WO2019178055A1 - Vésicules extracellulaires provenant de burkholderia - Google Patents

Vésicules extracellulaires provenant de burkholderia Download PDF

Info

Publication number
WO2019178055A1
WO2019178055A1 PCT/US2019/021792 US2019021792W WO2019178055A1 WO 2019178055 A1 WO2019178055 A1 WO 2019178055A1 US 2019021792 W US2019021792 W US 2019021792W WO 2019178055 A1 WO2019178055 A1 WO 2019178055A1
Authority
WO
WIPO (PCT)
Prior art keywords
burkholderia
carcinoma
pharmaceutical composition
cancer
bacteria
Prior art date
Application number
PCT/US2019/021792
Other languages
English (en)
Inventor
Brian Goodman
Christopher J. H. DAVITT
Original Assignee
Evelo Biosciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Evelo Biosciences, Inc. filed Critical Evelo Biosciences, Inc.
Publication of WO2019178055A1 publication Critical patent/WO2019178055A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • 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 comprising
  • Burkholderia bacteria and/or a product of such bacteria e.g. extracellular vesicles (EVs) (i.e., EVs generated by or isolated from bacteria of the genus Burkholderia) and/or pharmaceutically active biomasses (PhABs)) useful for the treatment and/or prevention of cancer), as well as methods of making and/or identifying such EVs, and methods of using such pharmaceutical compositions (e.g., for the treatment of cancers, either alone or in combination with other therapeutics).
  • the pharmaceutical compositions comprise both
  • Burkholderia EVs and whole Burkholderia bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria).
  • provided herein are pharmaceutical compositions comprising Burkholderia bacteria in the absence of Burkholderia EVs.
  • the pharmaceutical compositions comprise Burkholderia EVs in the absence of Burkholderia bacteria.
  • the pharmaceutical compositions comprise Burkholderia EVs and/or Burkholderia bacteria of the species Burkholderia ambifaria, Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia mallei, Burkholderia multivorans, Burkholderia
  • the bacterial and/or pharmaceutical composition comprises EVs, and/or PhABs (e.g., whole cells, fractions of cells, supernatant from fermentation, fractions of supernatant and/or extracellular vesicles) derived from the EVs, and/or PhABs (e.g., whole cells, fractions of cells, supernatant from fermentation, fractions of supernatant and/or extracellular vesicles) derived from the
  • extracellular vesicles produced by and/or generated by and/or isolated from Burkholderia bacteria provided herein.
  • the bacterial compositions comprise both Burkholderia EVs and whole
  • Burkholderia bacteria e.g., live bacteria, killed bacteria, attenuated bacteria.
  • Burkholderia EVs e.g., live bacteria, killed bacteria, attenuated bacteria.
  • the pharmaceutical compositions comprise Burkholderia EVs in the absence (or substantially in the absence) of Burkholderia bacteria.
  • PhABs derived from and/or comprising a Burkholderia genus provided herein.
  • the PhABs comprise whole cells, fractions of cells, supernatant from fermentation, fractions of supernatant and/or extracellular vesicles derived from bacteria described herein.
  • the bacterial cells comprise whole cells, fractions of cells, supernatant from fermentation, fractions of supernatant and/or extracellular vesicles derived from bacteria described herein.
  • compositions provided herein comprise a PhAB derived from Burkholderia genus provided herein.
  • the pharmaceutical composition comprises a specific ratio of Burkholderia bacteria to Burkholderia EV particles.
  • the pharmaceutical composition comprises at least 1 Burkholderia bacterium for every 1, 1.1, 1.2,
  • the pharmaceutical composition comprises about 1 Burkholderia bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,
  • the pharmaceutical composition comprises no more than 1 Burkholderia bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1,
  • the pharmaceutical composition comprises about 1 Burkholderia 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.
  • the pharmaceutical composition comprises no more than 1 Burkholderia 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,
  • the Burkholderia EVs are from an engineered Burkholderia bacteria that is modified to enhance certain desirable properties.
  • the engineered Burkholderia bacteria are modified to increase production of Burkholderia EVs.
  • the engineered Burkholderia bacteria are modified to produce Burkholderia EVs with enhanced oral delivery (e.g ., by improving acid resistance, muco-adherence and/or penetration and/or resistance to bile acids, resistance to anti-microbial peptides and/or antibody neutralization), to target desired cell types (e.g.
  • M-cells goblet cells, enterocytes, dendritic cells, macrophages
  • an appropriate niche e.g., mesenteric lymph nodes, Peyer’s patches, lamina basement, tumor draining lymph nodes, and/or blood
  • to enhance the immunomodulatory and/or therapeutic effect of th e Burkholderia EVs they produce e.g., either alone or in combination with another therapeutic agent
  • to enhance immune activation by the Burkholderia EVs they produce and/or to improve Burkholderia bacterial and/or Burkholderia EV manufacturing e.g., greater stability, improved freeze-thaw tolerance, shorter generation times.
  • provided herein are methods of making such Burkholderia EVs and Burkholderia bacteria.
  • provided herein are methods of treating a subject who has cancer comprising administering to the subject a pharmaceutical composition described herein.
  • 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 method further comprises the administration of an additional therapeutic bacterial composition and/or EV.
  • compositions comprising Burkholderia bacteria (e.g., killed, live, and/or attenuated bacteria, and/or a combination thereof) and/or a product of such bacteria (e.g., extracellular vesicles (EVs) and/or pharmaceutically active biomasses (PhABs)).
  • Burkholderia bacteria e.g., killed, live, and/or attenuated bacteria, and/or a combination thereof
  • a product of such bacteria e.g., extracellular vesicles (EVs) and/or pharmaceutically active biomasses (PhABs)
  • EVs extracellular vesicles
  • PhABs pharmaceutically active biomasses
  • 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 Burkholderia bacteria.
