the patients may have tumors that show enhanced macropinocytosis with the underlying genomics of this process including Ras activation.
  • patients suffer from other cancers.
  • the subject has undergone a cancer therapy.
  • 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.
  • compositions comprising Paraclostridium benzoelyticum and/or a derivative of such bacteria (e.g., extracellular vesicles (EVs) and/or pharmacologically active biomasses (PhABs)), and methods of using bacterial compositions comprising Paraclostridium benzoelyticum.
  • the Paraclostridium benzoelyticum bacteria is AgaParaclostridium benzoelyticum thobaculumStmin A.
  • the, Paraclostridium benzoelyticum is a strain comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g ., 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 (e.g., genomic sequence, 16S sequence, CRISPR sequence) of Paraclostridium benzoelyticum Strain A.
  • sequence identity e.g., 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
  • the Paraclostridium benzoelyticum is a strain comprising a 16S sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., 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 SEQ ID NO: 1 (i.e., as provided in Table 1).
  • sequence identity e.g., 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
  • benzoelyticum Strain A was deposited on _ , with the American Type Culture Collection
  • 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).
  • the bacteria described herein are modified to improve bacterial manufacturing (e.g ., higher oxygen tolerance, improved freeze-thaw tolerance, shorter generation times).
  • primers or probes capabable of detecting and/or distinguishing the Paraclostridium benzoelyticum strain.
  • the primer or probe comprises a nucleotide sequence complementary to a nucleic acid sequence (e.g., a genomic sequence, a 16S sequence, a CRISPR sequence) of Paraclostridium
  • the probe or primer comprises a sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length that is complementary a nucleic acid sequence (e.g., a genomic sequence, a 16S sequence, a CRISPR sequence) of Paraclostridium benzoelyticum Strain A.
  • a nucleic acid sequence e.g., a genomic sequence, a 16S sequence, a CRISPR sequence
  • the probe or primer comprises a nucleic acid sequence that is complementary to a sequence of SEQ ID NO: 1. In some embodiments, the probe or primer comprises a sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length that is complementary to SEQ ID NO: 1. In some embodiments, provided herein is a method of detecting a Paraclostridium benzoelyticum strain comprising contacting a sample with a probe or primer provided herein.
  • th e, Paraclostridium benzoelyticum EVs described herein can be prepared using any method known in the art.
  • the, Paraclostridium benzoelyticum EVs are prepared without an EV purification step.
  • Paraclostridium benzoelyticum bacteria described herein are killed using a method that leaves the
  • Paraclostridium benzoelyticum EVs intact and the resulting bacterial components, including the EVs, are used in the methods and compositions described herein.
  • the Paraclostridium benzoelyticum bacteria are killed using an antibiotic (e.g., using an antibiotic described herein).
  • the Paraclostridium benzoelyticum bacteria are killed using UV irradiation.
  • the Paraclostridium benzoelyticum bacteria are heat-killed.
  • the EVs described herein are purified from one or more other bacterial components. Methods for purifying EVs from bacteria are known in the art.
  • EVs are prepared from bacterial cultures using methods described in S.
  • the bacteria are cultured to high optical density and then centrifuged to pellet bacteria (e.g . , at 10,000 x g for 30 min at 4°C, at 15,500 x g for 15 min at 4°C).
  • the culture supernatants are then passed through filters to exclude intact bacterial cells (e.g., a 0.22 pm filter).
  • the supernatants are then subjected to tangential flow filtration, during which the supernatant is concentrated, species smaller than 100 kDa are removed, and the media is partially exchanged with PBS.
  • filtered supernatants are centrifuged to pellet bacterial EVs (e.g., at 100,000-150,000 x g for 1-3 hours at 4°C, at 200,000 x g for 1-3 hours at 4°C).
  • the EVs are further purified by resuspending the resulting EV pellets (e.g., in PBS), and applying the resuspended EVs to an Optiprep (iodixanol) gradient or gradient (e.g., a 30-60% discontinuous gradient, a 0-45% discontinuous gradient), followed by centrifugation (e.g., at 200,000 x g for 4-20 hours at 4°C).
  • EV bands can be collected, diluted with PBS, and centrifuged to pellet the EVs (e.g., at 150,000 x g for 3 hours at 4°C, at 200,000 x g for 1 hour at 4°C).
  • the purified EVs can be stored, for example, at -80°C or - 20°C until use.
  • the EVs are further purified by treatment with DNase and/or proteinase K.
  • cultures of Paraclostridium benzoelyticum bacteria disclosed herein can be centrifuged at 11,000 x g for 20-40 min at 4°C to pellet the bacteria.
  • Culture supernatants may be passed through a 0.22 pm filter to exclude intact bacterial cells.
  • Filtered supernatants may then be concentrated using methods that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration.
  • ammonium sulfate precipitation 1.5-3 M ammonium sulfate can be added to filtered supernatant slowly, while stirring at 4°C.
  • Precipitations can be incubated at 4°C for 8-48 hours and then centrifuged at 11,000 x g for 20-40 min at 4°C. The resulting pellets contain Paraclostridium benzoelyticum EVs and other debris. Using ultracentrifugation, filtered supernatants can be centrifuged at 100,000-200,000 x g for 1-16 hours at 4°C. The pellet of this centrifugation contains Paraclostridium benzoelyticum EVs and other debris such as large protein complexes.
  • supernatants can be filtered so as to retain species of molecular weight > 50 or 100 kDa.
  • EVs can be obtained from Paraclostridium benzoelyticum cultures continuously during growth, or at selected time points during growth, for example, by connecting a bioreactor to an alternating tangential flow (ATF) system (e.g ., XCell ATF from Repligen).
  • ATF alternating tangential flow
  • the ATF system retains intact cells (>0.22 um) in the bioreactor, and allows smaller components (e.g., EVs, free proteins) to pass through a filter for collection.
  • the system may be configured so that the ⁇ 0.22 um filtrate is then passed through a second filter of 100 kDa, allowing species such as EVs between 0.22 um and 100 kDa to be collected, and species smaller than 100 kDa to be pumped back into the bioreactor.
  • the system may be configured to allow for medium in the bioreactor to be replenished and/or modified during growth of the culture. EVs collected by this method may be further purified and/or concentrated by ultracentrifugation or filtration as described above for filtered supernatants.
  • EVs obtained by methods provided herein may be further purified by size-based column chromatography, by affinity chromatography, by ion-exchange chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column.
  • Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in PBS and 3 volumes of 60% Optiprep are added to the sample. In some embodiments, if filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep.
  • Samples are applied to a 0-45% discontinuous Optiprep gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C, e.g. 4-24 hours at 4°C.
  • Samples are applied to a 0-45% discontinuous Optiprep gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C, e.g. 4-24 hours at 4°C.
  • EVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 um filter to exclude intact cells. To further increase purity, isolated EVs may be DNase or proteinase K treated.
  • EVs are processed as described previously (G. Norheim, et al. PLoS ONE. 10(9): e0l34353 (2015)). Briefly, after sucrose gradient centrifugation, bands containing EVs are resuspended to a final concentration of 50 pg/mL in a solution containing 3% sucrose or other solution suitable for in vivo injection known to one skilled in the art. This solution may also contain adjuvant, for example aluminum hydroxide at a concentration of 0-0.5% (w/v). In some embodiments, for preparation of EVs used for in vivo injections, EVs in PBS are sterile-filtered to ⁇ 0.22 um.
  • samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 using filtration (e.g. Amicon Eiltra columns), dialysis, or
  • the sterility of the EV preparations can be confirmed by plating a portion of the EVs onto agar medium used for standard culture of the bacteria used in the generation of the EVs and incubating using standard conditions.
  • select EVs are isolated and enriched by chromatography and binding surface moieties on EVs.
  • select EVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.
  • the PhABs described herein can be prepared using any method known in the art.
  • the PhABs described herein are prepared by fractionation.
  • Bacterial cells and/or supernatants from cultured bacteria cells are fractionated into various pharmacologically active biomass (PhABs) and/or products derived therefrom. Bacterial cells and/or supernatants are fractionated using materials and methods known in the art (see e.g.
  • PhABs obtained by methods provided herein may be further purified by size based column chromatography, by affinity chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation is used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration is used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Eiltra column.
  • Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation is used to concentrate the filtered supernatants, pellets are resuspended in 35% Optiprep in PBS. In some embodiments, if filtration is used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C.
  • PhABs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 um filter to exclude intact cells. To further increase purity, isolated PhABs may be DNase or proteinase K treated.
  • PhABs used for in vivo injections purified PhABs are processed as described previously (G. Norheim, et al. PLoS ONE. 10(9): eO 134353 (2015)). Briefly, after sucrose gradient centrifugation, bands containing PhABs are resuspended to a final concentration of 50 pg/mL in a solution containing 3% sucrose or other solution suitable for in vivo injection known to one skilled in the art. This solution may also contain adjuvant, for example aluminum hydroxide at a concentration of 0-0.5% (w/v).
