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WO2020006216A1 - Compositions et méthodes de traitement du cancer faisant appel à des bactéries neisseria - Google Patents

Compositions et méthodes de traitement du cancer faisant appel à des bactéries neisseria Download PDF

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Publication number
WO2020006216A1
WO2020006216A1 PCT/US2019/039482 US2019039482W WO2020006216A1 WO 2020006216 A1 WO2020006216 A1 WO 2020006216A1 US 2019039482 W US2019039482 W US 2019039482W WO 2020006216 A1 WO2020006216 A1 WO 2020006216A1
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WIPO (PCT)
Prior art keywords
pharmaceutical composition
carcinoma
mage
cancer
protein
Prior art date
Application number
PCT/US2019/039482
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English (en)
Inventor
Brian Goodman
Christopher J.H. DAVITT
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Evelo Biosciences, Inc.
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Filing date
Publication date
Application filed by Evelo Biosciences, Inc. filed Critical Evelo Biosciences, Inc.
Publication of WO2020006216A1 publication Critical patent/WO2020006216A1/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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 e.g ., bacterial compositions, pharmaceutical compositions
  • a pharmaceutical composition comprising Neisseria (e.g., Neisseria Meningitidis) bacteria and/or a derivative of such bacteria (e.g., extracellular vesicles (EVs) and/or pharmaceutically active biomasses (PhABs)).
  • Neisseria e.g., Neisseria Meningitidis
  • EVs extracellular vesicles
  • PhABs pharmaceutically active biomasses
  • the pharmaceutical compositions comprise monoclonal microbial populations (i.e., microbial populations having a threshold heterogeneity index, e.g., a heterogeneity index greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more than about 99%).
  • microbial populations having a threshold heterogeneity index, e.g., a heterogeneity index greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more than about 99%.
  • such compositions include a population of Neisseria (e.g., Neisseria Meningitidis) bacteria derived from a limited number of clones, e.g., ⁇ 10, 9, 8, 7, 6, 5, 4, 3, 2 or derived from a single clone.
  • bioreactors comprising such bacteria.
  • the administration of the pharmaceutical composition induces an immune response against a tumor in the subject.
  • the administration of the pharmaceutical composition treats the cancer in the subject.
  • the administration augments a tumor microenvironment in the subject.
  • the cancer is a colorectal carcinoma.
  • EVs extracellular vesicles
  • the pharmaceutical compositions comprise both Neisseria (e.g., Neisseria Meningitidis) EVs and whole Neisseria (e.g., Neisseria Meningitidis) bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria).
  • Neisseria e.g., Neisseria Meningitidis
  • whole Neisseria e.g., Neisseria Meningitidis
  • pharmaceutical compositions comprising Neisseria (e.g ., Neisseria
  • compositions comprise Neisseria (e.g., Neisseria Meningitidis) EVs in the absence of Neisseria bacteria.
  • Neisseria e.g., Neisseria Meningitidis
  • compositions comprising Neisseria (e.g., Neisseria Meningitidis) bacteria (e.g., killed, live and/or attenuated bacteria) and/or a derivative of such bacteria (e.g., extracellular vesicles (EVs) and/or pharmaceutically active biomasses (PhABs)).
  • Neisseria e.g., Neisseria Meningitidis
  • bacteria e.g., killed, live and/or attenuated bacteria
  • a derivative of such bacteria e.g., extracellular vesicles (EVs) and/or pharmaceutically active biomasses (PhABs)
  • EVs extracellular vesicles
  • PhABs pharmaceutically active biomasses
  • at least 50%, 60%, 70%, 80%, 85%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bacteria in the pharmaceutical composition are Neisseria (e.g.
  • the bacterial formulation comprises at least 1 x 10 5 , 5 x l0 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 ,
  • the pharmaceutical composition comprises EVs and/or PhABs (e.g., whole cells, fractions of cells, supernatant from fermentation, fractions of supernatant and/or extracellular vesicles) made from Neisseria (e.g., Neisseria Meningitidis) bacteria.
  • EVs and/or PhABs e.g., whole cells, fractions of cells, supernatant from fermentation, fractions of supernatant and/or extracellular vesicles
  • Neisseria e.g., Neisseria Meningitidis
  • the pharmaceutical composition is administered orally, intravenously, intratumorally, subtumorally, intradermally, intraperitoneally, or subcutaneously. In some embodiments, the pharmaceutical composition is administered in 2 or more doses (e.g., one 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,
  • a second bacterial strain and/or genus is administered as part of an ecological consortium.
  • the composition comprises a specific ratio of Neisseria
  • Meningitidis bacteria to Neisseria e.g., Neisseria Meningitidis
  • the pharmaceutical composition comprises at least 1 Neisseria (e.g., Neisseria Meningitidis) bacteriaium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,
  • the pharmaceutical composition comprises about 1 Neisseria (e.g., Neisseria Meningitidis) bacteriaium 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,
  • Neisseria e.g., Neisseria Meningitidis
  • the pharmaceutical composition comprises no more than 1 Neisseria (e.g., Neisseria Meningitidis) bacteriaium for every 1 , 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1 ,
  • Neisseria e.g., Neisseria Meningitidis
  • the pharmaceutical composition comprises at least 1 Neisseria (e.g., Neisseria Meningitidis) 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,
  • Neisseria e.g., Neisseria Meningitidis
  • the pharmaceutical composition comprises about 1 Neisseria (e.g., Neisseria Meningitidis) 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,
  • Neisseria e.g., Neisseria Meningitidis
  • Neisseria e.g ., Neisseria Meningitidis
  • the pharmaceutical composition comprises no more than 1 Neisseria (e.g., Neisseria Meningitidis)
  • Neisseria e.g ., Neisseria Meningitidis
  • bacteriaium e.g ., Neisseria Meningitidis
  • kits for treating a subject who has cancer comprising administering to the subject a pharmaceutical composition comprising Neisseria (e.g., Neisseria Meningitidis) bacteria (e.g., killed, live and/or attenuated
  • Neisseria e.g., Neisseria Meningitidis
  • bacteria e.g., killed, live and/or attenuated
  • 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 Neisseria (e.g., Neisseria Meningitidis) bacteria .
  • Neisseria e
  • the method further comprises administering to the subject an antibiotic.
  • the method further comprises administering to the subject one or more other cancer therapies (e.g., surgical removal of a tumor, the administration of a chemotherapeutic agent, the administration of radiation therapy, and/or the administration of a cancer immunotherapy, such as 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, and/or an adjuvant).
  • cancer therapies e.g., surgical removal of a tumor, the administration of a chemotherapeutic agent, the administration of radiation therapy, and/or the administration of a cancer immunotherapy, such as 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, and/or
  • the administration of the pharmaceutical composition reduces the dose of the cancer therapy (e.g., immune checkpoint inhibitor) that needs to be administered to the subject to achieve therapeutic efficacy.
  • the subject is administered a dose of the immune checkpoint inhibitor that is lower than the therapeutically effective dose of the immune checkpoint inhibitor when administered without the pharmaceutical composition.
  • the subject is administered a dose of the immune checkpoint inhibitor that is no more than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the therapeutically effective dose of the immune checkpoint inhibitor when administered without the pharmaceutical composition.
  • the dose is no more than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.9 mg/kg, 0.8 mg/kg, 0.7 mg/kg 0.6 mg/kg 0.5 mg/kg, 0.4 mg/kg, 0.3 mg/kg, 0.2 mg/kg or 0. lmg/kg.
  • the cancer is treated by the dose of the immune checkpoint inhibitor that is lower than the therapeutically effective dose of the immune checkpoint inhibitor when administered without administering the pharmaceutical composition.
  • the subject experiences fewer and/or less severe adverse reactions following administration of the lower dose of the immune checkpoint inhibitor compared to subjects who are administered the therapeutically effective dose of the immune checkpoint inhibitor when administered without the pharmaceutical composition.
  • 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 Neisseria Meningitidis EVs from the Bexsero® vaccine 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 Neisseria Meningitidis EVs from the Bexsero® vaccine compared to that of intraperitoneally (i.p.) administered anti-PD-l or vehicle in a mouse colorectal carcinoma model at day 11.
  • a subject e.g., a human subject
  • administering a pharmaceutical composition comprising Neisseria (e.g ., Neisseria Meningitidis) bacteria and/or a derivative of such bacteria (e.g., extracellular vesicles (EVs) and/or
  • Neisseria e.g ., Neisseria Meningitidis
  • a derivative of such bacteria e.g., extracellular vesicles (EVs) and/or
  • PhABs pharmaceutically 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
  • intradermal e.g
  • 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 (F) 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.
  • Cellular augmentation broadly refers to the influx of cells or expansion of cells in an environment that are not substantially present in the environment prior to administration of a composition and not present in the composition itself.
  • Cells that augment the environment include immune cells, stromal cells, bacterial and fungal cells. Environments of particular interest are the microenvironments where cancer cells reside or locate.
  • the microenvironment is a tumor microenvironment or a tumor draining lymph node.
  • the microenvironment is a pre-cancerous tissue site or the site of local administration of a composition or a site where the composition will accumulate after remote administration.
  • Clade refers to the OTUs or members of a phylogenetic tree that are
  • the clade comprises a set of terminal leaves in the phylogenetic tree that is a distinct monophyletic evolutionary unit and that share some extent of sequence similarity.“Operational taxonomic units,” OTU (or plural, “OTUs” ⁇ 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 (MUST), specific genes, or sets of genes may be genetically compared.
  • MUST multilocus sequence tags
  • OTUs that share 397% average nucleotide identity across the entire 16S or some variable region of the 16S are considered the same QTU (see e.g. C!aesson M .1 Wang Q, O'Sullivan O, Greene-Diniz R, Cole J R, Ros R P, and O'Toole P W. 2010. Comparison of two next-generation sequencing technologi es for resolving highly complex microbiota composition using tandem variable I6S rRNA gene regions. Nucleic Acids Res 38: e200.
  • O TUs 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. Such characterization employs, e.g., WGS data or a whole genome sequence.
  • A“combination” of two or more monoclonal microbial strains includes the physical co-existence of the two monoclonal microbial strains, either in the same material or product or in physically connected products, as well as the temporal co-administration or co localization of the monoclonal microbial strains.
  • 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.
  • 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.
  • engineered bacteria are any bacteria that have been genetically altered from their natural state by human intervention and the progeny of any such bacteria.
  • Engineered bacteria include, for example, the products of targeted genetic modification, the products of random mutagenesis screens and the products of directed evolution.
  • 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.
  • extracellular vesicle refers to a composition derived from a bacteria that comprises bacterial lipids, and bacterial proteins and/or bacterial nucleic acids and/or carbohydrate moieties contained in a nanoparticle. These EVs may contain 1, 2, 3, 4, 5, 10, or more than 10 different lipid species. EVs may contain 1, 2, 3, 4, 5, 10, or more than 10 different protein species. EVs may contain 1, 2, 3, 4, 5, 10, or more than 10 different nucleic acid species. EVs may contain 1, 2, 3, 4, 5, 10, or more than 10 different carbohydrate species.
  • 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.
  • Pharmaceutical compositions and the microbial components thereof are generally purified from residual habitat products.