  • 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 Burkholderia bacteria.
  • EVs and/or PhABs e.g., whole cells, fractions of cells, supernatant from fermentation, fractions of supernatant and/or extracellular vesicles
  • the bacteria are frozen, irradiated or heat-inactivated.
  • PhABs made from and/or comprising bacteria from the genus Burkholderia.
  • the PhABs comprise whole cells, fractions of cells, supernatant from fermentation, fractions of supernatant and/or extracellular vesicles made from bacteria described herein.
  • the pharmaceutical compositions provided herein comprise Burkholderia bacteria.
  • Figure 1 shows the efficacy of Burkholderia pseudomallei EVs compared to
  • intravenously i.v. administered anti-PD-l or vehicle in a mouse colorectal carcinoma model.
  • Figure 2 shows the efficacy of Burkholderia pseudomallei EVs compared to
  • a subject e.g ., a human subject
  • a bacterial composition comprising bacteria of genus Burkholderia and/or a derivative of such bacteria (e.g., extracellular vesicles (EVs) and/or pharmacologically active biomasses (PhABs)), as well as methods of making and/or identifying such bacteria and/or bacterially-derived products.
  • a bacterial composition comprising bacteria of genus Burkholderia and/or a derivative of such bacteria (e.g., extracellular vesicles (EVs) and/or pharmacologically active biomasses (PhABs)
  • EVs extracellular vesicles
  • PhABs pharmacologically active biomasses
  • Adjuvant or“Adjuvant therapy” broadly refers to an agent that affects an
  • 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.
  • IV intravenous
  • IM intramuscular
  • IT intratumoral
  • SC subcutaneous
  • the pharmaceutical compositions described herein can be administered in any form by any effective route, including but not limited to intratumoral, oral, parenteral, enteral, intravenous,
  • transdermal e.g., using any standard patch
  • intradermal e.g., using any standard patch
  • intradermal e.g., using any standard patch
  • intradermal e.g., using any standard patch
  • intradermal e.g., using any standard patch
  • intradermal e.g., using any standard patch
  • ophthalmic e.g., using any standard patch
  • intradermal e.g., using any standard patch
  • intradermal ophthalmic
  • intrasally 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
  • implanted intravesical, intrapulmonary, intraduodenal, intragastrical,
  • the pharmaceutical compositions described herein are administered orally, rectally, intratumorally, topically, intravesically, by injection into or adjacent to a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously.
  • the term“antibody” may refer to both an intact antibody and an antigen binding fragment thereof.
  • Intact antibodies are glycoproteins that include at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • VH and VL regions can be further subdivided into regions of
  • variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • 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.
  • antigen binding fragment and“antigen-binding portion” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen.
  • binding fragments encompassed within the term "antigen-binding fragment” of an antibody include Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chain antibodies, NANOBODIES®, isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of an intact antibody.
  • These antibody fragments can be obtained using conventional recombinant and/or enzymatic techniques and can be screened for antigen binding in the same manner as intact antibodies.
  • 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.
  • 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 downstream of a statistically valid node in a phylogenetic tree.
  • 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 ail 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.
  • MMT multilocus sequence tags
  • I 6S embodiments OTUs that share 397% average nucleotide identity across die entire 16S or some variable region of the 16S are considered the same OTU (see e g. Claesson M J, Wang Q, O'Sullivan O, Greene-Diniz R, Cole J R, Ros R P, and O'Toole P W. 2010.
  • 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, m 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 EVs from two or more microbial strains includes the physical co existence of the two EVs, either in the same material or product or in physically connected products, as well as the temporal co-administration or co-localization of the EVs from the two 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.
  • the term“dysbiosis” refers to a state in which the synergy between microbes and the tumor is broken such as the microbes no longer support the nucleation, maintenance, progression or spread or metastasis of a tumor.
  • 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 or T cell receptor.
  • 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 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 disease (e.g., cancer) and includes, for example, checkpoint inhibitors, cancer vaccines, cytokines, cell therapy, CAR-T cells, and dendritic cell therapy.
  • disease e.g., cancer
  • 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, RLRs, C-type lectin receptors, STING-cGAS Pathway components, inflammasome complexes.
  • 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, EV or other entity or substance that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by the hand of man. Isolated microbes may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated.
  • isolated microbes are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • a substance is“pure” if it is substantially free of other components.
  • the terms“purify,”“purifying” and“purified” refer to a microbe or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated ( e.g ., whether in nature or in an experimental setting), or during any time after its initial production.
  • a microbe or a microbial population may be considered purified if it is isolated at or after production, such as from a material or environment containing the microbe or microbial population, and a purified microbe or microbial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered“isolated.”
  • purified microbes or microbial population are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • the one or more microbial types present in the composition can be independently purified from one or more other microbes produced and/or present in the material or environment containing the microbial type.
  • Microbial compositions and the microbial components thereof are generally purified from residual habitat products.
  • ‘Metabolite” as used herein 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 a bacterium/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.
  • 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.
  • 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 cancer- associated bacterial strains are present in a sample.
  • the microbiome profile indicates the relative or absolute amount of each bacterial strain detected in the sample.
  • the microbiome profile is a cancer-associated microbiome profile.
  • a cancer- associated microbiome profile is a microbiome profile that occurs with greater frequency in a subject who has cancer than in the general population.
  • the cancer- associated microbiome profile comprises a greater number of or amount of cancer-associated bacteria than is normally present in a microbiome of an otherwise equivalent tissue or sample taken from an individual who does not have cancer.
  • ‘‘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 the bacteria in such a way that it increases or decreases 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 the 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.