  • adjuvant for example aluminum hydroxide at a concentration of 0-0.5% (w/v).
  • samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 using filtration (e.g. Amicon Ultra columns), dialysis, or
  • the sterility of the PhAB preparations can be confirmed by plating a portion of the PhABs onto agar medium used for standard culture of the bacteria used in the generation of the PhABs and incubating using standard conditions.
  • select PhABs are isolated and enriched by chromatography and binding surface moieties on PhABs.
  • select PhABs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.
  • compositions comprising Paraclostridium benzoelyticum provided herein (e.g. Paraclostridium benzoelyticum Strain A) and/or a derivative of such bacteria (e.g., extracellular vesicles (EVs) and/or pharmacologically active biomasses (PhABs)).
  • Paraclostridium benzoelyticum e.g. Paraclostridium benzoelyticum Strain A
  • a derivative of such bacteria e.g., extracellular vesicles (EVs) and/or pharmacologically active biomasses (PhABs)
  • EVs extracellular vesicles
  • PhABs pharmacologically active biomasses
  • Paraclostridium benzoelyticum bacteria is Paraclostridium benzoelyticum Strain A.
  • the Paraclostridium benzoelyticum is a strain comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., 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 (e.g., genomic sequence, 16S sequence, CRISPR sequence) of Paraclostridium benzoelyticum Strain A.
  • sequence identity e.g., 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
  • the Paraclostridium benzoelyticum is a strain comprising a 16S sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., 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 SEQ ID NO: 1.
  • the bacterial formulation comprises a bacterium and/or a combination of bacteria described herein and a pharmaceutically acceptable carrier.
  • the bacteria in the bacterial and/or pharmaceutical composition are the Paraclostridium benzoelyticum strain.
  • substantially all of the bacteria in the bacterial composition are the
  • the bacterial 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 bacterial and/or pharmaceutical compositions described herein may include only the Paraclostridium benzoelyticum strain or may include two or more species or strains of bacteria, including the, Paraclostridium benzoelyticum strain.
  • the compositions provided herein can be included in the compositions provided herein.
  • the bacterial and/or pharmaceutical composition comprises a killed bacterium, a live bacterium and/or an attenuated bacterium.
  • Bacteria may be heat-killed by pasteurization, sterilization, high temperature treatment, spray cooking and/or spray drying (heat treatments can be performed at 50°C, 65°C, 85°C or a variety of other temperatures and/or a varied amount of time).
  • Bacteria may also be killed or inactivated using g-irradiation (gamma irradiation), exposure to UV light, formalin-inactivation, and/or freezing methods, or a combination thereof.
  • the bacteria may be exposed to 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, or 50kGy of radiation prior to administration.
  • bacteria are killed using gamma irradiation.
  • the bacteria are killed or inactivated using electron irradiation (e.g., beta radiation) or x-ray irradiation.
  • the bacteria in the bacterial and/or pharmaceutical composition described herein are killed using a method that leaves the disease modulating activity of the bacteria intact and the resulting bacterial components are used in the methods and compositions described herein.
  • the bacteria in the composition described herein are killed using an antibiotic (e.g., using an antibiotic described herein).
  • the bacteria in the composition described herein are killed using UV irradiation.
  • Bacteria may be grown to various growth phases and tested for efficacy at different dilutions and at different points during the growth phase. For example, bacteria may be tested for efficacy following administration at stationary phase (including early or late stationary phase), or at various timepomts during exponential phase. In addition to inactivation by various methods, bacteria may be tested for efficacy using different ratios of live versus inactivated cells, or different ratios of cells at various growth phases, and/or EVs prepared from bacteria harvested during one or more of the growth phases.
  • bacterial and/or pharmaceutical compositions comprising Paraclostridium benzoelyticum EVs and/ or Paraclostridium benzoelyticum bacteria, provided herein (e.g ., an EV composition), such as those disclosed in ET.S. Provisional Patent Application No. 62/578,559, hereby incorporated by reference in its entirety.
  • the EV composition comprises an EV and/or a combination of EVs described herein and a pharmaceutically acceptable carrier.
  • the bacterial and/or pharmaceutical compositions comprise
  • Paraclostridium benzoelyticum EVs substantially or entirely free of bacteria.
  • the pharmaceutical compositions comprise both Paraclostridium benzoelyticum EVs and whole Paraclostridium benzoelyticum bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria).
  • the pharmaceutical compositions comprise Paraclostridium benzoelyticum bacteria that is substantially or entirely free of EVs.
  • the bacterial and/or pharmaceutical composition comprises at least 1 Paraclostridium benzoelyticum bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
  • the bacterial and/or pharmaceutical composition comprises about 1 Paraclostridium benzoelyticum EV particles.
  • the bacterial and/or pharmaceutical composition comprises about 1 Paraclostridium benzoelyticum EV particles.
  • the bacterial and/or pharmaceutical composition comprises a certain ratio of Paraclostridium benzoelyticum bacteria particles to Paraclostridium benzoelyticum EV particles.
  • the number of Paraclostridium benzoelyticum bacteria particles can be based on actual particle number or (if the bacteria is live) the number of CFUs.
  • the particle number can be established by combining a set number of purified Paraclostridium benzoelyticum EVs with a set number of purified Paraclostridium benzoelyticum bacterium, by modifying the growth conditions under which the Paraclostridium benzoelyticum bacteria are cultured, or by modifying the Paraclostridium benzoelyticum bacteria itself to produce more or fewer
  • EVs and/ or Paraclostridium benzoelyticum bacteria present in a bacterial sample electron microscopy (e.g EM of ultrathin frozen sections) can be used to visualize the vesicles and bacteria and count their relative numbers.
  • electron microscopy e.g EM of ultrathin frozen sections
  • combinations of nanoparticle tracking analysis (NTA), Coulter counting, and dynamic light scattering (DLS) or a combination of these techniques can be used.
  • NTA and the Coulter counter count particles and show their sizes. DLS gives the size distribution of particles, but not the concentration. Bacteria frequently have diameters of 1-2 um. The full range is 0.2-20 um. Combined results from Coulter counting and NTA can reveal the numbers of bacteria in a given sample.
  • Coulter counting reveals the numbers of particles with diameters of 0.7-10 um.
  • NTA reveals the numbers of particles with diameters of 50-1400 nm.
  • the Coulter counter alone can reveal the number of bacteria in a sample.
  • EVs are 20-250 nm in diameter. NTA will allow us to count the numbers of particles that are 50-250 nm in diameter.
  • DLS reveals the distribution of particles of different diameters within an approximate range of 1 nm - 3 um.
  • the bacterial and/or pharmaceutical composition comprises no more than 1 Paraclostridium benzoelyticum bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
  • the bacterial and/or pharmaceutical composition comprises at least 1 Paraclostridium benzoelyticum EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8.
  • the bacterial and/or pharmaceutical composition comprises about 1 Paraclostridium benzoelyticum EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
  • the bacterial and/or pharmaceutical composition comprises no more than 1 Paraclostridium benzoelyticum EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5,
  • the protein in the bacterial and/or pharmaceutical composition is Paraclostridium benzoelyticum bacteria protein.
  • composition is Paraclostridium benzoelyticum EV protein.
  • composition is Paraclostridium benzoelyticum bacteria protein.
  • lipids in the bacterial and/or pharmaceutical composition are Paraclostridium benzoelyticum EV lipids.
  • lipids in the bacterial and/or pharmaceutical composition are Paraclostridium benzoelyticum bacteria lipids.
  • lipids in the bacterial and/or pharmaceutical composition are Paraclostridium benzoelyticum EV lipids.
  • lipids in the bacterial and/or pharmaceutical composition are Paraclostridium benzoelyticum bacteria lipids.
  • compositions are Paraclostridium benzoelyticum EV lipids.
  • compositions are Paraclostridium benzoelyticum bacteria lipids.
  • the Paraclostridium benzoelyticum EVs in the bacterial and/or pharmaceutical composition are purified from one or more other bacterial components.
  • the bacterial and/or pharmaceutical composition further comprises other bacterial components.
  • the bacterial and/or pharmaceutical composition comprise bacteria cells.
  • the bacterial and/or pharmaceutical compositions disclosed herein may be specially formulated for administration in solid or liquid form, including those adapted for oral or rectal administration.
  • the composition described herein may be a bacterial and/or pharmaceutical composition, a dietary supplement, and/or a food product (e.g., a food or beverage).
  • a food product e.g., a food or beverage
  • the food product is an animal feed.
  • the bacterial and/or pharmaceutical composition for oral administration described herein comprises an additional component that enables efficient delivery of the bacteria to the colon.
  • bacterial and/or pharmaceutical preparation that enables the delivery of the bacteria to the colon can be used.