  • an other entity or substance includes EVs.
  • LPS mutant or lipopolysaccharide 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 colistin).
  • Metal refers to any and all molecular compounds, compositions, molecules, ions, co-factors, catalysts or nutrients used as substrates in any cellular or microbial metabolic reaction or resulting as product compounds, compositions, molecules, ions, co-factors, catalysts or nutrients from any cellular or microbial metabolic reaction.
  • ‘Microbe” refers to any natural or engineered organism characterized as bacteria, fungus, microscopic alga, protozoan, and the stages of development or life cycle stages (e.g ., vegetative, spore (including sporulation, dormancy, and germination), latent, biofilm) associated with the organism.
  • gut microbes examples include: Actinomyces graevenitzii, Actinomyces odontolyticus, Akkermansia muciniphila, Bacteroides caccae, Bacteroides fragilis, Bacteroides putredinis, Bacteroides thetaiotaomicron, Bacteroides vultagus, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bilophila wadsworthia, Lactococcus lactis, Butyrivibrio,
  • Clostridia cluster III Clostridia cluster IV
  • Clostridia cluster IX (Acidaminococcaceae group)
  • Clostridia cluster XIV Clostridia cluster XV, Collinsella aerofaciens, Coprococcus, Corynebacterium sunsvallense, Desulfomonas pigra, Dorea formicigenerans, Dorea
  • Microbiome broadly refers to the microbes residing on or in body site of a subject or patient.
  • Microbes in a microbiome may include bacteria, viruses, eukaryotic microorganisms, and/or viruses.
  • Individual microbes in a microbiome may be metabolically active, dormant, latent, or exist as spores, may exist planktonically or in biofilms, or may be present in the microbiome in sustainable or transient manner.
  • the microbiome may be a commensal or healthy-state microbiome or a disease-state microbiome.
  • the microbiome may be native to the subject or patient, or components of the microbiome may be modulated, introduced, or depleted due to changes in health state (e.g ., precancerous or cancerous state) or treatment conditions (e.g., antibiotic treatment, exposure to different microbes).
  • the microbiome occurs at a mucosal surface.
  • the microbiome is a gut microbiome.
  • the microbiome is a tumor microbiome.
  • A‘microbiome profile” or a“microbiome signature” of a tissue or sample refers to an at least partial characterization of the bacterial makeup of a microbiome.
  • a microbiome profile indicates whether at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bacterial strains are present or absent in a microbiome.
  • Modified in reference to a bacteria broadly refers to a bacteria that has undergone a change from its wild-type form.
  • bacterial modifications include genetic modification, gene expression, phenotype modification, formulation, chemical modification, and dose or concentration. Examples of improved properties are described throughout this specification and include, e.g., attenuation, auxotrophy, homing, or antigenicity.
  • Phenotype modification might include, by way of example, bacteria growth in media that modify the phenotype of bacteriathat increase or decrease virulence.
  • 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.
  • polynucleotide and“nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • loci locus
  • polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide may be further modified, such as by conjugation with a labeling component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.
  • “Operational taxonomic units” 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.
  • pharmacologically active biomass or“PhABs” broadly refer to a composition containing pharmacologically active bacterial components, for example, derived from lysed or otherwise disrupted cells.
  • purify refers 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 preparation. It also refers to a composition that has been significantly enriched or concentrated. In some embodiments the EVs are concentrated by 2 fold, 3-fold, 4-fold, 5-fold, lO-fold, lOO-fold, lOOO-fold, 10, 000-fold or more than 10,000 fold.
  • 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.
  • “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.
  • Neisseria e.g., Neisseria Meningitidis
  • a derivative of such bacteria e.g., extracellular vesicles (EVs) and/or pharmaceutically active biomasses (PhABs)
  • EVs extracellular vesicles
  • PhABs pharmaceutically active biomasses
  • 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).
  • the bacteria is selected or engineered for reduced endotoxin content.
  • the bacteria is selected or engineered for reduced inflammatory molecules.
  • the Neisseria (e.g., Neisseria Meningitidis) bacteria can be cultured according to methods known in the art.
  • the Neisseria (e.g., Neisseria Meningitidis) bacteria can be grown in ATCC Medium 2722, ATCC Medium 1490, or other medium using methods disclosed, for example in Caballero et al, 2017.“Cooperating Commensals Restore Colonization Resistance to Vancomycin-Resistant Enterococcus faecium” Cell Host & Microbe 21 : 592-602, which is hereby incorporated by reference in its entirety.
  • the Neisseria (e.g., Neisseria Meningitidis) bacteria EVs described herein can be prepared using any method known in the art.
  • Neisseria Meningitidis bacteria EVs are isolated from the
  • Bexsero® vaccine such as those described in El.S. Patent 9,259,461, which is hereby
  • EVs also known as outer membrane vesicles, microvesicles or blebs
  • blebs outer membrane vesicles, microvesicles or blebs
  • the Neisseria (e.g., Neisseria Meningitidis) bacteria EVs are prepared without an EV purification step.
  • Neisseria (e.g., Neisseria Meningitidis) bacteria comprising the EVs described herein are killed using a method that leaves the Neisseria (e.g., Neisseria Meningitidis) bacteria EVs intact and the resulting bacterial components, including the EVs, are used in the methods and compositions described herein.
  • the Neisseria (e.g., Neisseria Meningitidis) bacteria bacteria are killed using an antibiotic (e.g., using an antibiotic described herein).
  • the Neisseria (e.g ., Neisseria Meningitidis) bacteria bacteria are killed using UV irradiation.
  • 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. Bin Park, et al. PLoS ONE. 6(3):el7629 (2011) or G. Norheim, et al. PLoS ONE. 10(9): e0l34353 (2015), each of which is hereby incorporated by reference in its entirety.
  • 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).
  • the culture supernatants are then passed through filter to exclude intact bacterial cells (e.g., a 0.22 pm filter).
  • filtered supernatants are centrifuged to pellet bacterial EVs (e.g., at 100,000-150,000 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 sucrose gradient (e.g., a 30-60% discontinuous sucrose gradient), followed by centrifugation (e.g., at 200,000 x g for 20 hours at 4°C).
  • EV bands can be collected, washed with (e.g., with PBS), and centrifuged to pellet the EVs (e.g., at 150,000 x g for 3 hours at 4°C).
  • the purified EVs can be stored, for example, at -80°C until use.
  • the EVs are further purified by treatment with DNase and/or proteinase K.
  • cultures of Neisseria e.g., Neisseria
  • Meningitidis bacteria disclosed herein can be centrifuged at 11,000 x g for 20-40 min at 4°C to pellet 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 Neisseria (e.g., Neisseria Meningitidis) bacteria EVs and other debris.
  • 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 Neisseria (e.g., Neisseria Meningitidis) bacteria EVs and other debris.
  • supernatants can be filtered so as to retain species of molecular weight > 50 or 100 kDa.
  • EVs can be obtained from Neisseria (e.g ., Neisseria Meningitidis) bacteria cultures continuously during growth, or at selected time points during growth, 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, 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
  • 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.
  • 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).
  • 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 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 derivatives 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/or 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 35% Optiprep in PBS. 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 35-60%
  • 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 ultracentrifugation (200,000 x g, > 3 hours, 4°C) and resuspension.
  • filtration e.g. Amicon Ultra columns
  • dialysis e.g. dialysis
  • ultracentrifugation 200,000 x g, > 3 hours, 4°C
  • 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 a
  • Neisseria e.g., Neisseria Meningitidis bacteria and/or a derivative of such bacteria (e.g., extracellular vesicles (EVs) and/or pharmaceutically active biomasses (PhABs)
  • the bacterial formulation comprises bacteriaand/or a combination of bacterial strains described herein and a pharmaceutically acceptable carrier (e.g., a pharmaceutical composition).
  • Neisseria e.g., Neisseria Meningitidis
  • substantially all of the bacteria in the pharmaceutical composition are Neisseria (e.g., Neisseria Meningitidis) bacteria .
  • the pharmaceutical composition comprises at least 1 x 10 3 colony forming units (CFUs), 1 x 10 4 colony forming units (CFUs), 1 x 10 5 colony forming units (CFUs), 5 x 10 5 colony forming units (CFUs), 1 x 10 6 colony forming units (CFUs), 2 x 10 6 colony forming units (CFUs), 3 x 10 6 colony forming units (CFUs), 4 x 10 6 colony forming units (CFUs), 5 x 10 6 colony forming units (CFUs), 6 x 10 6 colony forming units (CFUs), 7 x 10 6 colony forming units (CFUs), 8 x 10 6 colony forming units (CFUs), 9 x 10 6 colony forming units (CFUs), 1 x 10 7 colony forming units (CFUs), 2 x 10 7 colony forming units (CFUs), 3 x 10 7 colony forming units (CFUs), 4 x 10 6 colony
  • 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition are selected from among the bacterial species described herein. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition are selected from among the bacterial strains described herein.
  • compositions described herein may include only one species of bacteria described herein or may include two or more species of the bacteria described herein.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 species or strains, in any combination can be included in the compositions provided herein.
  • the bacterial and/or pharmaceutical composition comprises killed, live and/or attenuated bacteria.
  • 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 attenuated.
  • one or more mutations are introduced in the bacteria rendering it non-pathogenic.
  • Non-pathogenic mutations are generated using methods known to those skilled in the art (Propst KL et al. Infect Immun. 2010 Jul;78(7):3l36- 43).
  • Neisseria strain is fully attenuated.
  • 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.
  • the bacteria in the composition described herein are killed using heat (temperature) sterilization, filtration, and radiation using methods known to those skilled in the art (Garg M., see the World Wide Web at
  • the bacteria may be killed via E-beam using methods known to those skilled in the art (SiLiNDIR M. et al, FAB AD J. Pharm.
  • the bacteria in the composition described herein are killed and/or attenuated by a chemical agent, for example, aldehydes, e.g., formaldehyde, glutaraldehyde, and the like; food preservative agents such as SO2, sorbic acid, benzoic, acid, nitrate, and nitrite salts; gases such as ethylene oxide; halogens, such as iodine, chlorine, and the like; peroxygens, such as ozone, peroxide, peracetic acid; bisphenols; phenols; phenolics; biguanides, e.g., chlorhexidine; and the like.
  • aldehydes e.g., formaldehyde, glutaraldehyde, and the like
  • food preservative agents such as SO2, sorbic acid, benzoic, acid, nitrate, and nitrite salts
  • gases such as ethylene oxide
  • halogens such as iodine,
  • Bacteria may he 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 timepoints during exponential phase. In addition to inactivation by various methods, bacteria may be tested for efficacy using ifferent ratios of live versus inactivated cells, or different ratios of cells at various growth phases.
  • 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 timepo ts 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.
  • compositions comprising Neisseria (e.g ., Neisseria Meningitidis) EVs and/or Neisseria (e.g., Neisseria Meningitidis) bacteria provided herein (e.g., an EV composition), such as those disclosed in U.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 pharmaceutical compositions comprise Neisseria (e.g., a)
  • Neisseria Meningitidis EVs substantially or entirely free of bacteria.
  • the pharmaceutical compositions comprise both Neisseria (e.g., Neisseria Meningitidis) EVs and whole Neisseria (e.g., Neisseria Meningitidis) bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria).