  • An“oncobiome” as used herein comprises pathogenic, tumorigenic and/or cancer- associated microbiota, wherein the microbiota comprises one or more of a virus, a
  • bacterium/bacteria a fungus, a protist, a parasite, or another microbe.
  • Oncotrophic or“oncophilic” microbes and bacteria are microbes that are highly associated or present in a cancer microenvironment. They may be preferentially selected for within the environment, preferentially grow in a cancer microenvironment or hone to a said environment.
  • “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.
  • MLSTs 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.
  • 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.
  • 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), micro RNA (miRNA), silencing RNA (siRNA), 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.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • 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.
  • U nucleotides are interchangeable with T nucleotides.
  • pharmacologically active biomass or“PhABs” broadly refer to a composition containing pharmacologically active bacterial components, for example, derived from lysed or otherwise disrupted cells.
  • a substance is“pure” if it is substantially free of other components.
  • the terms“purify,”“purifying” and“purified” refer to EVs or other material that have 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.
  • ‘Residual habitat products” refers to material derived from the habitat for microbiota within or on a subject.
  • microbes live in feces in the gastrointestinal tract, on the skin itself, in saliva, mucus of the respiratory tract, or secretions of the genitourinary tract (i.e., biological matter associated with the microbial community).
  • Substantially free of residual habitat products means that the microbial composition no longer contains the biological matter associated with the microbial environment on or in the human or animal subject and is 100% free, 99% free, 98% free, 97% free, 96% free, or 95% free of any contaminating biological matter associated with the microbial community.
  • Residual habitat products can include abiotic materials (including undigested food) or it can include unwanted microorganisms. Substantially free of residual habitat products may also mean that the microbial composition contains no detectable cells from a human or animal and that only microbial cells are detectable. In one embodiment, substantially free of residual habitat products may also mean that the microbial composition contains no detectable viral (including microbial viruses (e.g., phage)), fungal, mycoplasmal contaminants.
  • microbial viruses e.g., phage
  • contamination may be reduced by isolating desired constituents through multiple steps of streaking to single colonies on solid media until replicate (such as, but not limited to, two) streaks from serial single colonies have shown only a single colony morphology.
  • reduction of contamination can be accomplished by multiple rounds of serial dilutions to single desired cells (e.g., a dilution of 10-8 or 10-9), such as through multiple 10-fold serial dilutions. This can further be confirmed by showing that multiple isolated colonies have similar cell shapes and Gram staining behavior.
  • Other methods for confirming adequate purity include genetic analysis (e.g., PCR, DNA sequencing), serology and antigen analysis, enzymatic and metabolic analysis, and methods using instrumentation such as flow cytometry with reagents that distinguish desired constituents from contaminants.
  • “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.
  • compositions that comprise Burkholderia bacteria and/or Burkholderia EVs and/or pharmaceutically active biomasses (PhABs) made from bacteria.
  • the pharmaceutical compositions comprise Burkholderia EVs and/or Burkholderia bacteria of the species Burkholderia ambifaria,
  • Burkholderia cenocepacia Burkholderia cepacia, Burkholderia mallei, Burkholderia multivorans, Burkholderia oklahomensis, Burkholderia pseudomallei, Burkholderia rhizoxinica, Burkholderia sp. 383, Burkholderia xenovorans, Burkholderiales bacterium 1 1 47,
  • the Burkholderia bacteria from which the EVs are obtained are modified to enhance EV production, to enhance oral delivery of the produced EVs (e.g ., by improving acid resistance, muco-adherence and/or penetration and/or resistance to bile acids, digestive enzymes, resistance to anti-microbial peptides and/or antibody neutralization), to target desired cell types (e.g.
  • the engineered Burkholderia bacteria described herein are modified to improve Burkholderia bacterial and/or EV manufacturing (e.g., higher oxygen tolerance, stability, improved freeze-thaw tolerance, shorter generation times).
  • the engineered Burkholderia bacteria described include bacteria harboring one or more genetic changes, such change being an insertion, deletion, translocation, or substitution, or any combination thereof, of one or more nucleotides contained on the bacterial chromosome or endogenous plasmid and/or one or more foreign plasmids, wherein the genetic change may result in the overexpression and/or underexpression of one or more genes.
  • the engineered microbe(s) may be produced using any technique known in the art, including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, or any combination thereof.
  • site-directed mutagenesis including site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, or any combination thereof.
  • the Burkholderia bacteria are modified such that they have attenuated
  • the Burkholderia bacteria from which the EVs are obtained are modified to enhance modulation of CD8 function (e.g., to enhance activation, to lower inhibitory marker expression, to up-regulate PD-l).
  • the Burkholderia EVs and/or Burkholderia bacteria described herein are modified such that they comprise, are linked to, and/or are bound by a therapeutic moiety.
  • the therapeutic moiety is a cancer-specific moiety.
  • the cancer-specific moiety has binding specificity for a cancer cell (e.g., has binding specificity for a cancer-specific antigen).
  • the cancer-specific moiety comprises an antibody or antigen binding fragment thereof.
  • the cancer-specific moiety comprises a T cell receptor or a chimeric antigen receptor (CAR).
  • the cancer-specific moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof.
  • the cancer-specific moiety is a bipartite fusion protein that has two parts: a first part that binds to and/or is linked to the Burkholderia bacterium and a second part that is capable of binding to a cancer cell ( e.g ., by having binding specificity for a cancer-specific antigen).
  • the first part is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP.
  • the first part has binding specificity for the Burkholderia EV (e.g., by having binding specificity for a Burkholderia bacterial antigen).
  • the first and/or second part comprises an antibody or antigen binding fragment thereof.
  • the first and/or second part comprises a T cell receptor or a chimeric antigen receptor (CAR).