  • formulations include pH sensitive compositions, such as buffered sachet formulations or enteric polymers that release their contents when the pH becomes alkaline after the enteric polymers pass through the stomach.
  • pH sensitive composition can be a polymer whose pH threshold of the decomposition of the composition is between about 6.8 and about 7.5.
  • a bacterial and/or pharmaceutical composition useful for delivery of the bacteria to the colon is one that ensures the delivery to the colon by delaying the release of the bacteria by approximately 3 to 5 hours, which corresponds to the small intestinal transit time.
  • the pharmaceutical composition for delayed release includes a hydrogel shell. The hydrogel is hydrated and swells upon contact with gastrointestinal fluid, with the result that the contents are effectively released (released predominantly in the colon).
  • Delayed release dosage units include bacteria-containing compositions having a material which coats or selectively coats the bacteria. Examples of such a selective coating material include in vivo degradable polymers, gradually hydrolyzable polymers, gradually water-soluble polymers, and/or enzyme degradable polymers.
  • a wide variety of coating materials for efficiently delaying the release is available and includes, for example, cellulose-based polymers such as
  • hydroxypropyl cellulose acrylic acid polymers and copolymers such as methacrylic acid polymers and copolymers, and vinyl polymers and copolymers such as polyvinylpyrrolidone.
  • composition enabling the delivery to the colon further include bioadhesive compositions which specifically adhere to the colonic mucosal membrane (for example, a polymer described in the specification of Ei.S. Pat. No. 6,368,586, hereby incorporated by reference) and compositions into which a protease inhibitor is incorporated for protecting particularly a biopharmaceutical preparation in the gastrointestinal tracts from decomposition due to an activity of a protease.
  • bioadhesive compositions which specifically adhere to the colonic mucosal membrane
  • compositions into which a protease inhibitor is incorporated for protecting particularly a biopharmaceutical preparation in the gastrointestinal tracts from decomposition due to an activity of a protease for example, a polymer described in the specification of Ei.S. Pat. No. 6,368,586, hereby incorporated by reference
  • An example of a system enabling the delivery to the colon is a system of delivering a composition to the colon by pressure change in such a way that the contents are released by utilizing pressure change caused by generation of gas in bacterial fermentation at a distal portion of the stomach.
  • a system is not particularly limited, and a more specific example thereof is a capsule which has contents dispersed in a suppository base and which is coated with a hydrophobic polymer (for example, ethyl cellulose).
  • Another example of the system enabling the delivery to the colon is a system of delivering a composition to the colon, the system being specifically decomposed by an enzyme (for example, a carbohydrate hydrolase or a carbohydrate reductase) present in the colon.
  • an enzyme for example, a carbohydrate hydrolase or a carbohydrate reductase
  • Such a system is not particularly limited, and more specific examples thereof include systems which use food components such as non-starch polysaccharides, amylose, xanthan gum, and azopolymers.
  • Probiotic formulations containing Paraclostridium benzoelyticum are provided as encapsulated, enteric coated, or powder forms, with doses ranging up to 10 11 cfu (e.g., up to 10 10 cfu).
  • the composition comprises 5 x 10 11 cfu of the Paraclostridium benzoelyticum strain and 10% (w/w) corn starch in a capsule.
  • the capsule is enteric coated for duodenal release at pH5.5
  • the capsule is enteric coated for duodenal release at pH 5.5.
  • the composition comprises a powder of freeze-dried Paraclostridium benzoelyticum which is deemed“Qualified
  • the composition is stable at frozen or refrigerated temperature.
  • Methods for producing bacterial and/or pharmaceuticalcompositions may include three main processing steps. The steps are: organism banking, organism production, and preservation. In certain embodiments, a sample that contains an abundance of Paraclostridium benzoelyticum may be cultured by avoiding an isolation step.
  • the strains included in the bacterial and/or pharmaceutical 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 bacterial 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. 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 bacterial 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).
  • 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.
  • Bacterial production may be conducted using similar culture steps to banking, including medium composition and culture conditions described above. It may be conducted at larger scales of operation, especially for clinical development or commercial production. At larger scales, there may be several subcultivations 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 bacterial composition formulated as provided herein.
  • a filler such as microcrystalline cellulose for consistency and ease of handling, and the bacterial composition formulated as provided herein.
  • bacterial compositions for administration subjects are provided.
  • the bacterial compositions 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, 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,
  • poly ethyleneglycol 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.
  • 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 bacterial composition comprises an enteric coating and/or microcapsule(s) 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. Pub. 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
  • a health food or beverage e.g. a food or beverage
  • a food or beverage for infants e.g., a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group
  • a functional food e.g., a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.
  • 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 which 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. Administration
  • a method of delivering a bacterium, bacterial derivative (e.g ., EVs, and/or PhABs), a bacterial composition and/or a pharmaceutical composition described herein to a subject In some embodiments of the methods provided herein, the bacteria, bacterial derivative and/or composition are administered in conjunction with the administration of a cancer therapeutic. In some embodiments, the bacteria and/or bacterial derivative is co-formulated in a pharmaceutical composition with the cancer therapeutic. In some embodiments, the bacteria and/or bacterial derivative is 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 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, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours after, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days after).
  • the same mode of delivery is used to deliver both the bacteria and/or bacterial derivative and the cancer therapeutic.
  • different modes of delivery are used to administer and/or bacterial derivative the bacteria and the cancer therapeutic.
  • the bacteria and/or bacterial derivative is 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 according to known clinical factors. As is known in the medical arts, dosages for any one patient can depend on many factors, including the subject's species, size, body surface area, age, sex, immunocompetence, and general health, the particular microorganism to be administered, duration and route of administration, the kind and stage of the disease, for example, tumor size, and other compounds such as drugs being administered concurrently. In addition to the above factors, such levels can be affected by the infectivity of the microorganism, and the nature of the microorganism, as can be determined by one skilled in the art.
  • appropriate minimum dosage levels of microorganisms can be levels sufficient for the microorganism to survive, grow and replicate in a tumor or metastasis.
  • the methods of treatment described herein may be suitable for the treatment of a primary tumor, a secondary tumor or metastasis, as well as for recurring tumors or cancers.
  • 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 prevent cancer, delay its onset, or slow or stop its progression or prevent a relapse of a cancer.
  • 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 by conventional range finding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached.
  • An effective dosage and treatment protocol can be determined by routine and conventional means, starting e.g., with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Animal studies are commonly used to determine the maximal tolerable dose ("MTD”) of bioactive agent per kilogram weight. Those skilled in the art regularly extrapolate doses for efficacy, while avoiding toxicity, in other species, including humans.
  • MTD maximal tolerable dose
  • the dosages of the active agents used in accordance with the invention vary depending on the active agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage.
  • the dose should be sufficient to result in slowing, and preferably regressing, the growth of the tumors and most preferably causing complete regression of the cancer.
  • Separate administrations can include any number of two or more administrations
  • the doses may be separated by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
  • the methods provided herein include methods of providing to the subject one or more administrations of a bacterium, where the number of administrations can be determined by monitoring the subject, and, based on the results of the monitoring, determining whether or not to provide one or more additional administrations. Deciding on whether or not to provide one or more additional administrations can be based on a variety of monitoring results, including, but not limited to, indication of tumor growth or inhibition of tumor growth, appearance of new metastases or inhibition of metastasis, the subject's anti-bacterium antibody titer, the subject's anti-tumor antibody titer, the overall health of the subject and/or the weight of the subject.
  • the time period between administrations can be any of a variety of time periods.
  • 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 response; for example, the time period can be more than the time period for a subject to mount an immune response, 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 response, 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 delivery of a cancer therapeutic in combination with the bacteria and/or bacterial derivative described herein reduces the adverse effects and/or improves the efficacy of the cancer therapeutic.
  • the effective dose of a cancer therapeutic described herein is the amount of the therapeutic agent that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, with the least toxicity to the patient.
  • the effective dosage level can be identified using the methods described herein and will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions administered, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • an effective dose of a cancer therapy will be the amount of the therapeutic agent which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the toxicity of a cancer therapy is the level of adverse effects experienced by the subject during and following treatment.
  • Adverse events associated with cancer therapy toxicity include, but are not limited to, abdominal pain, acid indigestion, acid reflux, allergic reactions, alopecia, anaphylaxis, anemia, anxiety, lack of appetite, arthralgias, asthenia, ataxia, azotemia, loss of balance, bone pain, bleeding, blood clots, low blood pressure, elevated blood pressure, difficulty breathing, bronchitis, bruising, low white blood cell count, low red blood cell count, low platelet count, cardiotoxicity, cystitis, hemorrhagic cystitis, arrhythmias, heart valve disease, cardiomyopathy, coronary artery disease, cataracts, central neurotoxicity, cognitive impairment, confusion, conjunctivitis, constipation, coughing, cramping, cystitis, deep vein thrombosis, dehydration, depression, diarrhea, dizziness, dry mouth, dry skin, dyspepsia
  • hyperchloremia hyperglycemia, hyperkalemia, hyperlipasemia, hypermagnesemia,
  • hypernatremia hyperphosphatemia, hyperpigmentation, hypertriglyceridemia, hyperuricemia, hypoalbuminemia, hypocalcemia, hypochloremia, hypoglycemia, hypokalemia,
  • the administration of the bacterial and/or pharmaceutical composition treats the cancer.