  • the pharmaceutical compositions comprise Neisseria (e.g., Neisseria Meningitidis) bacteria that is substantially or entirely free of EVs.
  • the pharmaceutical composition comprises at least 1
  • Neisseria e.g., Neisseria Meningitidis
  • Neisseria Meningitidis bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8.
  • the pharmaceutical composition comprises about 1
  • Neisseria e.g., Neisseria Meningitidis
  • Neisseria Meningitidis bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8.
  • Neisseria e.g., Neisseria Meningitidis
  • the pharmaceutical composition comprises a certain ratio of Neisseria (e.g., Neisseria Meningitidis) bacteria particles to Neisseria (e.g., Neisseria
  • Neisseria Meningitidis e.g., Neisseria Meningitidis
  • the number of Neisseria (e.g., Neisseria Meningitidis) 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 Neisseria (e.g., Neisseria Meningitidis) EVs with a set number of purified Neisseria (e.g., Neisseria
  • Neisseria Meningitidis bacteria, by modifying the growth conditions under which the Neisseria (e.g., Neisseria Meningitidis) bacteria are cultured, or by modifying the Neisseria (e.g., Neisseria Meningitidis) bacteria itself to produce more or fewer Neisseria (e.g., Neisseria Meningitidis) EVs.
  • Neisseria e.g., Neisseria Meningitidis
  • Neisseria Meningitidis e.g., Neisseria Meningitidis
  • Meningitidis EVs and/or Neisseria (e.g., Neisseria Meningitidis) bacteria present in a bacterial sample
  • electron microscopy e.g., EM of ultrathin frozen sections
  • NTA nanoparticle tracking analysis
  • Coulter counting Coulter counting
  • DLS dynamic light scattering
  • 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 pharmaceutical composition comprises no more than 1
  • Neisseria e.g., Neisseria Meningitidis
  • Neisseria Meningitidis bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8.
  • the pharmaceutical composition comprises at least 1
  • Neisseria e.g., Neisseria Meningitidis
  • EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
  • the pharmaceutical composition comprises about 1
  • Neisseria e.g., Neisseria Meningitidis
  • EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
  • the pharmaceutical composition comprises no more than 1 Neisseria (e.g., Neisseria Meningitidis) 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.
  • Neisseria e.g., Neisseria Meningitidis
  • Neisseria e.g . , Neisseria Meningitidis
  • Neisseria e.g ., Neisseria Meningitidis
  • Neisseria e.g., Neisseria Meningitidis
  • Neisseria e.g., Neisseria Meningitidis
  • EV protein e.g., Neisseria Meningitidis
  • Neisseria e.g., Neisseria Meningitidis
  • the Neisseria (e.g., Neisseria Meningitidis) EVs in the pharmaceutical composition are purified from one or more other bacterial components.
  • the pharmaceutical composition further comprises other bacterial components.
  • the pharmaceutical composition comprise bacteria cells.
  • compositions that comprise monoclonal microbials that comprise monoclonal Neisseria (e.g., Neisseria Meningitidis) bacteria populations and a pharmaceutically acceptable carrier.
  • monoclonal microbials that comprise monoclonal Neisseria (e.g., Neisseria Meningitidis) bacteria populations and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions comprise monoclonal microbials substantially or entirely free of other microbes.
  • the pharmaceutical compositions comprise whole monoclonal microbials (e.g., live bacteria, killed bacteria, attenuated bacteria).
  • the pharmaceutical composition comprises a certain number monoclonal microbial particles. The number of monoclonal microbial particles can be based on actual particle number or (if the bacteria is live) the number of CFUs.
  • 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 pharmaceutical composition, a dietary supplement, 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 pharmaceutical composition for oral administration described herein comprises an additional component that enables efficient delivery of the bacteria to the colon.
  • pharmaceutical preparation that enables the delivery of the bacteria to the colon can be used.
  • examples of such 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.
  • the 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 pharmaceutical composition useful for delivery of the bacteria and/or bacterial derivative 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 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 U.S. Pat. No. 6,368,586, hereby
  • 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 are 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.
  • 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 a bacteria described herein 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 a bacteria described herein and 10% (w/w) corn starch in a capsule.
  • the capsule is enteric coated for duodenal release at pH 5.5
  • the capsule is enteric coated for duodenal release at pH 5.5.
  • the composition comprises a powder of freeze-dried bacteria of a bacteria described herein which is deemed“Qualified Presumption of Safety”
  • the composition is stable at frozen or refrigerated temperature.
  • Methods for producing pharmaceutical compositions 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 the bacterial strain described herein may be cultured by avoiding an isolation step.
  • the strains included in the 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 pharmaceutical composition may be cultivated alone, as a subset of the pharmaceutical composition, or as an entire collection comprising the pharmaceutical 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.
  • pharmaceutical compositions for human use this is often at 37°C temperature, pH, and other parameter with values similar to the normal human niche.
  • the environment may be actively controlled, passively controlled (e.g., via buffers), or allowed to drift.
  • an anoxic/reducing environment may be employed for anaerobic bacterial compositions. This can be accomplished by the 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).
  • Dried preservation removes water from the culture by evaporation (in the case of spray drying or‘cool drying’) or by sublimation (e.g., for freeze drying, spray freeze drying). Removal of water improves long-term pharmaceutical composition storage stability at temperatures elevated above cryogenic conditions. If the pharmaceutical composition comprises, for example, spore forming species and results in the production of spores, the final composition may be purified by additional means such as density gradient centrifugation. Pharmaceutical composition banking may be done by culturing and preserving the strains individually, or by mixing the strains together to create a combined bank.
  • a pharmaceutical 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 pharmaceutical 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
  • a pharmaceutical composition may be cultivated to a concentration of 10 10 CFU/mL, then concentrated 20-fold by tangential flow microfiltration; the spent medium may be exchanged by diafiltering with a preservative medium consisting of 2% gelatin, 100 mM trehalose, and 10 mM sodium phosphate buffer. The suspension can then be freeze-dried to a powder and titrated.
  • the powder may be blended to an appropriate potency, and mixed with other cultures and/or a filler such as microcrystalline cellulose for consistency and ease of handling, and the pharmaceutical composition formulated as provided herein.
  • compositions for administration subjects are combined with additional active and/or inactive materials in order to produce a final product, which may be in single dosage unit or in a multi-dose format.
  • the composition comprises at least one carbohydrate.
  • a “carbohydrate” refers to a sugar or polymer of sugars. The terms“saccharide,”“polysaccharide,” “carbohydrate,” and“oligosaccharide” may be used interchangeably. Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one on each carbon atom of the molecule. Carbohydrates generally have the molecular formula CnTknOn. 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 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 pharmaceutical composition comprises an enteric coating and/or microcapsules that dissolves at a pH associated with a particular region of the gastrointestinal tract.
  • the enteric coating and/or microcapsules dissolve at a pH of about 5.5 - 6.2 to release in the duodenum, at a pH value of about 7.2 - 7.5 to release in the ileum, and/or at a pH value of about 5.6 - 6.2 to release in the colon.
  • Exemplary enteric coatings and microcapsules are described, for example, in U.S. Pat. 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
  • 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.
  • cultures e.g., large scale cultures, of monoclonal bacteria.
  • Such cultures have a threshold heterogeneity index, e.g., a heterogeneity index greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more than about 99%).
  • Monoclonal microbial cultures may comprise, e.g., at least 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or l0 14 bacteria.
  • monoclonal microbial cultures may comprise volumes of at least 200pL, 300pL, 400pL, 500pL, 600pL, 700pL, 800pL, 900pL, lOOOpL, 2mL , 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, lOmL , 25mL, 50mL, 75mL, lOOmL, 200mL, 300mL, 400mL, 500mL, 600mL, 700mL, 800mL, 900mL, lOOOmL, 2L , 3L, 4L, 5L,
  • a bioreactor comprising a monoclonal bacteria composition having a threshold heterogeneity index, e.g., a heterogeneity index greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more than about 99%.
  • the bioreactor may be bubble columns, steel tank bioreactors, or any other commercial or large scale bioreactor known in the arts.
  • such cultures include a population of bacteria derived from a limited number of clones, e.g., ⁇ 10, 9, 8, 7, 6, 5, 4, 3, 2 or derived from a single clone
  • a monoclonal bacteria culture is generated by the process of: isolating a bacterial clone for culture by serial dilution or some other process of isolating a single bacterial clone; growing such clone into a resultant bacterial culture until it reaches a desired volume, density, or number of bacteria; evaluating the heterogeneity index of the resultant culture; and preparing a pharmaceutical preparation from the culture (e.g, formulating the culture as a drug product) if the heterogeneity index of the culture meets a predetermined threshold.
  • the predetermined threshold is greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more than about 99%.
  • the culture comprises at least at least 200m1 at a density optimum for bacterial cell growth.
  • monoclonal microbials are generated by dilution of bacterial cells comprising a bacteria of interest.
  • bacterial cells may be diluted in a dilution series until only approximately one cell is present in a given culture, such cell then being grown in culture to produce a monoclonal bacterial population.
  • monoclonal microbials are generated by serial plating. For example, a sample containing a bacteria of interest is streaked on an agar plate, allowed to to grow, and subsequently a colony of material is selected for streaking to repeat the process.
  • monoclonal microbials e.g., monoclonal bacteria
  • monoclonal microbials are generated by a combination of dilution and serial plating.
  • selected monoclonal microbials are isolated and 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 monoclonal microbials described herein may be indexed using the heterogeneity /diversity metric disclosed below.
  • this metric may help differentiate products made via different processes and from different sources (e.g., the same strain produced by two different methods, strains grown in a fermenter and passaged through a host).
  • H Length of all high-scoring segment pairs (HSPs)
  • the SNPs, insertions and deletions, noted above are defined by comparison to a reference genome with consistent polymorphisms at a sight above a critical threshold.
  • the numeric value, three is a weighting parameter that is set in the index above but can be adjusted empirically looking at various uses of this parameter.
  • the monoclonal microbials described herein may be indexed using transcriptomics in different conditions to identify key transcripts to measure subsequently. For example, sequencing the reference genome of a product strain with illumine and pac bio sequencing, identifying core SNPs that differentiate a monoclonal microbial strain from closely related strains, sequencing subsequent preparations of monoclonal microbial from master cell bank, working cell bank, production batchs, conversion of mutational hot spots to targeted sequencing regions for faster measure of diversity index, or calculating heterogeneity index in various conditions
  • provided herein is a method of delivering bacteriaand/or a pharmaceutical composition described herein to a subject.
  • the bacteria are administered in conjunction with the administration of an additional therapeutic.
  • the bacteria is co-formulated in a pharmaceutical composition with the additional therapeutic.
  • the bacteria and/or bacterial derviative is co-administered with the additional therapeutic.
  • the additional 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,
  • the additional therapeutic is administered to the subject after administration of the bacteria (e.g., about 1, 2, 3,
  • the same mode of delivery is used to deliver both the bacteria and/or bacterial derviative and the additional therapeutic.
  • different modes of delivery are used to administer the bacteria and/or bacterial derviative and the additional therapeutic.