  • the first and/or second part comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof.
  • co-administration of the cancer-specific moiety with the Burkholderia EVs increases the targeting of the Burkholderia EVs to the cancer cells.
  • the Burkholderia EVs described herein is modified such that they comprise, are linked to, and/or are bound by a magnetic and/or paramagnetic moiety (e.g., a magnetic bead).
  • the magnetic and/or paramagnetic moiety is comprised by and/or directly linked to the Burkholderia bacteria.
  • the magnetic and/or paramagnetic moiety is linked to and/or a part of an EV-binding moiety that that binds to the EV.
  • the EV-binding moiety is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP.
  • the EV-binding moiety has binding specificity for the EV (e.g., by having binding specificity for a Burkholderia bacterial antigen).
  • the EV-binding moiety comprises an antibody or antigen binding fragment thereof.
  • the EV-binding moiety comprises a T cell receptor or a chimeric antigen receptor (CAR).
  • the EV-binding moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor binding fragment thereof.
  • co-administration of the magnetic and/or paramagnetic moiety with the EVs can be used to increase the targeting of the EVs to cancer calls and/or a part of a subject where cancer cells are present.
  • the Burkholderia EVs described herein can be prepared using any method known in the art, such as for example, the methods disclosed in Nieves et al., Vaccine, 2011 Oct 26;29(46):838l-9; Baker et al, Vaccines (Basel), 2017 Dec 9;5(4); Petersen et al., Procedia Vaccinol. 2014 Jan l ;8:38-42; and Nieves et al., Clin Vaccine Immunol. 2014
  • the Burkholderia EVs are prepared without an EV purification step.
  • Burkholderia bacteria comprising the EVs described herein are killed using a method that leaves the Burkholderia bacterial EVs intact and the resulting bacterial components, including the EVs, are used in the methods and compositions described herein.
  • the Burkholderia bacteria are killed using an antibiotic (e.g . , using an antibiotic described herein).
  • the Burkholderia bacteria are killed using LTV 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 Burkholderia 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
  • 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 Burkholderia bacterial EVs and other debris.
  • a filtration technique such as through the use of an Amicon Ultra spin filter or by tangential flow filtration, supernatants can be filtered so as to retain species of molecular weight > 50 or 100 kDa.
  • EVs can be obtained from Burkholderia 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).
  • 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 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% 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.
  • purified 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 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 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. Production of PhABs
  • the PhABs described herein can be prepared using any method known in the art.
  • the PhABs described herein are prepared by fractionation.
  • Bacterial cells and/or supernatants from cultured bacteria cells are fractionated into various pharmacologically active biomass (PhABs) and/or products derived therefrom. Bacterial cells and/or supernatants are fractionated using materials and methods known in the art (see e.g.
  • PhABs obtained by methods provided herein may be further purified by size-based column chromatography, by affinity chromatography, and by gradient
  • ultracentrifugation using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation 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.
  • pellets are resuspended in 35% Optiprep in PBS.
  • 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): e0l34353 (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.
  • the methods provided herein are pharmaceutical compositions comprising Burkholderia EVs and/or Burkholderia bacteria (e.g., an EV composition) and/or pharmaceutically active biomasses (PhABs) provided herein.
  • the EV composition comprises an EV and/or a combination of EVs described herein and a
  • the pharmaceutical compositions comprise Burkholderia EVs substantially or entirely free of bacteria. In some embodiments, the pharmaceutical compositions comprise both Burkholderia EVs and whole Burkholderia bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria). In certain embodiments, the pharmaceutical compositions comprise Burkholderia bacteria that is substantially or entirely free of EVs. [75] In some embodiments, the bacterial and/or pharmaceutical composition comprises killed bacteria, live bacteria and/or attenuated bacteria, and/or any combination thereof.
  • 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. For example, 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. In some embodiments, bacteria are killed using gamma irradiation. In some embodiments, the bacteria are killed or inactivated using electron irradiation (e.g., beta radiation) or x-ray irradiation.
  • g- irradiation gamma irradiation
  • exposure to UV light e.g., UV light
  • formalin-inactivation
  • 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).
  • Burkholderia pseudomallei mutant Bp82 of the l026b 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 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 timepoints during exponential phase. In addition to inactivation by various methods, bacteria may be tested for efficacy using different ratios of live versus inactivated cells, or different ratios of cells at various growth phases, and/or EVs prepared from bacteria harvested during one or more of the growth phases.
  • compositions comprising Burkholderia EVs and/or Burkholderia bacteria, provided herein (e.g., an EV composition), such as those disclosed in U.S. Provisional Patent Application No.
  • the EV composition comprises an EV and/or a combination of EVs described herein and a
  • the bacterial and/or pharmaceutical compositions comprise Burkholderia EVs substantially or entirely free of bacteria.
  • the pharmaceutical compositions comprise both Burkholderia EVs and whole Burkholderia bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria).
  • the bacteria and/or pharmaceutical compositions comprise Burkholderia EVs substantially or entirely free of bacteria.
  • the pharmaceutical compositions comprise both Burkholderia EVs and whole Burkholderia bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria).
  • the pharmaceutical compositions comprise both Burkholderia EVs and whole Burkholderia bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria).
  • compositions comprise Burkholderia bacteria that is substantially or entirely free of EVs.
  • the bacterial and/or pharmaceutical composition comprises at least 1 Burkholderia bacterium for every 1 , 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4,
  • the bacterial and/or pharmaceutical composition comprises about 1 Burkholderia bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4,
  • the bacterial and/or pharmaceutical composition comprises a certain ratio of Burkholderia bacteria particles to Burkholderia EV particles.