  • the bacterial composition induces an anti tumor immune response in the subject.
  • the methods provided herein include the administration to a subject of a bacterium, bacterial derivative a bacterial composition and/or a pharmaceutical composition described herein (e.g., a Paraclostridium benzoelyticum -containing bacterial composition) either alone or in combination with another cancer therapeutic.
  • the other cancer therapeutic may include e.g, surgical resection, radiotherapy, or a cancer therapeutic agent.
  • the bacterial composition and the other cancer therapy can be administered to the subject in any order.
  • the bacterial and/or pharmaceutical composition and the other cancer therapy are administered conjointly.
  • the bacterial and/or 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
  • the bacterial and/or 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,
  • the bacterial and/or 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 subject is administered an antibiotic before the bacterial and/or pharmaceutical compositionis 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,
  • the subject is administered an antibiotic after the bacterial and/or pharmaceutical compositionis 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 bacterial and/or pharmaceutical compositionis 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,
  • the bacterial and/or pharmaceutical compositionand 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 X-rays, UV-irradiation, microwaves, electronic emissions, and radioisotopes.
  • the localized tumor site may be irradiated, including by one or more the above described forms of radiation. All of these factors may effect a broad range of damage on 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 angu
  • 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-l l); 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; vinor
  • 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-l), Pembrolizumab (Merck, anti-PD-l), 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, Gpl00:209-2l7, AGS-003, DCVax-L, Algenpantucel-L, Tergenpantucel-L, TG4010, ProstAtak, Prostvac-V/R-TRICOM, Rindopepimul, E75 peptide acetate, IMA901, POL-103A,
  • tumor vaccines such as Gardail, Cervarix, BCG, sipulencel-T, Gpl00:209-2l7, AGS-003, DCVax-L, Algenpantucel-L, Tergenpantucel-L, TG4010, ProstAtak, Prostvac-V/R-TRICOM, Rindopepimul, E75 peptide acetate, IMA901, POL-103A,
  • 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-l, 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.
  • 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-0020718C, AUR-012 and STI-A1010.
  • 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.
  • each strand can be between 21 and 25 nucleotides in length.
  • 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.
  • 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.
  • 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.
  • RNA 7 1509-1521 ; Hutvagner G et al., RNAi: Nature abhors a double-strand. Curr. Opin. Genetics & Development 12: 225-232; Brummelkamp, 2002, A system for stable expression of short interfering RNAs in mammalian cells. Science 296: 550-553; Lee NS, Dohjima T, Bauer G, Li H, Li M-J, Ehsani A, Salvaterra P, and Rossi J. (2002).
  • RNAs small interfering RNAs targeted against HIV-l rev transcripts in human cells. Nature Biotechnol. 20:500-505; Miyagishi M, and Taira K. (2002).
  • U6-promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells. Nature Biotechnol. 20:497-500; Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, and Conklin DS. (2002).
  • Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes & Dev. 16:948-958; Paul CP, Good PD, Winer I, and Engelke DR. (2002).
  • 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(l3):el09 (2004); Hanai et al. Ann NY Acad Sci., 1082:9-17 (2006); and Kawata et al. Mol Cancer Ther., 7(9):2904-l2 (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, ALDH1A1, alpha- actinin-4, alpha-fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-l, 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-l, 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 coli 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, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-l, 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-l, CPSF, CSNK1A1, CTAG1, CTAG2, cyclm 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, EZH2,
  • the cancer immunotherapy comprises administration of a cancer-specific chimeric antigen receptor (CAR).
  • CAR cancer-specific chimeric antigen receptor
  • 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.
  • 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-l”), 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-l”), Interferon gamma ("IFN-gamma”), Interlukin-l alpha (“IL-l alpha”), Interlukin-l beta (“IL-l beta”), Interleukin 1 receptor antagonist (“IL-l ra”), Interleukin-2 (“IL-2”),
  • BLC B lymphocyte chemoattractant
  • Eotaxin-2 Eosinophil chemotact
  • 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-l l”), Subunit beta of Interleukin- 12 (“IL-12 p40” or “IL-12 p70”),
  • Interleukin- 13 Interleukin- 15
  • Interleukin- 16 Interleukin- 16
  • Interleukin- 17 Interleukin- 17
  • Chemokine (C-C motif) Ligand 2 MCP-l "
  • MCP-l Macrophage colony-stimulating factor
  • MIG Monokine induced by gamma interferon
  • Chemokine (C-C motif) ligand 2 MIP-l alpha
  • PDGF-BB Platelet-derived growth factor subunit B
  • Chemokine (C-C motif) ligand 5 Regulated on Activation, Normal T cell Expressed and Secreted
  • RANTES TIMP metallopeptidase inhibitor 1
  • TIMP-2 TIMP metallopeptidase inhibitor 2
  • PIGF Phosphatidylinositol-glycan biosynthesis
  • Skp Cullin, F-box containing complex
  • SCF Stem cell factor receptor
  • SCF R Stem cell factor receptor
  • TGFalpha Transforming growth factor alpha
  • TGF beta-l Transforming growth factor beta-1
  • TGF beta-3 Transforming growth factor 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 LEO
  • Betacellulin BTC
  • Mucosae- associated epithelial chemokine CL28
  • C-C motif Chemokine
  • CXCL16 Chemokine (C-X-C motif) ligand 16
  • CXCL16 C-X-C motif chemokine 5
  • MCP-4 Macrophage-derived chemokine
  • MDC Macrophage-derived chemokine
  • MIF Macrophage migration inhibitory factor
  • C-C motif ligand 20 MIP-3 alpha
  • C-C motif chemokine 19 MIP-3 beta
  • Chemokine (C-C motif) ligand 23 MSPalpha
  • NAP- 2 Nucleosome assembly protein l-like 4
  • ErbB3 Endothelial-leukocyte adhesion molecule 1
  • E- Selectin' 1 Apoptosis antigen 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-l R4 IL-l 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
  • NRGl NRGl
  • VEGFRlAdiponectin Adipsin
  • AFP Alpha-fetoprotein
  • ANGPTL4 Angiopoietin-like 4
  • B2M Basal cell adhesion molecule
  • BCAM Basal cell adhesion molecule
  • CA125 Carbohydrate antigen 125
  • CA15-3 Cancer Antigen 15-3
  • CEA Carcinoembryonic antigen
  • CPP cAMP receptor protein
  • ErbB2 Human Epidermal Growth Factor Receptor 2
  • FoUistatin Follicle-stimulating hormone
  • FSH Follicle-stimulating hormone
  • FSH Follicle-stimulating hormone
  • GRO alpha Chemokine (C-X-C motif) ligand 1
  • GRO alpha Chemokine (C-X-C motif) ligand 1
  • beta HCG Insulin-like growth factor 1 receptor
  • IGF-l sR Insulin-like growth factor 1 receptor
  • IGF-l sR Insulin-like growth
  • 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-l 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-l”),
  • Tumor necrosis factor receptor superfamily member 13B (“TACI”), Tissue factor pathway inhibitor (“TFPI”), TSP-l, Tumor necrosis factor receptor superfamily, member lOb (“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- signaling pathway protein 1 (“WISP-l "), 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-5l, Cs-l3l, Ce-l34, Se-75, Ru-97, 1-125, Eu-l49, Os-l89m, Sb-l l9, 1-123, Ho-161, Sb-l l7, Ce-l39, In-l l l, Rh-l03m, Ga-67, T1-201, Pd-l03, Au-l95, Hg-l97, Sr-87m, Pt-l9l, P-33, Er-l69, Ru-l03, Yb- 169, Au-l99, Sn-l2l, Tm-l67, Yb-l75, In-l l3m, Sn-l l3, Lu-l77, Rh-l05, Sn-l l7m, Cu-67, Sc- 47, P
  • 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, bpopeptides, 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 cob, Klebsiella, Pseudomonas aeruginosa, and Francisella tularensis, and against certain aerobic bacteria but less effective against obbgate/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, Cefazobn, 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, Borrelia, and Treponema. 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, Levofloxacin, Lomefloxacin, 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, Demeclocy cline, 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, ole
  • 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 leuk
  • 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, plasmacytoma, colorectal cancer, rectal cancer, Merkel Cell carcinoma, salivary gland carcinoma, 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 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 which 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
  • Kupffer cell sarcoma Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.
  • 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.