  • the bacteria and/or bacterial derviative is administered orally while the additional 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-immune disorder treatment. 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.
  • the methods of treatment described herein may be suitable for the treatment of an immune disorder (e.g ., an autoimmune disease, an inflammatory disease, an allergy).
  • 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 the immune disorder, delay its onset, or slow or stop its progression or prevent a relapse of the immune disorder.
  • 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 therapy, among other factors affecting the selected dosage.
  • the dose should be sufficient to result in slowing, and preferably regressing, the advancement of an immune disorder.
  • 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 bacteria, 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 an immune disorder therapeutic in combination with the bacteria and/or bacterial dervative described herein reduces the adverse effects and/or improves the efficacy of the immune disorder therapeutic.
  • the effective dose of an immune disorder therapeutic described herein is the amount of 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 an immune disorder therapy will be the amount of 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 an immune disorder therapy is the level of adverse effects experienced by the subject during and following treatment.
  • Adverse events associated with immune disorder 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, dyspepsi
  • hyperkalemia hyperlipasemia, hypermagnesemia, hypernatremia, hyperphosphatemia, hyperpigmentation, hypertriglyceridemia, hyperuricemia, hypoalbuminemia, hypocalcemia, hypochloremia, hypoglycemia, hypokalemia, hypomagnesemia, hyponatremia,
  • hypophosphatemia impotence, infection, injection site reactions, insomnia, iron deficiency, itching, joint pain, kidney failure, leukopenia, liver dysfunction, memory loss, menopause, mouth sores, mucositis, muscle pain, myalgias, myelosuppression, myocarditis, neutropenic fever, nausea, nephrotoxicity, neutropenia, nosebleeds, numbness, ototoxicity, pain, palmar- plantar erythrodysesthesia, pancytopenia, pericarditis, peripheral neuropathy, pharyngitis, photophobia, photosensitivity, pneumonia, pneumonitis, proteinuria, pulmonary embolus, pulmonary fibrosis, pulmonary toxicity, rash, rapid heart beat, rectal bleeding, restlessness, rhinitis, seizures, shortness of breath, sinusitis, thrombocytopenia, tinnitus, urinary tract infection, vaginal bleeding, vaginal dryness, vertigo, water retention, weakness, weight
  • the administration of the pharmaceutical composition treats the disease (e.g ., cancer, auto-immune disease, inflammatory disease, metabolic disease).
  • the disease e.g ., cancer, auto-immune disease, inflammatory disease, metabolic disease.
  • the methods provided herein include the administration to a subject of bacteria, bacterial derivative and/or a pharmaceutical composition described herein pharmaceutical compositioneither alone or in combination with another therapeutic.
  • the pharmaceutical composition and the other therapy can be administered to the subject in any order.
  • the pharmaceutical composition and the other therapy are administered conjointly.
  • the bacteriais administered to the subject before the additional therapeutic is administered (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days before).
  • the bacteriais administered to the subject after the additional therapeutic is administered (e.g, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours after or at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
  • the bacteriaand the additional 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 bacteriais administered to the subject (e.g., at least 1, 2, 3,
  • the subject is administered an antibiotic after the bacteriais 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 bacteriais administered to the subject (e.g ., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours after or at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • the bacteriaand 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 additional therapeutic is an antibiotic.
  • antibiotics can be administered to eliminate the immune-disorder-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 bioavailability, or their spectrum of target microbe (e.g ., Gram-negative vs. Gram-positive bacteria, aerobic vs. anaerobic bacteria, etc.) and these may be used to kill specific bacteria in specific areas of the host (“niches”) (Leekha, et al 2011. General Principles of Antimicrobial Therapy. Mayo Clin Proc. 86(2): 156-167). In certain embodiments, antibiotics can be used to selectively target bacteria of a specific niche.
  • target microbe e.g ., Gram-negative vs. Gram-positive bacteria, aerobic vs. anaerobic bacteria, etc.
  • antibiotics known to treat a particular infection that includes an immune disorder niche may be used to target immune-disorder-associated microbes, including immune-disorder- 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. Furthermore, while 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. In certain treatment conditions, bacteriostatic antibiotics inhibit the activity of bactericidal antibiotics. Thus, in certain embodiments, bactericidal and bacteriostatic antibiotics are not combined.
  • Antibiotics include, but are not limited to aminoglycosides, ansamycins, carbacephems, carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptide antibiotics, quinolones, fluoroquinolone, sulfonamides, tetracyclines, and anti-mycobacterial compounds, and combinations thereof.
  • Aminoglycosides include, but are not limited to Amikacin, Gentamicin,
  • Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, and Spectinomycin are examples of the compounds listed in the following paragraphs.
  • Aminoglycosides are effective, e.g., against Gram-negative bacteria, such as Escherichia coli, Klebsiella, Pseudomonas aeruginosa, and Francisella tularensis, and against certain aerobic bacteria but less effective against obligate/facultative anaerobes. Aminoglycosides are believed to bind to the bacterial 30S or 50S ribosomal subunit thereby inhibiting bacterial protein synthesis.
  • Ansamycins include, but are not limited to, Geldanamycin, Herbimycin, Rifamycin, and Streptovaricin.
  • Geldanamycin and Herbimycin are believed to inhibit or alter the function of Heat Shock Protein 90.
  • Carbacephems include, but are not limited to, Loracarbef. Carbacephems are believed to inhibit bacterial cell wall synthesis.
  • Carbapenems include, but are not limited to, Ertapenem, Doripenem,
  • Carbapenems are bactericidal for both Gram-positive and Gram-negative bacteria as broad-spectrum antibiotics. Carbapenems are believed to inhibit bacterial cell wall synthesis.
  • Cephalosporins include, but are not limited to, Cefadroxil, Cefazolin, Cefalotin, Cefalothin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil,and Ceftobiprole. Selected
  • Cephalosporins are effective, e.g., against Gram-negative bacteria and against Gram-positive bacteria, including Pseudomonas, certain Cephalosporins are effective against methicillin- resistant Staphylococcus aureus (MRSA). Cephalosporins are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Glycopeptides include, but are not limited to, Teicoplanin, Vancomycin, and Telavancin. Glycopeptides are effective, e.g, against aerobic and anaerobic Gram-positive bacteria including MRSA and Clostridium difficile. Glycopeptides are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Lincosamides include, but are not limited to, Clindamycin and Lincomycin.
  • Lincosamides are effective, e.g, against anaerobic bacteria, as well as Staphylococcus, and Streptococcus. Lincosamides are believed to bind to the bacterial 50S ribosomal subunit thereby inhibiting bacterial protein synthesis.
  • Lipopeptides include, but are not limited to, Daptomycin. Lipopeptides are effective, e.g, against Gram-positive bacteria. Lipopeptides are believed to bind to the bacterial membrane and cause rapid depolarization.
  • Macrolides include, but are not limited to, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, and Spiramycin. Macrolides are effective, e.g, against Streptococcus and Mycoplasma. Macrolides are believed to bind to the bacterial 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 methods provided herein further comprise administering another cancer therapeutic to the subject.
  • the additional therapeutic is a cancer therapeutic.
  • the cancer therapeutic is a chemotherapeutic agent. Examples of such
  • chemotherapeutic agents include, but are not limited to, alkylating agents such as
  • 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 triethiylenethiophosphoramide and trimethylolomelamine
  • acetogenins especially bullatacin and bullatacinone
  • a camptothecin including the synthetic analogue topotecan
  • bryostatin especially the synthetic analogue topotecan
  • 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 gamma II 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, cyanomorphobno-doxorubicin, 2-pyrrobno- doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenob
  • demecolcine diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;
  • losoxantrone podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;
  • pipobroman gacytosine; arabinoside ("Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine;
  • methotrexate platinum coordination complexes such as cisplatin, oxabplatin 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, oxabplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincris
  • 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.
  • 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).
  • 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. It is not necessary that there be perfect complementarity between the siRNA molecule and the target, but the
  • the sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double-strand character of the molecule.
  • an siRNA molecule may be modified or include nucleoside surrogates.
  • Single stranded regions of an siRNA molecule may be modified or include nucleoside surrogates, e.g., the unpaired region or regions of a hairpin structure, e.g., a region which links two complementary regions, can have modifications or nucleoside surrogates.
  • Modification to stabilize one or more 3'- or 5'-terminus of an siRNA molecule, e.g., against exonucleases, or to favor the antisense siRNA agent to enter into RISC are also useful.
  • Modifications can include C3 (or C6, C7, Cl 2) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, Cl 2, abasic, tri ethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that come as phosphoramidites and that have another DMT- protected hydroxyl group, allowing multiple couplings during RNA synthesis.
  • Each strand of an siRNA molecule can be equal to or less than 35, 30, 25, 24, 23, 22, 21, or 20 nucleotides in length. In some embodiments, the strand is at least 19 nucleotides in length. For example, each strand can be between 21 and 25 nucleotides in length. In some embodiments, siRNA agents have a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more overhangs, such as one or two 3' overhangs, of 2-3 nucleotides.
  • the immune checkpoint inhibitor is a shRNA molecule.
  • a “small hairpin RNA” or“short hairpin RNA” or“shRNA” includes a short RNA sequence that makes a tight hairpin turn that can be used to silence gene expression via RNA interference.
  • the shRNAs provided herein may be chemically synthesized or transcribed from a transcriptional cassette in a DNA plasmid. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • shRNAs are about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, or are about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g ., each complementary sequence of the double-stranded shRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, or about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded shRNA is about 15- 60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, or about 18-22, 19-20, or 19-21 base pairs in length).
  • shRNA duplexes may comprise 3’ overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides on the antisense strand and/or 5’-phosphate termini on the sense strand.
  • the shRNA comprises a sense strand and/or antisense strand sequence of from about 15 to about 60 nucleotides in length (e.g., about 15-60, 15-55, 15- 50, 15-45, 15-40, 15-35, 15-30, or 15-25 nucleotides in length), or from about 19 to about 40 nucleotides in length (e.g., about 19-40, 19-35, 19-30, or 19-25 nucleotides in length), or from about 19 to about 23 nucleotides in length (e.g., 19, 20, 21, 22, or 23 nucleotides in length).
  • Non-limiting examples of shRNA include a double-stranded polynucleotide molecule assembled from a single-stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; and a double-stranded polynucleotide molecule with a hairpin secondary structure having self-complementary sense and antisense regions.
  • the sense and antisense strands of the shRNA are linked by a loop structure comprising from about 1 to about 25 nucleotides, from about 2 to about 20 nucleotides, from about 4 to about 15 nucleotides, from about 5 to about 12 nucleotides, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides.
  • 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.
  • oligonucleotides may have about or at least about 70% sequence complementarity, e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity, between the oligonucleotide and the target sequence.
  • Oligonucleotide backbones that are less susceptible to cleavage by nucleases are discussed herein.
  • Mismatches are typically less destabilizing toward the end regions of the hybrid duplex than in the middle.
  • the number of mismatches allowed will depend on the length of the oligonucleotide, the percentage of G:C base pairs in the duplex, and the position of the mismatch(es) in the duplex, according to well understood principles of duplex stability.