  • the number of Burkholderia 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 Burkholderia EVs with a set number of purified Burkholderia bacterial cells, by modifying the growth conditions under which the Burkholderia bacteria are cultured, or by modifying the Burkholderia bacteria itself to produce more or fewer Burkholderia EVs.
  • NTA nanoparticle tracking analysis
  • DLS dynamic light scattering
  • the pharmaceutical composition comprises at least 1
  • Burkholderia bacterium for every 1 , 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1 , 2.2, 2.3, 2.4,
  • the pharmaceutical composition comprises about 1 Burkholderia bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8.
  • the pharmaceutical composition comprises a certain ratio of Burkholderia bacteria particles to Burkholderia EV particles.
  • the number of Burkholderia 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
  • Burkholderia EVs with a set number of purified Burkholderia bacteria, by modifying the growth conditions under which the Burkholderia bacteria are cultured, or by modifying the Burkholderia bacteria itself to produce more or fewer Burkholderia EVs.
  • NTA nanoparticle tracking analysis
  • 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 Burkholderia bacterium for every 1 , 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,
  • the pharmaceutical composition comprises at least 1 Burkholderia
  • the pharmaceutical composition comprises about 1 Burkholderia 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,
  • the pharmaceutical composition comprises no more than 1 Burkholderia EV particle for every 1 , 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2,
  • the Burkholderia EVs in the pharmaceutical composition are purified from one or more other bacterial components. In some embodiments, the
  • composition further comprises other bacterial components.
  • the pharmaceutical composition comprise bacteria cells.
  • compositions for administration subjects are provided.
  • the pharmaceutical compositions are combined with additional active and/or inactive materials in order to produce a final product, which may be in single dosage unit or in a multi-dose format.
  • the composition comprises at least one carbohydrate.
  • a “carbohydrate” refers to a sugar or polymer of sugars. The terms“saccharide,”“polysaccharide,” “carbohydrate,” and“oligosaccharide” may be used interchangeably. Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one on each carbon atom of the molecule. Carbohydrates generally have the molecular formula CnEhnOn. 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), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6) (DHA), and t
  • the composition comprises at least one supplemental mineral or mineral source.
  • supplemental mineral or mineral source examples include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium.
  • Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.
  • the composition comprises at least one supplemental vitamin.
  • the at least one vitamin can be fat-soluble or water-soluble vitamins.
  • Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin.
  • Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.
  • the composition comprises an excipient.
  • suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.
  • the excipient is a buffering agent.
  • suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.
  • the excipient comprises a preservative.
  • suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.
  • the composition comprises a binder as an excipient.
  • suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof.
  • the composition comprises a lubricant as an excipient.
  • suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc,
  • polyethyleneglycol sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil.
  • the composition comprises a dispersion enhancer as an excipient.
  • suitable dispersants include starch, alginic acid,
  • polyvinylpyrrolidones 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 composition is a food product (e.g a food or beverage) such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.
  • a food product e.g a food or beverage
  • a health food or beverage e.g. a food or beverage
  • a food or beverage for infants e.g., a food or beverage for infants
  • a food or beverage for pregnant women e.g., athletes, senior citizens or other specified group
  • a functional food e.g., a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.
  • the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, and Chinese soups; soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products, including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, carb
  • the composition is a food product for animals, including humans.
  • the animals, other than humans, are not particularly limited, and the composition can be used for various livestock, poultry, pets, experimental animals, and the like.
  • Specific examples of the animals include pigs, cattle, horses, sheep, goats, chickens, wild ducks, ostriches, domestic ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, and the like, but the animals are not limited thereto.
  • the methods provided herein include the administration to a subject of a pharmaceutical composition described herein either alone or in combination with an additional therapeutic.
  • the additional therapeutic is an
  • immunosuppressant a steroid, and/or a cancer therapeutic.
  • the Burkholderia EV is administered to the subject before the 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 Burkholderia EV is administered to the subject after the therapeutic is administered (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours after or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27.
  • the Burkholderia EV and the 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 Burkholderia EV is administered to the subject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 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 Burkholderia EV is administered to the subject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days after).
  • the Burkholderia EV and the antibiotic are administered to the subject simultaneously or nearly simultaneously (e.g., administrations occur within an hour of each other).
  • 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 thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide,
  • triethiylenethiophosphoramide and trimethylolomelamine 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 gammall and calicheamicin omegall; dynemicin, including dynemicin A;
  • bisphosphonates such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophibn, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrobno- doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid
  • 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; vincristine;
  • 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.
  • 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.
  • cancer-associated antigens include, but are not limited to, 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, 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, FGF5, FLT
  • 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,
  • Triosephosphate isomerase TRP-l/gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-lb/GAGED2a.
  • the antigen is a neo-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
  • Adjuvant 65 an immune modulatory protein
  • Adjuvant 65 a-GalCer
  • aluminum phosphate aluminum hydroxide
  • calcium phosphate calcium phosphate
  • b-Glucan Peptide CpG ODN DNA
  • GPI-0100 lipid A
  • lipopolysaccharide lipovant
  • Montanide N-acetyl-muramyl-L-alanyl- D-isoglut
  • enterotoxigenic Escherichia coli including derivatives of these (CTB, mmCT, CTA1-DD, LTB, LTK63, LTR72, dmLT) and trehalose dimycolate.