  • 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, plasmacytoma, rectal cancer, Merkel Cell carcinoma, salivary gland carcinoma, 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,
  • 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 myelocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, mast cell leukemia, multiple myeloma, myeloid lymphoma, Hodgkin' s lymphoma, non-Hodgkin' s lymphoma.
  • 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.
  • 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.
  • Example 1 Orally Administered Paraclostridium benzoelyticum Strain A inhibits colorectal carcinoma tumor growth
  • mice Female 6-8 week old Balb/c mice were obtained from Taconic (Germantown, NY). 100,000 CT-26 colorectal tumor cells (ATCC CRL-2638) were resuspended in sterile PBS and inoculated in the presence of 50% Matrigel. CT-26 tumor cells were subcutaneously injected into one hind flank of each mouse. When tumor volumes reached an average of lOOmm 3 (approximately 10-12 days following tumor cell inoculation), animals were distributed into the following groups: 1) Vehicle; 2) anti-PD-l antibody; and 3) Paraclostridium benzoelyticum Strain A.
  • Antibodies were administered intraperitoneally (i.p.) at 200ug/mouse (lOOul final volume) every four days, starting on day 1 , and Paraclostridium benzoelyticum Strain A bacteria (5.5xl0 7 ) were administered by oral gavage (p.o.) daily, starting on day 1 until the conclusion of the study.
  • the Paraclostridium benzoelyticum Strain A group showed tumor growth inhibition comparable to that seen in the anti-PD-l group ( Figures 1, 2, 3, and 4).
  • CT-26 colorectal tumor cells (ATCC CRL-2638) were resuspended in sterile PBS and inoculated in the presence of 50% Matrigel.
  • CT-26 tumor cells were subcutaneously injected into one hind flank of each mouse. When tumor volumes reached an average of lOOmm 3 (approximately 10-12 days following tumor cell inoculation), animals were distributed into the following groups: 1)
  • Paraclostridium benzoelyticum Strain A and anti-PD-l antibody were administered intraperitoneally (i.p.) at 200ug/mouse (lOOul final volume) every four days, starting on day 1 , and Paraclostridium benzoelyticum Strain A bacteria (5.5xl0 7 ) were administered by oral gavage (p.o.) daily, starting on day 1 until the conclusion of the study.
  • the Paraclostridium benzoelyticum Strain A group showed tumor growth inhibition comparable to that seen in the anti-PD-l group ( Figures 5 and 6).
  • mice may be treated with Paraclostridium
  • benzoelyticum bacteria live, killed, irradiated or lyophilized
  • EVs e.g. EVs
  • PhABs e.g. anti-PD-l
  • Example 2 Paraclostridium benzoelyticum in a mouse lung cancer model
  • Paraclostridium benzoelyticum Strain A is tested for its efficacy in the mouse lung cancer model, either alone or in combination with other cancer therapies, including checkpoint inhibitor(s). Mice are divided into groups receiving Paraclostridium benzoelyticum Strain A, with or without checkpoint inhibitor treatment. Paraclostridium benzoelyticum Strain A is administered at varied doses at defined intervals. For example, some mice receive
  • mice Paraclostridium benzoelyticum Strain A (p.o.) on the day following tumor cell injection (day 1). Some mice receive seven (7) consecutive doses of Paraclostridium benzoelyticum Strain A (one dose per day on days 14-21). Other mice receive daily dosing or, alternatively, some mice receive dosing every other day. Other mice are treated with Paraclostridium benzoelyticum bacteria (live, killed, irradiated or lyophilized), EVs, and/or PhABs. Alternatively, mice are randomized into various treatment groups at a defined timepoint (e.g. on day 13) or when the tumors reach a certain size (e.g. 100 mm 3 ) and treatment is then initiated accordingly.
  • a defined timepoint e.g. on day 13
  • a certain size e.g. 100 mm 3
  • lxl 0 6 LLC1 cells or an appropriate number of lung cancer cells from another lung cancer cell line, are injected into the hind flank of syngeneic mice. Tumors from the various treatment groups are measured with calipers at regular intervals. Some mice are sacrificed for ex vivo tumor analysis using flow cytometry. Other mice may be rechallenged with tumor cell injection into the contralateral flank to determine the impact of the immune system’s memory response on tumor growth.
  • Example 3 Paraclostridium benzoelyticum in a mouse breast cancer model
  • Parados tridium benzoelyticum Strain A is tested for its efficacy in the mouse breast cancer model, either alone or in combination with other cancer therapies, including checkpoint inhibitor(s). Mice are divided into groups receiving Paradostridium benzoelyticum Strain A, with or without checkpoint inhibitor treatment. Paradostridium benzoelyticum Strain A is administered at varied doses at defined intervals. For example, some mice receive
  • Paradostridium benzoelyticum Strain A (p.o.) on the day following tumor cell injection (day 1). Some mice receive seven (7) consecutive doses of Paradostridium benzoelyticum Strain A (one dose per day on days 14-21). Other mice receive daily dosing or, alternatively, some mice receive dosing every other day. Other mice are treated with Paradostridium benzoelyticum bacteria (live, killed, irradiated or lyophilized), EVs, and/or PhABs. Alternatively, mice are randomized into various treatment groups at a defined timepoint (e.g. on day 13) or when the tumors reach a certain size (e.g. 100 mm 3 ) and treatment is then initiated accordingly.
  • a defined timepoint e.g. on day 13
  • a certain size e.g. 100 mm 3
  • 4T1 mouse mammary carcinoma cells are obtained from ATCC and lxl 0 6 cells in
  • 50ul PBS are injected subcutaneously into one or both hind limbs of Balb/c female mice (as described by Wang et al. 2003, Systemic dissemination of viral vectors during intratumoral injection. Molecular Cancer Therapeutics; 2(11)).
  • EMT6 mouse mammary carcinoma cells are obtained from ATCC and lxl 0 6 cells in 50m1 PBS are injected
  • mice 6-8 weeks old subcutaneously into one or both of the hind limbs of Balb/c female mice 6-8 weeks old (as described by Guo et al. 2014, Combinatorial Photothermal and Immuno Cancer Therapy Using Chitosan-Coated Hollow Copper Sulfide Nanoparticles. ASC Nano.; 8(6): 5670-5681).
  • other available mouse mammary cell lines may be used.
  • Tumors from the various treatment groups are measured with calipers at regular intervals. Paradostridium benzoelyticum Strain A is administered at varied doses at defined intervals. For example, some mice are sacrificed for ex vivo tumor analysis using flow cytometry. Other mice may be rechallenged with tumor cell injection into the contralateral flank to determine the impact of the immune system’s memory response on tumor growth.
  • 4T1 cells can be used in an orthotopic murine model of breast cancer as described by Tao et al. (Tao et al. 2008. Imagable 4T1 model for the study of late stage breast cancer. 8: 288). Mice are sacrificed for ex vivo tumor analysis. Tumors are analyzed by flow cytometry and immunohistochemistry.
  • Example 4 Paraclostridium benzoelyticum Strain A in a mouse pancreatic cancer model
  • Paraclostridium benzoelyticum Strain A is tested for its efficacy in the mouse model of pancreatic cancer, either alone or in combination with other cancer therapies, including checkpoint inhibitor(s).
  • Mice are divided into groups receiving Paraclostridium benzoelyticum Strain A, with or without checkpoint inhibitor treatment. Some mice receive Paraclostridium benzoelyticum Strain A (p.o.) on the day following tumor cell injection (day 1). Some mice receive seven (7) consecutive doses of Paraclostridium benzoelyticum Strain A (one dose per day on days 14-21). Other mice receive daily dosing or, alternatively, some mice receive dosing every other day.
  • mice are treated with Paraclostridium benzoelyticum bacteria (live, killed, irradiated or lyophilized), EVs, and/or PhABs.
  • mice are randomized into various treatment groups at a defined timepoint (e.g . on day 13) or when the tumors reach a certain size ( e.g . 100 mm 3 ) and treatment is then initiated accordingly.
  • Panc02 cells are maintained in DMEM, supplemented with 10% fetal calf serum and 1% penicillin/streptomycin, and incubated at 37°C at 5% C02.
  • Female 8-10 week-old C57B1/6 mice are obtained from Charles River, Inc. or other certified vendor.
  • Female C57B1/6 mice are injected subcutaneously into the right hind flank with lxl 0 6 Panc02 cells. This protocol is based on standard Panc02 tumor models (Maletzki et al. 2008. Pancreatic cancer regression by intratumoral injection of live streptococcus pyogenes in a syngeneic mouse model. Gut. 57:483- 491). Tumors from the various treatment groups are measured with calipers at regular intervals. Some mice are sacrificed for ex vivo tumor analysis using flow cytometry, while other mice are rechallenged to determine the impact of the memory response on tumor growth.
  • Panc02, 6606PDA, or Capan-l cells lines can be used in an orthotopic murine model of pancreatic cancer as described by Partecke et al. (Partecke et al.