  • the inhibitory nucleic acid molecule can be prepared, for example, by chemical synthesis, in vitro transcription, or digestion of long dsRNA by Rnase III or Dicer. These can be introduced into cells by transfection, electroporation, or other methods known in the art. See Hannon, GJ, 2002, RNA Interference, Nature 418: 244-251; Bernstein E et al., 2002, The rest is silence. RNA 7: 1509-1521 ; Hutvagner G et al., RNAi: Nature abhors a double-strand. Curr. Opin.
  • Short hairpin RNAs induce sequence-specific silencing in mammalian cells. Genes & Dev. 16:948-958; Paul CP, Good PD, Winer I, and Engelke DR. (2002). Effective expression of small interfering RNA in human cells. Nature Biotechnol. 20:505-508; Sui G, Soohoo C, Affar E-B, Gay F, Shi Y, Forrester WC, and Shi Y. (2002). A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl.
  • RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells Proc. Natl. Acad. Sci. ETSA 99(9): 6047-6052.
  • the inhibitory nucleic acid molecule can be administered to the subject, for example, as naked nucleic acid, in combination with a delivery reagent, and/or as a nucleic acid comprising sequences that express an interfering nucleic acid molecule.
  • the nucleic acid comprising sequences that express the interfering nucleic acid molecules are delivered within vectors, e.g. plasmid, viral and bacterial vectors. Any nucleic acid delivery method known in the art can be used in the methods described herein. Suitable delivery reagents include, but are not limited to, e.g., the Mirus Transit TKO lipophilic reagent;
  • atelocollagen as a delivery vehicle for nucleic acid molecules is described in Minakuchi et al. Nucleic Acids Res., 32(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 8,324,366, each of which is hereby incorporated by reference in its entirety.
  • 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 cob Nissle 1917, Escherichia cob K-12/LLO, Listeria monocytogenes, and Shigella flexneri.
  • viral vectors include, but are not limited to, vaccinia, adenovirus, RNA viruses, and replicationdefective avipox, replication-defective fowlpox, replication-defective canarypox, replicationdefective 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,
  • Kallikrein 4 KIF20A, KK-LC-l, KKLC1, KM-HN-l, KMHN1 also known as CCDC110, LAGE-l, LDLR-fucosyltransferaseAS fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE- A10, MAGE-A12, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A/MART-l, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-l, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR-l, NY-
  • the cancer immunotherapy comprises administration of a cancer-specific chimeric antigen receptor (CAR).
  • CAR cancer-specific chimeric antigen receptor
  • the CAR is administered on the surface of a T cell.
  • 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 THl 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 ODN DNA, GPI-0100, lipid A, lipopolysaccharide, Lipovant, Montanide, N-acetyl- muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A , cholera toxin (CT) and heat- labile toxin from enterotoxigenic Escherichia coli (LT) including derivatives of these (CTB, mmCT, CTA1- DD, LTB, LTK63, LTR72, dmLT) and trehalose dimycolate.
  • an immune modulatory protein Adjuvant 65, a-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, b-Glucan Peptide, CpG ODN DNA
  • the immunotherapy agent is an immune modulating protein to the subject.
  • the immune modulatory protein is a cytokine or chemokine.
  • 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”),
  • GM-CSF Granulocyte macrophage colony-stimulating factor
  • IMM-l Intercellular Adhesion Molecule 1
  • IFN-alpha Interferon alpha
  • IFN-beta Interferon beta
  • IFN-gamma Interferon gamma
  • IL-l alpha Interlukin-l alpha
  • IL-l beta Interleukin-l beta
  • IL-1 receptor antagonist Interleukin 1 receptor antagonist
  • IL-2 Interleukin-2
  • IL-4 Interleukin-4
  • IL-5 Interleukin-6
  • IL-6 soluble receptor Interleukin-7
  • IL-8 Interleukin-8
  • Interleukin- 10 Interleukin- 11
  • IL-12 p40 Interleukin- 11
  • IL-12 p70 Interleukin- 12
  • TIMP metallopeptidase inhibitor 2 TIMP metallopeptidase inhibitor 2
  • TNF alpha Tumor necrosis factor
  • TNF beta Tumor necrosis factor
  • Soluble TNF receptor type 1 sTNFRI
  • sTNFRIIAR Brain-derived neurotrophic factor
  • BDNF Brain-derived neurotrophic factor
  • BFGF Basic fibroblast growth factor
  • BMP-4 Bone morphogenetic protein 4
  • BMP-5" Bone morphogenetic protein 5
  • BMP-7 Bone morphogenetic protein 7
  • Nerve growth factor b-NGF
  • EGF Epidermal growth factor
  • EGFR Epidermal growth factor receptor
  • FGF-7 Endocrine- gland-derived vascular endothelial growth factor
  • FGF-7 Keratinocyte growth factor
  • GDF-15 Glial cell-derived neurotrophic factor
  • GDF-15 Glial cell-derived neurotrophic factor
  • GDNF Glial cell-derived neurotrophic factor
  • Osteoclastogenesis inhibitory factor (“Osteoprotegerin”), Platelet-derived growth factor receptors (“PDGF-AA”), Phosphatidylinositol-glycan biosynthesis (“PIGF”), Skp, Cullin, F-box containing comples (“SCF”), Stem cell factor receptor (“SCF R”), Transforming growth factor alpha ("TGFalpha”), Transforming growth factor beta-l (“TGF beta 1 "), Transforming growth factor beta-3 (“TGF beta 3”), Vascular endothelial growth factor (“VEGF”), Vascular endothelial growth factor receptor 2 (“VEGFR2”), Vascular endothelial growth factor receptor 3
  • VFGFR3 VEGF-D 6Ckine, Tyrosine-protein kinase receptor UFO (“Axl”), Betacellulin (“BTC”), Mucosae-associated epithelial chemokine (“CCL28”), Chemokine (C-C motif) ligand 27 (“CTACK”), Chemokine (C-X-C motif) ligand 16 (“CXCL16”), C-X-C motif chemokine 5 (“ENA-78”), Chemokine (C-C motif) ligand 26 (“Eotaxin-3”), Granulocyte chemotactic protein 2 ("GCP-2"), GRO, Chemokine (C-C motif) ligand 14 (“HCC-l”), Chemokine (C-C motif) ligand 16 (“HCC-4"), Interleukin-9 (“IL-9”), Interleukin- 17 F (“IL-17F”), Interleukin- 18-binding protein (“IL-18 BPa”), Interleukin-28
  • TGF beta 2 Transforming growth factor-beta 2
  • Tie-2 Tie-2
  • TPO Tumor necrosis factor receptor superfamily member 10D
  • TRAIL R4 Tumor necrosis factor receptor superfamily member 10D
  • TEM-l Tumor necrosis factor receptor superfamily member 10D
  • TEM-l Tumor necrosis factor receptor superfamily member 10D
  • TEM-l Triggering receptor expressed on myeloid cells 1
  • VEGF-C Vascular endothelial growth factor C
  • VEGFRlAdiponectin Adipsin ("AND), Alpha-fetoprotein (“AFP”), Angiopoietin-like 4 (“ANGPTL4"), Beta-2-microglobulin (“B2M”), Basal cell adhesion molecule (“BCAM”), Carbohydrate antigen 125 (“CA125”), Cancer Antigen 15-3 (“CA15-3"), Carcinoembryonic antigen (“CEA”), cAMP receptor protein (“CRP”), Human Epidermal Growth Factor Receptor 2 (“ErbB2”), Follistatin, Follicle-stimulating hormone (“FSH”), Chemokine (C-X-C motif) ligand 1 (“GRO alpha”), human chorionic gonadotropin (“beta HCG”), Insulin-like growth factor 1 receptor (“IGF-l sR”), IL-l sRII, IL-3, IL-18 Rb, IL-21, Leptin, Matrix metalloproteinase- 1 (“MMP-l
  • 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
  • GPCR-associated sorting protein 1 GASP-l
  • GPCR-associated sorting protein 2 GASP-2
  • GPCR-associated sorting protein 2 GASP-2
  • GSF 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 Pepsinogen I
  • RBP4 Retinol binding protein 4
  • SOST Heparan sulfate proteoglycan
  • TACI Tumor necrosis factor receptor superfamily member 13B
  • TACI Tumor necrosis factor receptor superfamily member 13B
  • TACI Tumor necrosis factor receptor superfamily member 13B
  • TACI Tumor necrosis factor receptor superfamily member 13B
  • TACI Tumor necrosis factor receptor superfamily member 13B
  • TACI Tumor necrosis factor receptor super
  • the cancer therapeutic agent is an anti-cancer compound.
  • anti-cancer compounds include, but are not limited to, Alemtuzumab (Campath®), Alitretinoin (Panretin®), Anastrozole (Arimidex®), Bevacizumab (Avastin®), Bexarotene (Targretin®), Bortezomib (Velcade®), Bosutinib (Bosulif®), Brentuximab vedotin (Adcetris®), Cabozantinib (CometriqTM), Carfilzomib (KyprobsTM), Cetuximab (Erbitux®), Crizotinib (Xalkori®), Dasatinib (Sprycel®), Denileukin diftitox (Ontak®), Erlotinib hydrochloride (Tarceva®), Everolimus (Afinitor®), Exe
  • Ipilimumab (YervoyTM), Lapatinib ditosylate (Tykerb®), Letrozole (Femara®), Nilotinib (Tasigna®), Ofatumumab (Arzerra®), Panitumumab (Vectibix®), Pazopanib hydrochloride (Votrient®), Pertuzumab (PerjetaTM), Pralatrexate (Folotyn®), Regorafenib (Stivarga®), Rituximab (Rituxan®), Romidepsin (Istodax®), Sorafenib tosylate (Nexavar®), Sunitinib malate (Sutent®), Tamoxifen, Temsirolimus (Torisel®), Toremifene (Fareston®), Tositumomab and l3 lI-tositumomab (Bexxar®), Tra
  • Exemplary anti-cancer compounds that modify the function of proteins that regulate gene expression and other cellular functions are Vorinostat (Zolinza®), Bexarotene (Targretin®) and Romidepsin (Istodax®), Alitretinoin (Panretin®), and Tretinoin (Vesanoid®).
  • Exemplary anti-cancer compounds that induce apoptosis are Bortezomib (Velcade®), Carfilzomib (KyprolisTM), and Pralatrexate (Folotyn®).
  • anti-cancer compounds that increase anti-tumor immune response are Rituximab (Rituxan®), Alemtuzumab (Campath®), Ofatumumab (Arzerra®), and Ipilimumab (YervoyTM).
  • exemplary anti-cancer compounds are small molecule inhibitors and conjugates thereof of, e.g., Janus kinase, ALK, Bcl-2, PARP, PI3K, VEGF receptor, Braf, MEK, CDK, and HSP90.
  • Exemplary platinum-based anti-cancer compounds include, for example, cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, Nedaplatin, Triplatin, and Lipoplatin.
  • Other metal-based drugs suitable for treatment include, but are not limited to ruthenium-based compounds, ferrocene derivatives, titanium-based compounds, and gallium-based compounds.
  • 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, Pt-l95m, Ce-l4l, 1-131, Tb-l6l, As-77, Pt-l97, Sm
  • 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.
  • the methods and compositions described herein relate to the treatment of cancer.
  • any cancer can be treated using the methods described herein.