  • 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
  • Macrophage inflammatory protein- 1 -delta Macrophage inflammatory protein- 1 -delta
  • PDGF-BB Platelet-derived growth factor subunit B
  • Chemokine (C-C motif) ligand 5 Regulated on Activation, Normal T cell Expressed and Secreted
  • RANTES Normal T cell Expressed and Secreted
  • ⁇ MR metallopeptidase inhibitor 1 ⁇ MR metallopeptidase inhibitor 1
  • 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 (“VEGFR3”), VEGF-D 6Ckine, Tyrosine-protein kinase receptor UFO (“Axl”), Betacellulin (“BTC”), Mucosae-associated epithelial chemokine (“CCF28”), Chemokine (C-C motif) ligand 27 (“CT
  • VEGFRlAdiponectin Adipsin
  • AFP Alpha-fetoprotein
  • ANGPTL4 Angiopoietin-like 4
  • B2M Basal cell adhesion molecule
  • BCAM Basal cell adhesion molecule
  • CA125 Carbohydrate antigen 125
  • CA15-3 Cancer Antigen 15-3
  • CEA Carcinoembryonic antigen
  • CPP cAMP receptor protein
  • ErbB2 Human Epidermal Growth Factor Receptor 2
  • FoUistatin Follicle-stimulating hormone
  • FSH Follicle-stimulating hormone
  • FSH Follicle-stimulating hormone
  • GRO alpha Chemokine (C-X-C motif) ligand 1
  • GRO alpha Chemokine (C-X-C motif) ligand 1
  • beta HCG Insulin-like growth factor 1 receptor
  • IGF-l sR Insulin-like growth factor 1 receptor
  • IGF-l sR Insulin-like growth
  • Interleukin 24 Interleukin 24
  • Interleukin 33 Interleukin 33
  • Kalbkrein 14 Asparaginyl endopeptidase
  • Legumain Oxidized low-density lipoprotein receptor 1
  • MBL Mannose-binding lectin
  • Neprilysin NEP
  • 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
  • FLR1 Furin
  • GASP-l GPCR-associated sorting protein 1
  • GASP-2 GPCR-associated sorting protein 2
  • GCSF R Granulocyte colony- stimulating factor receptor
  • HAI-2 Serine protease hepsin
  • IL-17B R Interleukin 17B Receptor
  • IL-27 Interleukin 27
  • LAG-3 Lymphocyte-activation gene 3
  • LDL R Apolipoprotein A-V
  • Pepsinogen I Pepsinogen I
  • Retinol binding protein 4 (“RBP4"), SOST, Heparan sulfate proteoglycan (“Syndecan-l”),
  • Tumor necrosis factor receptor superfamily member 13B (“TACI”), Tissue factor pathway inhibitor (“TFPI”), TSP-l, Tumor necrosis factor receptor superfamily, member lOb (“TRAIL R2”), TRANCE, Troponin I, Urokinase Plasminogen Activator (“uPA”), Cadherin 5, type 2 or VE-cadherin (vascular endothelial) also known as CD144 (“VE-Cadherin”), WNTl-inducible- signaling pathway protein 1 (“WISP-l "), and Receptor Activator of Nuclear Factor k B
  • the cancer therapeutic agent is an anti-cancer compound.
  • Exemplary 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 (KyprolisTM), Cetuximab (Erbitux®), Crizotinib (Xalkori®), Dasatinib (Sprycel®), Denileukin diftitox (Ontak®), Erlotinib hydrochloride (Tarceva®), Everolimus (Afinitor®), Exemestane (Aromasin®), Fulvestrant (Faslod
  • 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 l3lI-tositumomab (Bexxar®), Tras
  • 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,
  • the cancer therapeutic is an antibiotic.
  • antibiotics can be administered to eliminate the cancer-associated bacteria from the subject.
  • Antibiotics broadly refers to compounds capable of inhibiting or preventing a bacterial infection. Antibiotics can be classified in a number of ways, including their use for specific infections, their mechanism of action, their
  • antibiotics can be used to selectively target bacteria of a specific niche.
  • antibiotics known to treat a particular infection that includes a cancer niche may be used to target cancer-associated microbes, including cancer-associated bacteria in that niche.
  • antibiotics are administered after the bacterial treatment.
  • antibiotics are administered after the bacterial treatment to remove the engraftment.
  • antibiotics can be selected based on their bactericidal or bacteriostatic properties.
  • Bactericidal antibiotics include mechanisms of action that disrupt the cell wall (e.g., b-lactams), the cell membrane (e.g., daptomycin), or bacterial DNA (e.g., fluoroquinolones).
  • Bacteriostatic agents inhibit bacterial replication and include sulfonamides, tetracyclines, and macrolides, and act by inhibiting protein synthesis.
  • some drugs can be bactericidal in certain organisms and bacteriostatic in others, knowing the target organism allows one skilled in the art to select an antibiotic with the appropriate properties.
  • bacteriostatic antibiotics inhibit the activity of bactericidal antibiotics.
  • bactericidal and bacteriostatic antibiotics are not combined.
  • Antibiotics include, but are not limited to aminoglycosides, ansamycins, carbacephems, carbapenems, cephalosporins, glycopeptides, lincosamides, bpopeptides, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptide antibiotics, quinolones, fluoroquinolone, sulfonamides, tetracyclines, and anti-mycobacterial compounds, and combinations thereof.
  • Aminoglycosides include, but are not limited to Amikacin, Gentamicin,
  • Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, and Spectinomycin are examples of the compounds listed in the following paragraphs.
  • Aminoglycosides are effective, e.g., against Gram-negative bacteria, such as Escherichia cob, Klebsiella, Pseudomonas aeruginosa, and Francisella tularensis, and against certain aerobic bacteria but less effective against obbgate/facultative anaerobes. Aminoglycosides are believed to bind to the bacterial 30S or 50S ribosomal subunit thereby inhibiting bacterial protein synthesis.
  • Ansamycins include, but are not limited to, Geldanamycin, Herbimycin, Rifamycin, and Streptovaricin.
  • Geldanamycin and Herbimycin are believed to inhibit or alter the function of Heat Shock Protein 90.
  • Carbacephems include, but are not limited to, Loracarbef. Carbacephems are believed to inhibit bacterial cell wall synthesis.