  • Paraclostridium benzoelyticum Strain A is tested for its efficacy in the mouse model of hepatocellular carcinoma, either alone or in combination with other cancer therapies, including checkpoint inhibitor(s). Mice are divided into groups receiving Paraclostridium benzoelyticum Strain A, with or without checkpoint inhibitor treatment. Paraclostridium benzoelyticum Strain A is administered at varied doses at defined intervals. For example, some mice receive Paraclostridium benzoelyticum Strain A (p.o.) on the day following tumor cell injection (day 1). Some mice receive seven (7) consecutive doses of Paraclostridium
  • benzoelyticum Strain A one dose per day on days 14-21. Other mice receive daily dosing or, alternatively, some mice receive dosing every other day. Other mice are treated with
  • mice are randomized into various treatment groups at a defined timepoint (e.g . on day 13) or when the tumors reach a certain size (e.g. 100 mm 3 ) and treatment is then initiated accordingly.
  • Hepatocellular carcinoma is induced in mice by subcutaneous inoculation of lxl 0 6 Hepal29 cells (obtained from NCI or other source), or an appropriate number of cells from other hepatocellular carcinoma cell line (as described by Gonzalez-Carmona et al. 2008. CD40 ligand-expressing dendritic cells induce regression of hepatocellular carcinoma by activating innate and acquired immunity in vivo. Hepatology. 48(1): 157-168). Tumor cells are inoculated into one or both flanks. Tumors from the various treatment groups are measured with calipers at regular intervals. Some mice are sacrificed for ex vivo tumor analysis using flow cytometry, while other mice are rechallenged to determine the impact of the memory response on tumor growth.
  • Example 6 Paraclostridium benzoelyticum in a mouse lymphoma model
  • Paraclostridium benzoelyticum Strain A is tested for its efficacy in the mouse model of lymphoma, either alone or in combination with other cancer therapies, including checkpoint inhibitor(s). Mice are divided into groups receiving Paraclostridium benzoelyticum Strain A, with or without checkpoint inhibitor treatment. Paraclostridium benzoelyticum Strain A is administered at varied doses at defined intervals. For example, some mice receive Paraclostridium benzoelyticum Strain A (p.o.) on the day following tumor cell injection (day 1). Some mice receive seven (7) consecutive doses of Paraclostridium benzoelyticum Strain A (one dose per day on days 14-21).
  • mice receive daily dosing or, alternatively, some mice receive dosing every other day.
  • Other mice are treated with Paraclostridium benzoelyticum bacteria (live, killed, irradiated or lyophilized), EVs, and/or PhABs.
  • mice are randomized into various treatment groups at a defined timepoint (e.g. on day 13) or when the tumors reach a certain size (e.g. 100 mm 3 ) and treatment is then initiated accordingly.
  • lymphoma cell line is the A20 lymphoma, although other lymphoma cell lines may be used with syngeneic mice.
  • A20 lymphoma cells are obtained from ATCC and 5x10 6 cells in 50ul PBS are injected subcutaneously into one or both of the hind limbs of Balb/c female mice (as described by Houot et al. 2009. T-cell modulation combined with intratumoral CpG cures lymphoma in a mouse model without the need for chemotherapy. Blood. 113(15): 3546- 3552). Tumors from the various treatment groups are measured with calipers at regular intervals. Some mice are sacrificed for ex vivo tumor analysis using flow cytometry, while other mice are rechallenged to determine the impact of the memory response on tumor growth.
  • Example 7 Paraclostridium benzoelyticum in a mouse prostate cancer model
  • Paraclostridium benzoelyticum Strain A is tested for its efficacy in the mouse model of prostate cancer, either alone or in combination with other cancer therapies, including checkpoint inhibitor(s). Mice are divided into groups receiving Paraclostridium benzoelyticum Strain A, with or without checkpoint inhibitor treatment. Paraclostridium benzoelyticum Strain A is administered at varied doses at defined intervals. For example, some mice receive
  • mice Paraclostridium benzoelyticum Strain A (p.o.) on the day following tumor cell injection (day 1). Some mice receive seven (7) consecutive doses of Paraclostridium benzoelyticum Strain A (one dose per day on days 14-21). Other mice receive daily dosing or, alternatively, some mice receive dosing every other day. Other mice are treated with Paraclostridium benzoelyticum bacteria (live, killed, irradiated or lyophilized), EVs, and/or PhABs. Alternatively, mice are randomized into various treatment groups at a defined timepoint (e.g. on day 13) or when the tumors reach a certain size (e.g. 100 mm 3 ) and treatment is then initiated accordingly.
  • a defined timepoint e.g. on day 13
  • a certain size e.g. 100 mm 3
  • Mouse prostate cancer cells (lxl 0 5 RM-l cells or an appropriate number of cells from another prostate cancer cell line) are injected into syngeneic mice. Tumors from the various treatment groups are measured with calipers at regular intervals. Some mice are sacrificed for ex vivo tumor analysis using flow cytometry, while other mice are rechallenged to determine the impact of the memory response on tumor growth.
  • Example 8 Paraclostridium benzoelyticum in a mouse plasmacytoma model
  • Paraclostridium benzoelyticum Strain A is tested for its efficacy in the mouse model of plasmacytoma, either alone or in combination with other cancer therapies, including checkpoint inhibitor(s). Mice are divided into groups receiving Paraclostridium benzoelyticum Strain A, with or without checkpoint inhibitor treatment. Paraclostridium benzoelyticum Strain A is administered at varied doses at defined intervals. For example, some mice receive
  • mice Paraclostridium benzoelyticum Strain A (p.o.) on the day following tumor cell injection (day 1). Some mice receive seven (7) consecutive doses of Paraclostridium benzoelyticum Strain A (one dose per day on days 14-21). Other mice receive daily dosing or, alternatively, some mice receive dosing every other day. Other mice are treated with Paraclostridium benzoelyticum bacteria (live, killed, irradiated or lyophilized), EVs, and/or PhABs. Alternatively, mice are randomized into various treatment groups at a defined timepoint (e.g. on day 13) or when the tumors reach a certain size (e.g. 100 mm 3 ) and treatment is then initiated accordingly.
  • a defined timepoint e.g. on day 13
  • a certain size e.g. 100 mm 3
  • mice are injected intraperitoneally three times with 500ul of 2,6,l0,l2-tetramethylpentadecane (“pristane oil”) at various time points between 0 and 60 days, as described by Potter et al. 1983. Peritoneal plasmacytomagenesis in mice: comparison of different pristane dose regimens. J. Natl. Cancer Inst. 7l(2):39l-5 (see also Lattanzio et al. 1997. Defective Development of Pristane-Oil Induced Plasmacytomas in Interleukin-6-Deficient BALB/C Mice. Am. J. Pathology: 151(3):689696). Progression of disease is measured by the degree of abdominal swelling and immune cells and particles in the ascites. Ascites fluid is analyzed for immune cell phenotype by flow cytometry.
  • MOPC-104E cells or J558 plasmacytoma cells are injected subcutaneously into one or more hind flanks of Balb/c mice (5x10 6 cells), based on model described by Bhoopalam et al. 1980. Effect of dextran-S (alpha, 1-3 dextran) on the growth of plasmacytomas MOPC-104E and J558. J. Immunol. 125(4): 1454-8 (see also Wang et al. 2015.
  • IL-10 enhances CTL-mediated tumor rejection by inhibiting highly suppressive CD4+ T cells and promoting CTL persistence in a murine model of plasmacytoma.
  • Mice are divided into groups receiving Paraclostridium benzoelyticum Strain A by oral gavage, and with or without checkpoint inhibitor treatment. Tumors from the various treatment groups are measured with calipers at regular intervals. Some mice are sacrificed for ex vivo tumor analysis using flow cytometry, while other mice are rechallenged to determine the impact of the memory response on tumor growth.
  • Example 9 Paraclostridium benzoelyticum in a SC ID mouse model of mouse myeloma
  • Paraclostridium benzoelyticum Strain A is tested for its efficacy in the SCID mouse model of myeloma, either alone or in combination with other cancer therapies, including checkpoint inhibitor(s). Mice are divided into groups receiving Paraclostridium benzoelyticum Strain A, with or without checkpoint inhibitor treatment. Paraclostridium benzoelyticum Strain A is administered at varied doses at defined intervals. For example, some mice receive
  • mice Paraclostridium benzoelyticum Strain A (p.o.) on the day following tumor cell injection (day 1). Some mice receive seven (7) consecutive doses of Paraclostridium benzoelyticum Strain A (one dose per day on days 14-21). Other mice receive daily dosing or, alternatively, some mice receive dosing every other day. Other mice are treated with Paraclostridium benzoelyticum bacteria (live, killed, irradiated or lyophilized), EVs, and/or PhABs. Alternatively, mice are randomized into various treatment groups at a defined timepoint (e.g. on day 13) or when the tumors reach a certain size (e.g. 100 mm 3 ) and treatment is then initiated accordingly.