  • cancers that may treated by methods and compositions described herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular
  • adenocarcinoma adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp;
  • adenocarcinoma familial polyposis coli
  • solid carcinoma carcinoid tumor, malignant
  • branchiolo-alveolar adenocarcinoma branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;
  • acidophil carcinoma acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary
  • cystadenocarcinoma papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma;
  • rhabdomyosarcoma alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma;
  • mesothelioma malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant;
  • lymphangiosarcoma osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma;
  • chondroblastoma malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; n
  • 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, plasmacytoma, rectal cancer, Merkel Cell carcinoma, salivary gland carcinoma, 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, 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, leimyosarcoma, rhabdotheliosarcoma, invasive ductal carcinoma, papillary adenocarcinoma, melanoma, pulmonary squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (well differentiated, moderately differentiated, poorly differentiated or undifferentiated), bronchioloalveolar carcinoma, renal cell carcinoma, hypernephroma, hypernephroid adenocarcinoma, bile duct carcinoma,
  • Cancers treated in certain embodiments also include precancerous lesions, e.g., actinic keratosis (solar keratosis), moles (dysplastic nevi), acitinic chelitis (farmer's lip), cutaneous horns, Barrett's esophagus, atrophic gastritis, dyskeratosis congenita, sideropenic dysphagia, lichen planus, oral submucous fibrosis, actinic (solar) elastosis and cervical dysplasia.
  • precancerous lesions e.g., actinic keratosis (solar keratosis), moles (dysplastic nevi), acitinic chelitis (farmer's lip), cutaneous horns, Barrett's esophagus, atrophic gastritis, dyskeratosis congenita, sideropenic dysphagia, lichen
  • 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.
  • compositions for use of treating cancer are disclosed.
  • a pharmaceutical composition comprising Neisseria bacteria and a pharmaceutically acceptable carrier (e.g., as described herein) for use in treating cancer is described herein.
  • a pharmaceutical composition comprising isolated Neisseria extracellular vesicles (EVs) and a pharmaceutically acceptable carrier (e.g., as described herein) for use in treating cancer is described herein.
  • a pharmaceutical composition comprising Neisseria extracellular vesicles (EVs), Neisseria bacteria, and a pharmaceutically acceptable carrier (e.g., as described herein) for use in treating cancer is described herein.
  • a pharmaceutical composition comprising Neisseria bacteria isolated from EVs for use in treating cancer is described herein.
  • a pharmaceutical composition comprising a pharmaceutically active biomass (PhAB) derived from Neisseria bacteria for use in treating cancer is described herein.
  • a pharmaceutical composition comprising a pharmaceutically active biomass (PhAB) isolated from Neisseria bacteria for use in treating cancer is described herein.
  • a pharmaceutical composition comprising a monoclonal microbial population (monoclonal microbials) derived from Neisseria bacteria for use in treating cancer is described herein.
  • a pharmaceutical composition for the preparation of a medicament for treating cancer uses of a pharmaceutical composition for the preparation of a medicament for treating cancer.
  • use of a pharmaceutical composition for the preparation of a medicament for treating cancer wherein the pharmaceutical composition comprises Neisseria bacteria and a pharmaceutically acceptable carrier is described herein.
  • the pharmaceutical composition comprises isolated Neisseria extracellular vesicles (EVs) and a pharmaceutically acceptable carrier is described herein.
  • use of a pharmaceutical composition for the preparation of a medicament for treating cancer wherein the pharmaceutical composition comprises Neisseria extracellular vesicles (EVs), Neisseria bacteria, and a pharmaceutically acceptable carrier is described herein.
  • use of a pharmaceutical composition for the preparation of a medicament for treating cancer wherein the pharmaceutical composition comprises Neisseria bacteria isolated from EVs is described herein.
  • use of a pharmaceutical composition for the preparation of a medicament for treating cancer wherein the pharmaceutical composition comprises a pharmaceutically active biomass (PhAB) derived from Neisseria bacteria is described herein.
  • a pharmaceutical composition for the preparation of a medicament for treating cancer wherein the pharmaceutical composition comprises a pharmaceutically active biomass (PhAB) isolated from Neisseria bacteria is described herein.
  • a pharmaceutical composition for the preparation of a medicament for treating cancer wherein the pharmaceutical composition comprises a monoclonal microbial population (monoclonal microbials) derived from Neisseria bacteria is described herein.
  • Neisseria EVs are Neisseria EVs. In further embodiments, at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,
  • Neisseria bacteria 97%, 98% or 99% of the total Neisseria EV and bacteria particles in the pharmaceutical composition are Neisseria bacteria.
  • Neisseria EV protein 99% of the total Neisseria EV and Neisseria bacteria protein in the pharmaceutical composition is Neisseria EV protein.
  • Neisseria bacteria protein a Neisseria bacteria protein.
  • Neisseria EV and bacteria lipids in the pharmaceutical composition are Neisseria EV lipids.
  • Neisseria EV and Neisseria bacteria lipids in the pharmaceutical composition are Neisseria bacteria lipids.
  • the EVs and/or bacteria are from Neisseria Meningitidis.
  • the bacteria is selected for reduced endotoxin content.
  • the bacteria is engineered for reduced endotoxin content.
  • the bacteria is selected or engineered for reduced inflammatory molecules.
  • the cancer is selected from the group consisting of hematological malignancy, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukemia, le
  • 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, Rous sarcoma, serocystic sarcoma, synovial sarcoma, telangiectaltic sarcoma, 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, 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'
  • the cancer is prostate cancer, lung cancer, colon cancer, colorectal cancer, melanoma, breast cancer, pancreatic cancer, hepatocellular carcinoma, or lymphoma. In some embodiments, the cancer is prostate cancer.
  • the pharmaceutical composition is administered orally, rectally, intravenously, intratumorally, subtumorally, intradermally, intraperitoneally, or subcutaneously. In some embodiments, the pharmaceutical composition is administered in two or more doses. In some embodiments, the administration to the subject of the two or more doses are separated by at least 1 day. In some embodiments, the administration of the two or more doses are separated by at least 1 week. In some embodiments, the pharmaceutical composition comprises live bacteria. In some embodiments, the
  • composition comprises attenuated bacteria. In some embodiments, the pharmaceutical composition comprises killed bacteria. In some embodiments, the bacterial composition comprises irradiated bacteria. In some embodiments, the bacterial composition comprises gamma irradiated bacteria. In some embodiments, the Neisseria bacteria is resistant to polymyxin. In some embodiments, the polymyxin is polymyxin B or colistin. In some embodiments, the Neisseria bacteria comprises LPS mutant. In some embodiments, the LPS mutant is a mutation or disruption in a gene involved in lipid A biosynthesis. In some embodiments, the gene is lpxA, lpxC, or lpxD. In some embodiments, composition further comprises a cancer therapy.
  • the cancer therapy comprises a chemotherapy agent.
  • the chemotherapy agent is selected from the group consisting of thiotepa, cyclosphosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, altretamine, triethylenemelamine, trietylenephosphoramide,
  • triethiylenethiophosphoramide trimethylolomelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, callystatin, CC-1065, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil,
  • the cancer therapy comprises cancer immunotherapy.
  • the cancer immunotherapy comprises an immune checkpoint inhibitor.
  • the dose of the immune checkpoint inhibitor is no more than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.9 mg/kg, 0.8 mg/kg, 0.7 mg/kg 0.6 mg/kg 0.5 mg/kg, 0.4 mg/kg, 0.3 mg/kg, 0.2 mg/kg or 0. lmg/kg.
  • the cancer is treated by the dose of the immune checkpoint inhibitor that is lower than the therapeutically effective dose of the immune checkpoint inhibitor when administered without administering the pharmaceutical composition.
  • the immune checkpoint inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to an immune checkpoint protein.
  • the immune checkpoint inhibitor is an siRNA molecule, an shRNA molecule or an antisense RNA molecule that inhibits expression of an immune checkpoint protein.
  • the immune checkpoint protein is CTLA4, PD-l, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3 or VISTA.
  • the immune checkpoint inhibitor is atezolizumab, avelumab, durvalumab, ipilimumab, nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, BGB-A317, STI- A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0020718C, AUR-012 or STI- A1010.
  • the cancer immunotherapy comprises a cancer-specific antibody or antigen-binding fragment thereof. In some embodiments, the cancer-specific antibody or antigen-binding fragment thereof binds specifically to a cancer-associated antigen.
  • the cancer-associated antigen is selected from the group consisting of adipophilin, AIM-2, ALDH1A1, alpha-actinm-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, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-l, CPSF, CSNK1A1, CTAG1, CTAG2, cychn Dl, Cyclm-Al, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen (“ETA”), ETV6-AML1 fusion protein, EZH2,
  • Kallikrein 4 KIF20A, KK-LC-l, KKLC1, KM-HN-l, KMHN1 also known as CCDC110, LAGE-l, LDLR-fucosyltransferaseAS fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE- A10, MAGE-A12, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A/MART-l, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-l, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR-l, NY-
  • the cancer associated antigen is a neo-antigen.
  • the cancer immunotherapy agent comprises a cancer vaccine.
  • the cancer vaccine comprises a polypeptide comprising an epitope of a cancer-associated antigen.
  • the cancer-associated antigen is selected from the group consisting of 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, CALC A, carcinoembryonic antigen (“CEA”), CASP-5, CASP-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, EpCAM, EphA3, epithelial tumor antigen (“ETA”), ETV6- AML1 fusion protein, EZH2, FGF
  • the cancer-associated antigen is a neo-antigen.
  • the polypeptide is a fusion protein.
  • the cancer vaccine comprises a nucleic acid encoding an epitope of a cancer-associated antigen.
  • the cancer- associated antigen is selected from the group consisting of 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, EpCAM, EphA3, epithelial tumor antigen (“ETA”), ETV6-AML1 fusion protein, EZH2, FGF
  • the cancer-associated antigen is a neo-antigen.
  • the nucleic acid is DNA.
  • the nucleic acid is RNA.
  • the RNA is mRNA.
  • the nucleic acid is in a vector.
  • the vector is a bacterial vector.
  • the bacterial vector is selected from the group consisting of Mycobacterium bovis (BCG), Salmonella Typhimurium ssp., Salmonella Typhi ssp., Clostridium sp. spores,
  • the vector is a viral vector.
  • the viral vector is selected from the group consisting of vaccinia, adenovirus, RNA viruses, and replication-defective avipox, replication-defective fowlpox, replication-defective canarypox, replication-defective MVA and replication-defective adenovirus.
  • the immunotherapy agent comprises 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.
  • the cancer-specific antigen is selected from the group consisting of adipophilin, AIM-2, ALDH1A1, alpha-actinm-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, 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, EphA
  • the cancer- specific antigen is a neo-antigen.
  • the immunotherapy agent comprises a cancer-specific chimeric antigen receptor (CAR).
  • the CAR is administered on the surface of a T cell.
  • the CAR binds specifically to a cancer-associated antigen.
  • the cancer-associated antigen is selected from the group consisting of 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, F
  • Kallikrein 4 KIF20A, KK-LC-l, KKLC1, KM-HN-l, KMHN1 also known as CCDC110, LAGE-l, LDLR-fucosyltransferaseAS fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE- A10, MAGE-A12, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A/MART-l, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-l, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR-l, NY-
  • the cancer associated antigen is a neo-antigen.