  • Carbapenems include, but are not limited to, Ertapenem, Doripenem,
  • Carbapenems are bactericidal for both Gram-positive and Gram-negative bacteria as broad-spectrum antibiotics. Carbapenems are believed to inhibit bacterial cell wall synthesis.
  • Cephalosporins include, but are not limited to, Cefadroxil, Cefazobn, Cefalotin, Cefalothin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil,and Ceftobiprole. Selected
  • Cephalosporins are effective, e.g., against Gram-negative bacteria and against Gram-positive bacteria, including Pseudomonas, certain Cephalosporins are effective against methicillin- resistant Staphylococcus aureus (MRSA). Cephalosporins are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Glycopeptides include, but are not limited to, Teicoplanin, Vancomycin, and Telavancin. Glycopeptides are effective, e.g., against aerobic and anaerobic Gram-positive bacteria including MRSA and Clostridium difficile. Glycopeptides are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Lincosamides include, but are not limited to, Clindamycin and Lincomycin.
  • Lincosamides are effective, e.g., against anaerobic bacteria, as well as Staphylococcus, and Streptococcus. Lincosamides are believed to bind to the bacterial 50S ribosomal subunit thereby inhibiting bacterial protein synthesis.
  • Lipopeptides include, but are not limited to, Daptomycin. Lipopeptides are effective, e.g., against Gram-positive bacteria. Lipopeptides are believed to bind to the bacterial membrane and cause rapid depolarization.
  • Macrobdes include, but are not limited to, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, and Spiramycin. Macrobdes are effective, e.g., against Streptococcus and Mycoplasma. Macrobdes are believed to bind to the bacterial or 50S ribosomal subunit, thereby inhibiting bacterial protein synthesis.
  • Monobactams include, but are not limited to, Aztreonam. Monobactams are effective, e.g., against Gram-negative bacteria. Monobactams are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Nitrofurans include, but are not limited to, Furazolidone and Nitrofurantoin.
  • Oxazobdonones include, but are not limited to, Linezobd, Posizobd, Radezobd, and Torezolid. Oxazobdonones are believed to be protein synthesis inhibitors.
  • Penicillins include, but are not limited to, Amoxicillin, Ampicillin, Azlocillin, Carbenicilbn, Cloxacillin, Dicloxacilbn, Flucloxacillin, Mezlocillin, Methicillin, Nafcilbn, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Temocilbn and Ticarcillin. Penicillins are effective, e.g., against Gram-positive bacteria, facultative anaerobes, e.g., Streptococcus,
  • Penicillins are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Penicillin combinations include, but are not limited to, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam, and Ticarcillin/clavulanate.
  • Polypeptide antibiotics include, but are not limited to, Bacitracin, Colistin, and Polymyxin B and E.
  • Polypeptide Antibiotics are effective, e.g., against Gram-negative bacteria. Certain polypeptide antibiotics are believed to inhibit isoprenyl pyrophosphate involved in synthesis of the peptidoglycan layer of bacterial cell walls, while others destabilize the bacterial outer membrane by displacing bacterial counter-ions.
  • Quinolones and Fluoroquinolone include, but are not limited to, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, 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, Demeclocycline, Doxycycline, Minocycline, Oxytetracy cline, and Tetracycline. Tetracyclines are effective, e.g., against Gram negative bacteria. Tetracyclines are believed to bind to the bacterial 30S ribosomal subunit thereby inhibiting bacterial protein synthesis.
  • Anti-mycobacterial compounds include, but are not limited to, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, and Streptomycin.
  • Suitable antibiotics also include arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin/dalfopristin, tigecycline, tinidazole, trimethoprim amoxicillin/clavulanate, ampicillin/sulbactam, amphomycin ristocetin, azithromycin, bacitracin, buforin II, carbomycin, cecropin PI, clarithromycin, erythromycins, furazolidone, fusidic acid, Na fusidate, gramicidin, imipenem, indolicidin, josamycin, magainan II, metronidazole, nitroimidazoles, mikamycin, mutacin B-Ny266, mutacin B-JH1 140, mutacin J-T8, nisin, nisin A, novobiocin, ole
  • the additional therapeutic is an immunosuppressive agent, a DMARD, a pain-control drug, a steroid, a non-steroidal anti-inflammatory drug (NSAID), or a cytokine antagonist, and combinations thereof.
  • a DMARD a pain-control drug
  • a steroid a steroid
  • NSAID non-steroidal anti-inflammatory drug
  • cytokine antagonist a cytokine antagonist
  • Representative agents include, but are not limited to, cyclosporin, retinoids, corticosteroids, propionic acid derivative, acetic acid derivative, enolic acid derivatives, fenamic acid derivatives, Cox-2 inhibitors, lumiracoxib, ibuprophen, cholin magnesium salicylate, fenoprofen, salsalate, difunisal, tolmetin, ketoprofen, flurbiprofen, oxaprozin, indomethacin, sulindac, etodolac, ketorolac, nabumetone, naproxen, valdecoxib, etoricoxib, MK0966; rofecoxib, acetominophen, Celecoxib, Diclofenac, tramadol, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefanamic acid, meclofenamic acid
  • TNF alpha antagonists e.g., TNF alpha antagonists or TNF alpha receptor antagonists
  • ADALIMUMAB Humira®
  • ETANERCEPT Enbrel®
  • INFLIXIMAB (Remicade®; TA-650), CERTOLIZEMAB PEGOL (Cimzia®; CDP870), GOLIMUMAB (Simpom®; CNTO 148), ANAKINRA (Kineret®), RITUXIMAB (Rituxan®; MabThera®), ABATACEPT (Orencia®), TOCILIZUMAB (RoActemra /Actemra®), integrin antagonists (TYSABRI® (natalizumab)), IL-l antagonists (ACZ885 (Ilaris)), Anakinra
  • CD4 antagonists IL-23 antagonists
  • IL-20 antagonists IL-6 antagonists
  • BLyS antagonists e.g., Atacicept, Benlysta®/ LymphoStat-B® (belimumab)
  • p38 Inhibitors CD20 antagonists (Ocrelizumab, Ofatumumab (Arzerra®)), interferon gamma antagonists
  • MMP antagonists MMP antagonists, defensin antagonists, IL-l antagonists (including IL-l beta antagonsits), and IL-23 antagonists (e.g., receptor decoys, antagonistic antibodies, etc.).