  • a defined timepoint e.g. on day 13
  • a certain size e.g. 100 mm 3
  • Example 10 Paraclostridium benzoelyticum in a mouse renal cell carcinoma model
  • Paraclostridium benzoelyticum Strain A is tested for its efficacy in the mouse model of renal cell carcinoma, either alone or in combination with other cancer therapies, including checkpoint inhibitor(s). Mice are divided into groups receiving Paraclostridium benzoelyticum Strain A, with or without checkpoint inhibitor treatment. Paraclostridium benzoelyticum Strain A is administered at varied doses at defined intervals. For example, some mice receive Paraclostridium benzoelyticum Strain A (p.o.) on the day following tumor cell injection (day 1). Some mice receive seven (7) consecutive doses of Paraclostridium
  • benzoelyticum Strain A one dose per day on days 14-21. Other mice receive daily dosing or, alternatively, some mice receive dosing every other day. Other mice are treated with
  • mice are randomized into various treatment groups at a defined timepoint (e.g . on day 13) or when the tumors reach a certain size (e.g. 100 mm 3 ) and treatment is then initiated accordingly.
  • Renca cells ATCC CRL-2947
  • other renal cell carcinoma cells are injected subcutaneously into one or both flanks of 7-8 week old syngeneic Balb/c mice (5x10 6 in 0.1 ml PBS). Tumors from the various treatment groups are measured with calipers at regular intervals. Some mice are sacrificed for ex vivo tumor analysis using flow cytometry, while other mice are rechallenged to determine the impact of the memory response on tumor growth.
  • Example 11 Paraclostridium benzoelyticum in a mouse bladder cancer model
  • Parados tridium benzoelyticum Strain A is tested for its efficacy in the mouse model of bladder cancer, either alone or in combination with other cancer therapies, including checkpoint inhibitor(s). Mice are divided into groups receiving Paradostridium benzoelyticum Strain A, with or without checkpoint inhibitor treatment. Paradostridium benzoelyticum Strain A is administered at varied doses at defined intervals. For example, some mice receive
  • Paradostridium benzoelyticum Strain A (p.o.) on the day following tumor cell injection (day 1). Some mice receive seven (7) consecutive doses of Paradostridium benzoelyticum Strain A (one dose per day on days 14-21). Other mice receive daily dosing or, alternatively, some mice receive dosing every other day. Other mice are treated with Paradostridium benzoelyticum bacteria (live, killed, irradiated or lyophilized), EVs, and/or PhABs. Alternatively, mice are randomized into various treatment groups at a defined timepoint (e.g. on day 13) or when the tumors reach a certain size (e.g. 100 mm 3 ) and treatment is then initiated accordingly.
  • a defined timepoint e.g. on day 13
  • a certain size e.g. 100 mm 3
  • MBT-2 cells (or other bladder cancer cell line) are harvested and resuspended in 1 : 1 PBS/Matrigel mixture. 2x10 5 MBT-2 cells are suspended in 100 ul of mixture and injected subcutaneously into one or both hind flanks of syngeneic mice. Tumors are measured with calipers at regular intervals.
  • mice are sacrificed for ex vivo tumor analysis using flow cytometry, while other mice are rechallenged to determine the impact of the memory response on tumor growth.
  • Enriched media is used to grow and prepare the bacterium for in vitro and in vivo use.
  • media may contain sugar, yeast extracts, plant based peptones, buffers, salts, trace elements, surfactants, anti-foaming agents, and vitamins.
  • Composition of complex components such as yeast extracts and peptones may be undefined or partially defined (including approximate concentrations of amino acids, sugars etc.).
  • Microbial metabolism may be dependent on the availability of resources such as carbon and nitrogen. Various sugars or other carbon sources may be tested.
  • media may be prepared and the selected bacterium grown as shown by Saarela et al., J. Applied Microbiology . 2005. 99: 1330-1339, which is hereby incorporated by reference. Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze-drying survival, storage stability, and acid and bile exposure of the selected bacterium produced without milk-based ingredients.
  • Sterilization may be by Ultra High
  • UHT Ultra High Temperature
  • the UHT processing is performed at very high temperature for short periods of time.
  • the UHT range may be from l35-l80°C.
  • the medium may be sterilized from between 10 to 30 seconds at l35°C.
  • Inoculum can be prepared in flasks or in smaller bioreactors and growth is monitored.
  • the inoculum size may be between approximately 0.5 and 3% of the total bioreactor volume.
  • bioreactor volume can be at least 2L, 10L, 80L, 100L, 250L, 1000L, 2500L, 5000L, l0,000L.
  • the bioreactor Before the inoculation, the bioreactor is prepared with medium at desired pH, temperature, and oxygen concentration.
  • the initial pH of the culture medium may be different that the process set-point. pH stress may be detrimental at low cell centration; the initial pH could be between pH 7.5 and the process set-point. For example, pH may be set between 4.5 and 8.0.
  • the pH can be controlled through the use of sodium hydroxide, potassium hydroxide, or ammonium hydroxide.
  • the temperature may be controlled from 25°C to 45°C, for example at 37°C. Anaerobic conditions are created by reducing the level of oxygen in the culture broth from around 8mg/L to Omg/L.
  • nitrogen or gas mixtures may be used in order to establish anaerobic conditions.
  • no gases are used and anaerobic conditions are established by cells consuming remaining oxygen from the medium.
  • the bioreactor fermentation time can vary. For example, fermentation time can vary from approximately 5 hours to 48 hours.
  • Reviving microbes from a frozen state may require special considerations.
  • Production medium may stress cells after a thaw; a specific thaw medium may be required to consistently start a seed train from thawed material.
  • the kinetics of transfer or passage of seed material to fresh medium may be influenced by the current state of the microbes (ex. exponential growth, stationary growth, unstressed, stressed).
  • Inoculation of the production fermenter(s) can impact growth kinetics and cellular activity.
  • the initial state of the bioreactor system must be optimized to facilitate successful and consistent production.
  • the fraction of seed culture to total medium (e.g . a percentage) has a dramatic impact on growth kinetics.
  • the range may be 1-5% of the fermenter’s working volume.
  • the initial pH of the culture medium may be different from the process set-point. pH stress may be detrimental at low cell concentration; the initial pH may be between pH 7.5 and the process set-point. Agitation and gas flow into the system during inoculation may be different from the process set-points. Physical and chemical stresses due to both conditions may be detrimental at low cell concentration.
  • Process conditions and control settings may influence the kinetics of microbial growth and cellular activity. Shifts in process conditions may change membrane composition, production of metabolites, growth rate, cellular stress, etc.
  • Optimal temperature range for growth may vary with strain. The range may be 20-40 °C.
  • Optimal pH for cell growth and performance of downstream activity may vary with strain. The range may be pH 5-8. Gasses dissolved in the medium may be used by cells for metabolism. Adjusting concentrations of O2, CO2, and N2 throughout the process may be required. Availability of nutrients may shift cellular growth. Microbes may have alternate kinetics when excess nutrients are available.
  • microbes The state of microbes at the end of a fermentation and during harvesting may impact cell survival and activity. Microbes may be preconditioned shortly before harvest to better prepare them for the physical and chemical stresses involved in separation and
  • a change in temperature may reduce cellular metabolism, slowing growth (and/or death) and physiological change when removed from the fermenter.
  • Effectiveness of centrifugal concentration may be influenced by culture pH. Raising pH by 1-2 points can improve effectiveness of concentration but can also be detrimental to cells.
  • Microbes may be stressed shortly before harvest by increasing the concentration of salts and/or sugars in the medium. Cells stressed in this way may better survive freezing and lyophilization during downstream.
  • Separation methods and technology may impact how efficiently microbes are separated from the culture medium.
  • Solids may be removed using centrifugation techniques. Effectiveness of centrifugal concentration can be influenced by culture pH or by the use of flocculating agents. Raising pH by 1-2 points may improve effectiveness of concentration but can also be detrimental to cells.
  • Microbes may be stressed shortly before harvest by increasing the concentration of salts and/or sugars in the medium. Cells stressed in this way may better survive freezing and lyophilization during downstream. Additionally, Microbes may also be separated via filtration. Filtration is superior to centrifugation techniques for purification if the cells require excessive g-minutes to successfully centrifuge. Excipients can be added before after separation.
  • Excipients can be added for cryo protection or for protection during lyophilization.
  • Excipients can include, but are not limited to, sucrose, trehalose, or lactose, and these may be alternatively mixed with buffer and anti-oxidants.
  • droplets of cell pellets mixed with excipients are submerged in liquid nitrogen.
  • Harvesting can be performed by continuous centrifugation.
  • Product may be resuspended with various excipients to a desired final concentration.
  • Excipients can be added for cryo protection or for protection during lyophilization.