  • the immunotherapy agent comprises a cancer-specific T cell.
  • the T cell is a CD4+ T cell.
  • the CD4+ T cell is a TH1 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- associated antigen is selected from the group consisting of 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, EpCAM, EphA3, epithelial tumor antigen (“ETA”), ETV6-AML1 fusion protein, EZH2, FGF
  • G250/MN/CAIX GAGE-l,2,8, GAGE-3,4,5,6,7, GAS 7, glypican-3, GnTV, gpl00/Pmell7, GPNMB, HAUS3, Hepsin, HER-2/neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDOl, IGF2B3, ILl3Ralpha2, Intestinal carboxyl esterase, K-ras, Kallikrein 4,
  • the immunotherapy agent comprises an immune activating protein.
  • the immune activating protein is a cytokine or chemokine.
  • the immune activating protein is selected from the group consisting of 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 alpha (“IFN-alpha”), Interferon beta (“IFN-beta”), 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-l C-C motif chemokine 11
  • 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- 17A-F Interleukin-17A-F
  • Interleukin- 18 Interleukm-18l
  • IL-21 Interleukin-22
  • IL-23 Interleukin-23
  • IL-33 Interleukin-33
  • Chemokine (C-C motif) Lignad 2 MCP-l "), Macrophage colony-stimulating factor (“M-CSF”), Monokine induced by gamma interferon (“MIG”), Chemokine (C-C motif) ligand 2 (“MIP-l alpha”), Chemokine (C-C motif) ligand 4 (“MIP-l beta”), Macrophage inflammatory protein- 1 -delta (“MIP-l delta”), Platelet-derived growth factor subunit B (“PDGF-BB”), Chemokine (C-C motif)
  • RANTES Normal T cell Expressed and Secreted
  • TNF alpha lymphotoxin-alpha
  • TNF beta Tumor necrosis factor
  • Soluble TNF receptor type 1 sTNFRI
  • sTNFRIIAR Brain-derived neurotrophic factor
  • BDNF Basic fibroblast growth factor
  • BMP-4 Bone morphogenetic protein 4
  • BMP-5" Bone morphogenetic protein 5
  • BMP-7 Bone morphogenetic protein 7
  • Nerve growth factor b-NGF
  • EGF Epidermal growth factor
  • EGFR Epidermal growth factor receptor
  • EG-VEGF Endocrine-gland-derived vascular endothelial growth factor
  • Fibroblast growth factor 4 (“FGF-4"), Keratinocyte growth factor (“FGF-7”), Growth
  • GDF-15 Glial cell-derived neurotrophic factor
  • GDNF Glial cell-derived neurotrophic factor
  • HGF Hepatocyte growth factor
  • IGFBP-l Insulin-like growth factor binding protein 1
  • IGFBP-2 Insulin-like growth factor binding protein 2
  • IGFBP-3 Insulin-like growth factor binding protein 3
  • IGFBP-4 Insulin-like growth factor binding protein 4
  • IGFBP-6 Insulin-like growth factor binding protein 6
  • IGFBP-6 Insulin-like growth factor 1
  • IGF-l Insulin, Macrophage colony- stimulating factor
  • NGF R Nerve growth factor receptor
  • NGF R Neurotrophin-3
  • Neurotrophin-4 Neurotrophin-4
  • Osteoclastogenesis inhibitory factor Osteoclastogenesis inhibitory factor
  • PDGF-AA Platelet- derived growth factor receptors
  • PDGF-AA Phosphatidylinositol-
  • Chemokine (C-C motif) ligand 16 (“HCC-4"), Interleukin-9 (“IL-9”), Interleukin- 17 F (“IL- 17F”), Interleukin- l8-binding protein (“IL-18 BPa”), Interleukin-28 A (“IL-28A”), Interleukin 29 (“IL-29”), Interleukin 31 (“IL-31”), C-X-C motif chemokine 10 (“IP-10”), Chemokine receptor CXCR3 (“I-TAC”), Leukemia inhibitory factor (“LIF”), Light, Chemokine (C motif) ligand (“Lymphotactin”), Monocyte chemoattractant protein 2 (“MCP-2”), Monocyte chemoattractant protein 3 (“MCP-3”), Monocyte chemoattractant protein 4 (“MCP-4"),
  • Macrophage-derived chemokine MDC
  • Macrophage migration inhibitory factor MIF
  • Chemokine (C-C motif) ligand 20 MIP-3 alpha
  • C-C motif chemokine 19 MIP-3 beta
  • Chemokine (C-C motif) ligand 23 MSPalpha
  • MSPalpha Macrophage stimulating protein alpha chain
  • NAP-2 Nucleosome assembly protein l-like 4
  • Secreted phosphoprotein 1 (“Osteopontin”
  • PARC Pulmonary and activation-regulated cytokine
  • PARC Platelet factor 4
  • PF4 Platelet factor 4
  • Stroma cell-derived factor- 1 alpha SDF-l alpha
  • Chemokine (C-C motif) ligand 17 TARC
  • Thymus-expressed chemokine TECK
  • Thymic stromal lymphopoietin TSLP 4- IBB
  • TCB7 Thymic stromal lymphopoietin
  • Carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) ("CEACAM- 1"), Death Receptor 6 (“DR6"), Deoxythymidine kinase (“Dtk”), Type 1 membrane glycoprotein (“Endoglin”), Receptor tyrosine-protein kinase erbB-3 (“ErbB3”), Endothelial-leukocyte adhesion molecule 1 (“E-Selectin”), Apoptosis antigen 1 (“Fas”), Fms-like tyrosine kinase 3 (“Flt-3L”), Tumor necrosis factor receptor superfamily member 1 (“GITR”), Tumor necrosis factor receptor superfamily member 14 (“HVEM”), Intercellular adhesion molecule 3 (“ICAM- 3”), IL-l R4, IL-l RI, IL-10 Rbeta, IL-17R, IL-2Rgamma, IL-21R, Lysosome membrane protein 2
  • Angiopoietin 1 Angiostatin, Catheprin S, CD40, Cryptic family protein IB ("Cripto-l "), DAN, Dickkopf-related protein 1 ("DKK-l "), E-Cadherin, Epithelial cell adhesion molecule
  • EpCAM Fas Ligand
  • Fcg RIIB/C Follistatin
  • Galectin-7 Intercellular adhesion molecule 2
  • ICM-2 Intercellular adhesion molecule 2
  • IL-13 Rl Intercellular adhesion molecule 2
  • IL-13R2 Intercellular adhesion molecule 2
  • IL-17B Intercellular adhesion molecule 2
  • IL-2 Ra Intercellular adhesion molecule
  • IL-2 Rb Intercellular adhesion molecule
  • NrCAM Neuronal cell adhesion molecule
  • PKI-l Plasminogen activator inhibitor- 1
  • PDGF-AB Platelet derived growth factor receptors
  • SDF-l beta Resistin
  • SDF-l beta stromal cell-derived factor 1
  • sgpl30 Secreted frizzled-related protein 2
  • ShhN Sialic acid-binding immunoglobulin-type lectins
  • Siglec-5" Transforming growth factor-beta 2
  • TGF beta 2 Tie
  • the immunotherapy agent comprises an adjuvant.
  • the adjuvant is selected from the group consisting of 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,
  • the cancer therapeutic comprises an angiogenesis inhibitor.
  • the angiogenesis inhibitor is selected from the group consisting of Bevacizumab (Avastin®), Ziv-aflibercept (Zaltrap®), Sorafenib (Nexavar®), Sunitinib (Sutent®), Pazopanib (Votrient®), Regorafenib (Stivarga®), and
  • the method further comprises administering to the subject a second therapeutic bacteria.
  • the cancer therapy comprises administering an antibiotic to the subject.
  • the antibiotic is selected from the group consisting of aminoglycosides, ansamycins, carbacephems, carbapenems,
  • the cancer therapy comprises administering to the subject a second therapeutic bacteria.
  • the composition further comprises a prebiotic.
  • the prebiotic is a fructooligosaccharide, a galactooligosaccharide, a trans- galactooligosaccharide, a xylooligosaccharide, a chitooligosaccharide, a soy oligosaccharides, a gentiooligosaccharide, an isomaltooligosaccharide, a mannooligosaccharide, a
  • maltooligosaccharide a mannanoligosaccharide, lactulose, lactosucrose, palatinose, glycosyl sucrose, guar gum, gum Arabic, tagalose, amylose, amylopectin, pectin, xylan, or a cyclodextrin.
  • Example 1 Intraperitoneallv Administered Neisseria Meningitidis EVs 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) Neisseria Meningitidis EVs isolated from the Bexsero® vaccine; and 3) anti-PD-l antibody.
  • Antibodies were administered intraperitoneally (i.p.) at 200ug/mouse (lOOul final volume) every four days, starting on day 1, and Neisseria Meningitidis bacteria (about l . lxlO 2 ) were administered intraperitoneally (i.p.) daily, starting on day 1 until the conclusion of the study.
  • the Neisseria Meningitidis group showed tumor growth inhibition greater than that seen in the anti-PD-l group ( Figures 1, and 2).
  • Other mice are treated with Neisseria bacteria (live, killed, irradiatedor lyophilized), EVs, and/or PhABs, and/or some combination thereof.
  • Example 2 A mouse melanoma model
  • Neisseria Meningitidis EVs are tested for their efficacy in the mouse model of melanoma, either alone or in combination with whole bacterial cells, with or without the addition of other cancer therapies, including checkpoint inhibitor(s).
  • 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 Neisseria Meningitidis EVs 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
  • peritumoral or subtumoral or subcutaneous injection In order to assess the systemic anti-tumoral effects of Neisseria Meningitidis EV treatment, additional mice may be inoculated with tumor cells in the contralateral (untreated, second) flank prior to IT, peritumoral, or subtumoral treatment with Neisseria Meningitidis EV in the first flank.
  • mice may receive Neisseria Meningitidis EVs (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.
  • Other mice are treated with Neisseria bacteria (live, killed, irradiatedor lyophilized), EVs, and/or PhABs, and/or some combination thereof.
  • 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. For example, 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 70pm filter into a 50ml falcon tube and centrifuged at 1000 rpm for 10 minutes. Cells are resuspended in FACS buffer and washed to remove remaining debris. If necessary, samples are strained again through a second 70pm filter into a new tube. Cells are stained for analysis by flow cytometry using techniques known in the art.
  • 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.
  • Other 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, CTLA-4), and macrophage/myeloid markers (CDl lb, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-l).
  • serum cytokines are analyzed including, but not limited to, TNFa, IL-17, IL-13, IL-l2p70, ILl2p40, IL-10, IL-6, IL- 5, IL-4, IL-2, IL-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.
  • Example 3 A mouse lung cancer model
  • Neisseria Meningitidis EVs are tested for their efficacy in the mouse model of lung cancer, either alone or in combination with whole bacterial cells , with or without the addition of other cancer therapies, including checkpoint inhibitor(s).
  • Neisseria Meningitidis EVs 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 Neisseria Meningitidis EVs , with or without checkpoint inhibitor treatment. As described in Example 2, Neisseria Meningitidis is
  • mice receive a bacterial strain (p.o.) on the day following tumor cell injection (day 1). Some mice 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. Other mice are treated with Neisseria bacteria (live, killed, irradiatedor lyophilized), EVs, and/or PhABs, and/or some combination thereof.
  • Neisseria bacteria live, killed, irradiatedor lyophilized
  • EVs e.g. 100 mm 3
  • lxlO 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. As described in Example 2, 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 4 A mouse breast cancer model
  • Neisseria Meningitidis EVs are tested for their efficacy in the mouse model of breast cancer, either alone or in combination with whole bacterial cells, with or without the addition of other cancer therapies, including checkpoint inhibitor(s).Mice are divided into groups receiving Neisseria Meningitidis EVs, with or without checkpoint inhibitor treatment. As described in Example 2, a bacterial strain is administered at varied doses at defined intervals. For example, some mice receive a bacterial strain (p.o.) on the day following tumor cell injection (day 1). Some mice 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.
  • 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.
  • Other mice are treated with Neisseria bacteria (live, killed, irradiatedor lyophilized), EVs, and/or PhABs, and/or some combination thereof.
  • 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.
  • Neisseria Meningitidis EVsis administered at varied doses at defined 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.
  • 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 5 A mouse pancreatic cancer model
  • Neisseria Meningitidis EVs are tested for their efficacy in the mouse model of pancreatic cancer, either alone or in combination with whole bacterial cells, with or without the addition of other cancer therapies, including checkpoint inhibitor(s).Mice are divided into groups receiving EVs, with or without checkpoint inhibitor treatment. As described in Example 2, some mice receive Neisseria Meningitidis EVs(p.o.) on the day following tumor cell injection (day 1). Some mice 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 .
  • mice are treated with Neisseria bacteria (live, killed, irradiatedor lyophilized), EVs, and/or PhABs, and/or some combination thereof.
  • 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. As described in Example 2, 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.
  • Example 6 A mouse model of hepatocellular carcinoma
  • Neisseria Meningitidis EVs are tested for their efficacy in the mouse model of hepatocellular carcinoma, either alone or in combination with whole bacterial cells, with or without the addition of other cancer therapies, including checkpoint inhibitor(s).Mice are divided into groups receiving a EVs, with or without checkpoint inhibitor treatment. As described in Example 2, Neisseria Meningitidis EVs are administered at varied doses at defined intervals. For example, some mice receive Neisseria Meningitidis EVs (p.o.) on the day following tumor cell injection (day 1). Some mice receive seven (7) consecutive doses of a bacterial strain (one dose per day on days 14-21).
  • mice receive daily dosing or, alternatively, some mice receive dosing every other day.
  • 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.
  • Other mice are treated with Neisseria bacteria (live, killed, irradiatedor lyophilized), EVs, and/or PhABs, and/or some combination thereof.
  • 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. As described in Example 2, 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 A mouse lymphoma model
  • Neisseria Meningitidis EVs are tested for their efficacy in the mouse model of lymphoma, either alone or in combination with whole bacterial cells, with or without the addition of other cancer therapies, including checkpoint inhibitor(s).). For example, mice are divided into groups receiving Neisseria Meningitidis EVs , with or without checkpoint inhibitor treatment. As described in Example 2, EVs are administered at varied doses at defined intervals. For example, some mice receive EVs (p.o.) on the day following tumor cell injection (day 1). Some mice 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.
  • 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.
  • Other mice are treated with Neisseria bacteria (live, killed, irradiatedor lyophilized), EVs, and/or PhABs, and/or some combination thereof.
  • 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. As described in Example 2, 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 A mouse prostate cancer model
  • Neisseria Meningitidis EVs are tested for their efficacy in the mouse model of prostate cancer, either alone or in combination with whole bacterial cells, with or without the addition of other cancer therapies, including checkpoint inhibitor(s).Mice are divided into groups receiving Neisseria Meningitidis EVs , with or without checkpoint inhibitor treatment. As described in Example 2, Neisseria Meningitidis EVs is administered at varied doses at defined intervals. For example, some mice receive EVs (p.o.) on the day following tumor cell injection (day 1). Some mice receive seven (7) consecutive doses of Neisseria Meningitidis EVs (one dose per day on days 14-21).
  • mice receive daily dosing or, alternatively, some mice receive dosing every other day.
  • 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.
  • Other mice are treated with Neisseria bacteria (live, killed, irradiatedor lyophilized), EVs, and/or PhABs, and/or some combination thereof.
  • 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. As described in Example 2, 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 A mouse plasmacytoma model
  • Neisseria Meningitidis EVs are tested for their efficacy in the mouse model of plasmacytoma, either alone or in combination with whole bacterial cells , with or without the addition of other cancer therapies, including checkpoint inhibitor(s).). Mice are divided into groups receiving Neisseria Meningitidis EVs , with or without checkpoint inhibitor treatment. As described in Example 2, EVs are administered at varied doses at defined intervals. For example, some mice receive EVs (p.o.) on the day following tumor cell injection (day 1). Some mice receive seven (7) consecutive doses of Neisseria Meningitidis EVs (one dose per day on days 14- 21).
  • mice receive daily dosing or, alternatively, some mice receive dosing every other day.
  • 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.
  • Other mice are treated with Neisseria bacteria (live, killed, irradiatedor lyophilized), EVs, and/or PhABs, and/or some combination thereof.
  • mice are injected intraperitoneally three times with 500ul of 2,6,10,12- 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. 71 (2): 391 -5 (see also Lattanzio et al. 1997.
  • 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+
  • mice are divided into groups receiving Neisseria Meningitidis EVs by oral gavage, and with or without checkpoint inhibitor treatment. Tumors from the various treatment groups are measured with calipers at regular intervals. As described in Example 2, 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 10 A SC ID mouse model of mouse myeloma
  • Neisseria Meningitidis EVs are tested for their efficacy in the mouse model of myeloma, either alone or in combination with whole bacterial cells , with or without the addition of other cancer therapies, including checkpoint inhibitor(s).
  • Mice are divided into groups receiving Neisseria Meningitidis EVs , with or without checkpoint inhibitor treatment.
  • Neisseria Meningitidis EVs is administered at varied doses at defined intervals. For example, some mice receive EVs (p.o.) on the day following tumor cell injection (day 1). Some mice receive seven (7) consecutive doses of Neisseria Meningitidis EVs (one dose per day on days 14-21).
  • mice receive daily dosing or, alternatively, some mice receive dosing every other day.
  • 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.
  • Other mice are treated with Neisseria bacteria (live, killed, irradiatedor lyophilized), EVs, and/or PhABs, and/or some combination thereof.
  • Myeloma cells are injected subcutaneously into one or both hind flanks of SCID mice (See Caers et al. 2004. Of mice and men: disease models of multiple myeloma. Drug Discovery Today: Disease Models. l(4):373-380. Tumors from the various treatment groups are measured with calipers at regular intervals. As described in Example 2, 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 A mouse renal cell carcinoma model
  • Neisseria Meningitidis EVs are tested for their efficacy in the mouse model of renal cell carcinoma, either alone or in combination with whole bacterial cells , with or without the addition of other cancer therapies, including checkpoint inhibitor(s). Mice are divided into groups receiving Neisseria Meningitidis EVs , with or without checkpoint inhibitor treatment. As described in Example 2, Neisseria Meningitidis EVs may be administered at varied doses at defined intervals. For example, some mice receive EVs (p.o.) on the day following tumor cell injection (day 1). Some mice receive seven (7) consecutive doses of a Neisseria Meningitidis EVs bacterial strain (one dose per day on days 14-21).
  • mice receive daily dosing or, alternatively, some mice receive dosing every other day.
  • 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.
  • Other mice are treated with Neisseria bacteria (live, killed, irradiatedor lyophilized), EVs, and/or PhABs, and/or some combination thereof.
  • Renca cells (ATCC CRL-2947) or 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. As described in Example 2, 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 12 A mouse bladder cancer model
  • Neisseria Meningitidis EVs are tested for their efficacy in the mouse model of bladder cancer, either alone or in combination with whole bacterial cells, with or without the addition of other cancer therapies, including checkpoint inhibitor(s).Mice are divided into groups receiving Neisseria Meningitidis, with or without checkpoint inhibitor treatment. As described in Example 2, Neisseria Meningitidis EVs may be administered at varied doses at defined intervals. For example, some mice receive EVs (p.o.) on the day following tumor cell injection (day 1). Some mice 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.
  • 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.
  • Other mice are treated with Neisseria bacteria (live, killed, irradiatedor lyophilized), EVs, and/or PhABs, and/or some combination thereof.
  • 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.
  • Example 13 Preparation and purification of EVs from Neisseria Meningitidis bacteria.
  • Extracellular vesicles are prepared from Neisseria Meningitidis bacterial cultures using methods known to those skilled in the art (S. Bin Park, et al. PLoS ONE.
  • Neisseria Meningitidis bacterial cultures are centrifuged at 11,000 x g for 20-40 min at 4°C to pellet bacteria. Culture supernatants are then passed through a 0.22 pm filter to exclude intact bacterial cells. Filtered supernatants are concentrated using methods that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration. Briefly, for ammonium sulfate precipitation, 1.5-3 M ammonium sulfate is added to filtered supernatant slowly, while stirring at 4°C. Precipitations are incubated at 4°C for 8-48 hours and then centrifuged at 11,000 x g for 20-40 min at 4°C.
  • the pellets contain bacterial EVs and other debris.
  • filtered supernatants are centrifuged at 100,000-200,000 x g for 1-16 hours at 4°C.
  • the pellet of this centrifugation contains bacterial EVs and other debris.
  • supernatants are filtered so as to retain species of molecular weight > 50 or 100 kDa.
  • EVs are obtained from Neisseria Meningitidis bacterial cultures continuously during growth, or at selected time points during growth, by connecting a bioreactor to an alternating tangential flow (ATF) system (e.g., XCell ATF from Repligen) according to manufacturer’s instructions.
  • 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
  • EVs obtained by methods described above may be further purified 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 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 were used to concentrate the filtered supernatants, pellets are resuspended in 35% Optiprep in PBS. 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 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C.
  • EVs are serially diluted onto agar medium used for routine culture of the Neisseria Meningitidis 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.
  • samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 using filtration (e.g. Amicon Ultra columns), dialysis, or ultracentrifugation (200,000 x g, > 3 hours, 4°C) and resuspension.
  • filtration e.g. Amicon Ultra columns
  • dialysis e.g. dialysis
  • ultracentrifugation 200,000 x g, > 3 hours, 4°C
  • Enriched media is used to grow and prepare the bacteriafor 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 bacteriagrown 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 bacteriaproduced 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 2F, 10F, 80F, 100F, 250F, 1000F, 2500F, 5000F, l0,000F.
  • 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.

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Abstract

L'invention concerne des méthodes et des compositions associées à des bactéries Neisseria utiles en tant qu'agents thérapeutiques.
PCT/US2019/039482 2018-06-27 2019-06-27 Compositions et méthodes de traitement du cancer faisant appel à des bactéries neisseria WO2020006216A1 (fr)

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