  • Excipients can include, but are not limited to, sucrose, trehalose, or lactose, and these may be alternatively mixed with buffer and anti oxidants.
  • droplets of cell pellets mixed with excipients are submerged in liquid nitrogen.
  • Lyophilization of material begins with primary drying.
  • the ice is removed.
  • a vacuum is generated and an appropriate amount of heat is supplied to the material for the ice to sublime.
  • product bound water molecules are removed.
  • the temperature is raised higher than in the primary drying phase to break any physico-chemical interactions that have formed between the water molecules and the product material.
  • the pressure may also be lowered further to enhance desorption during this stage.
  • the chamber may be filled with an inert gas, such as nitrogen.
  • the product may be sealed within the freeze dryer under dry conditions, preventing exposure to atmospheric water and contaminants.
  • Antibiotic resistance can be classified as intrinsic, acquired, or adaptive. Adaptive resistance is defined as reduced antimicrobial killing in populations of bacteria that were originally susceptible to a particular antibiotic agent. It involves a transient increase in the ability of bacteria to survive the antibiotic, mainly because of alterations in gene and/or protein expression levels triggered by environmental conditions such as stress, nutrient conditions, and sub-inhibitory levels of the antibiotic. In contrast to intrinsic and acquired resistance
  • Adaptive resistance might be one of the reasons of the phenomenon that laboratory susceptibility results are not congruent with the clinical effectiveness of antibiotics.
  • Polymyxin resistance in bacteria is known to be adaptive, which is characterized by induction of resistance in the presence of drug and reversal to the susceptible phenotype in its absence.
  • LPS lipopoly saccharide
  • oligosaccharide components This results in a reduction of the net negative charge of the outer membrane.
  • the regulatory two-component systems (TCSs) PhoP-PhoQ (PhoPQ) and PmrA- PmrB (PmrAB) play important roles in lipid A modification, which subsequently results in bacteria becoming resistant to polymyxins. Further, the survival rates of bacteria comprising deletion mutants ⁇ IpxC and/or pmrB ), which are involved in LPS biosynthesis and modification, were decreased > 4-fold when colistin was present at sub- inhibitory concentrations, compared with those for their WT parents.
  • Paraclostridium benzoelyticum LPS mutants are generated by serial passages in the presence of colistin using methods known to those skilled in the art (JY Lee et al. Sci Rep. 2016 May 6;6:25543).
  • Paraclostridium benzoelyticum cultures are routinely grown on BRU agar plates (Anaerobic Systems) at 37C for 2-4 days.
  • Paraclostridium benzoelyticum cultures are grown in liquid medium in the presence of a range of colistin concentration (0.1 to 16 mg/L) for 1 to 3 days in order to determine minimum inhibitory concentration of colistin.
  • Paraclostridium benzoelyticum cultures are grown in liquid medium in the presence of sub- inhibitory colistin concentration (i.e. less than minimum inhibitory concentration) for 1 to 3 days to allow bacterial growth.
  • Grown cultures are diluted 25- to 50-fold in fresh medium containing 2-fold higher concentration of colistin.
  • Each bacterial culture media contained 0.13, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, or 256 mg/L colistin as bacterial cultures serially passaged for 10-14 generations.
  • cultures are plated on BRU agar plates and allowed to grow for 2-4 days.
  • Bacterial colonies are scooped from the plates to start cultures in liquid medium containing colistin at a concentration that allowed bacterial growth. Serial passages are repeated until bacteria are resistant to colistin at >250mg/L concentration. Following the last passage, bacterial colonies are grown on BRU plates containing 1 OOmg/L colistin and several individual colonies are selected for lipopolysaccharide (LPS) analysis.
  • LPS lipopolysaccharide
  • Colistin-resistant Paraclostridium benzoelyticum clones are selected for LPS analysis.
  • Analytical methods include SDS-PAGE analysis followed by ProQ LPS staining (Invitrogen), chromogenic LAL endotoxin assay (GeneScript), lipid A analysis by MALDI-TOF MS analysis (Bruker).
  • Example 14 A mouse melanoma model
  • mice Female 6-8 week old C57B1/6 mice are obtained from Taconic (Germantown, NY). 100,000 B16-F10 (ATCC CRL-6475) tumor cells are resuspended in sterile PBS containing 50% Matrigel and inoculated in a lOOul final volume into one hind flank (the first flank) of each mouse. Treatment with Paraclostridium benzoelyticum Strain A is initiated at some point following tumor cell inoculation at varied doses and at defined intervals. For example, some mice receive between 1-5c10 L 9 CFU (IOOmI final volume) per dose.
  • Possible routes of administration include oral gavage (p.o.), intravenous injection, intratumoral injection (IT) or peritumoral or subtumoral or subcutaneous injection.
  • I intratumoral injection
  • additional mice may be inoculated with tumor cells in the contralateral (untreated, second) flank prior to IT, peritumoral, or subtumoral treatment with Paraclostridium benzoelyticum in the first flank.
  • Other mice are treated ith Paraclostridium benzoelyticum bacteria (live, killed, irradiatedor lyophilized), EVs, and/or PhABs.
  • mice may receive Paraclostridium benzoelyticum (p.o.) on day 1 (the day following tumor cell injection). Other mice may receive seven (7) consecutive doses of a bacterial strain (one dose per day on days 14-21). Other mice receive daily dosing or, alternatively, some mice receive dosing every other day. Alternatively, mice are randomized into various treatment groups at a defined timepoint ( e.g . on day 13) or when the tumors reach a certain size (e.g. 100 mm 3 ) and treatment is then initiated accordingly.
  • a defined timepoint e.g . on day 13
  • a certain size e.g. 100 mm 3
  • mice when tumor volumes reach an average of lOOmm 3 (approximately 10-12 days following tumor cell inoculation), animals are distributed into groups and treated with either vehicle or a bacterial strain (p.o. or IT). Some additional groups of mice may be treated with an additional cancer therapeutic or appropriate control antibody.
  • a cancer therapeutic that may be administered is an inhibitor of an immune checkpoint, for example anti-PD-l, anti-PD-Ll, or other treatment that blocks the binding of an immune checkpoint to its ligand(s).
  • Checkpoint inhibitors anti-PD-l and anti-PD-Ll may be formulated in PBS and administered
  • mice are given lOOug of anti-PD-l (i.p.) every four days starting on day 1, and continuing for the duration of the study.
  • mice are treated with antibiotics prior to treatment.
  • antibiotics for example, vancomycin (0.5g/L), ampicillin (l .Og/L), gentamicin (l .Og/L) and amphotericin B (0.2g/L) are added to the drinking water, and antibiotic treatment is halted at the time of treatment or a few days prior to treatment.
  • Some mice are inoculated with tumor cells without receiving prior treatment with antibiotics.
  • mice are sacrificed and tumors, lymph nodes, or other tissues may be removed for ex vivo flow cytometric analysis using methods known in the art.
  • tumors are dissociated using a Miltenyi tumor dissociation enzyme cocktail according to the manufacturer’s instructions. Tumor weights are recorded and tumors are chopped then placed in 15ml tubes containing the enzyme cocktail and placed on ice. Samples are then placed on a gentle shaker at 37°C for 45 minutes and quenched with up to 15ml complete RPMI. Each cell suspension is strained through a 70mhi filter into a 50ml falcon tube and centrifuged at 1000 rpm for 10 minutes.
  • Staining antibodies can include anti-CDl lc (dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti- MHCII, anti-CD8a, anti-CD4, and anti-CD 103.
  • markers that may be analyzed include pan- immune cell marker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-l, CTFA-4), and macrophage/myeloid markers (CD1 lb, MHCII, CD206, CD40, CSF1R, PD-F1, Gr-l).
  • serum cytokines are analyzed including, but not limited to, TNFa, IF-17, IF-13, IF-l2p70, IFl2p40, IF-10, IP-6, IP- 5, IP-4, IP-2, IP- lb, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIPlb, RANTES, and MCP- 1.
  • Cytokine analysis may be carried out immune cells obtained from lymph nodes or other tissue, and/or on purified CD45+ tumor- infiltrated immune cells obtained ex vivo.
  • immunohistochemistry is carried out on tumor sections to measure T cells, macrophages, dendritic cells, and checkpoint molecule protein expression.
  • mice may be rechallenged with tumor cell injection into the contralateral flank (or other area) to determine the impact of the immune system’s memory response on tumor growth.

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Abstract

L'invention concerne des procédés et des compositions associés au Paraclostridium benzoelyticum utiles en tant qu'agents thérapeutiques.
PCT/US2019/020061 2018-02-28 2019-02-28 Compositions et méthodes de traitement du cancer à l'aide de paraclostridium benzoelyticum WO2019169138A1 (fr)

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CN114544955A (zh) * 2020-11-26 2022-05-27 四川大学华西医院 Gasp-2检测试剂在制备肺癌早期诊断和易感性检测试剂盒中的用途

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