  • IL-l antagonists including IL-l beta antagonsits
  • IL-23 antagonists e.g., receptor decoys, antagonistic antibodies, etc.
  • the agent is an immunosuppressive agent.
  • immunosuppressive agents include, but are not limited to, corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine, theophylline, cromolyn sodium, anti-leukotrienes, anti-cholinergic drugs for rhinitis, TLR antagonists, inflammasome inhibitors, anti-cholinergic decongestants, mast-cell stabilizers, monoclonal anti-IgE antibodies, vaccines (e.g., vaccines used for vaccination where the amount of an allergen is gradually increased), cytokine inhibitors, such as anti-IL-6 antibodies, TNF inhibitors such
  • provided herein is a method of delivering a pharmaceutical composition described herein to a subject.
  • the pharmaceutical composition is administered in conjunction with the administration of an additional therapeutic.
  • the pharmaceutical composition comprises Burkholderia EVs and/or bacteria co-formulated with the additional therapeutic.
  • the pharmaceutical composition is co-administered with the additional therapeutic.
  • the additional therapeutic is administered to the subject before
  • administration of the pharmaceutical composition 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,
  • the additional therapeutic is administered to the subject after administration of the pharmaceutical composition (e.g ., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes after, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours after, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days after).
  • the same mode of delivery are used to deliver both the
  • compositions and the additional therapeutic are used to administer the pharmaceutical composition and the additional therapeutic.
  • different modes of delivery are used to administer the pharmaceutical composition and the additional therapeutic.
  • the pharmaceutical composition 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-cancer treatment, such as, for example, radiation therapy and surgical resection of the tumor. These treatments may be applied as necessary and/or as indicated and may occur before, concurrent with or after administration of the pharmaceutical compositions, dosage forms, and kits described herein.
  • the dosage regimen can be any of a variety of methods and amounts, and can be determined by one skilled in the art according to known clinical factors. As is known in the medical arts, dosages for any one patient can depend on many factors, including the subject's species, size, body surface area, age, sex, immunocompetence, and general health, the particular microorganism to be administered, duration and route of administration, the kind and stage of the disease, for example, tumor size, and other compounds such as drugs being administered concurrently. In addition to the above factors, such levels can be affected by the infectivity of the microorganism, and the nature of the microorganism, as can be determined by one skilled in the art.
  • appropriate minimum dosage levels of microorganisms can be levels sufficient for the microorganism to survive, grow and replicate.
  • 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 disease (e.g ., cancer), delay its onset, or slow or stop its progression.
  • disease e.g ., cancer
  • dosage will depend upon a variety of factors including the strength of the particular compound employed, as well as the age, species, condition, and body weight of the subject.
  • the size of the dose will also be determined by the route, timing, and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound and the desired physiological effect.
  • Suitable doses and dosage regimens can be determined by conventional range-finding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached.
  • An effective dosage and treatment protocol can be determined by routine and conventional means, starting e.g., with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Animal studies are commonly used to determine the maximal tolerable dose ("MTD”) of bioactive agent per kilogram weight. Those skilled in the art regularly extrapolate doses for efficacy, while avoiding toxicity, in other species, including humans.
  • MTD maximal tolerable dose
  • the dosages of the active agents used in accordance with the invention vary depending on the active agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage.
  • the dose should be sufficient to result in slowing, and preferably regressing, the growth of the tumors and most preferably causing complete regression of the cancer.
  • Separate administrations can include any number of two or more administrations, including two, three, four, five or six administrations.
  • One skilled in the art can readily determine the number of administrations to perform or the desirability of performing one or more additional administrations according to methods known in the art for monitoring therapeutic methods and other monitoring methods provided herein.
  • the methods provided herein include methods of providing to the subject one or more administrations of an pharmaceutical composition, 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.
  • 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 EV 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 EV from normal tissue; for example, the time period can be more than the time period for a subject to clear the EV 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 additional therapeutic in combination with the pharmaceutical composition described herein reduces the adverse effects and/or improves the efficacy of the additional therapeutic.
  • the effective dose of an additional therapeutic described herein is the amount of the therapeutic agent that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, with the least toxicity to the patient.
  • the effective dosage level can be identified using the methods described herein and will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions administered, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • an effective dose of an additional therapy will be the amount of the therapeutic agent which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the toxicity of an additional therapy is the level of adverse effects experienced by the subject during and following treatment.
  • Adverse events associated with additional therapy toxicity include, but are not limited to, abdominal pain, acid indigestion, acid reflux, allergic reactions, alopecia, anaphylasix, anemia, anxiety, lack of appetite, arthralgias, asthenia, ataxia, azotemia, loss of balance, bone pain, bleeding, blood clots, low blood pressure, elevated blood pressure, difficulty breathing, bronchitis, bruising, low white blood cell count, low red blood cell count, low platelet count, cardiotoxicity, cystitis, hemorrhagic cystitis, arrhythmias, heart valve disease, cardiomyopathy, coronary artery disease, cataracts, central neurotoxicity, cognitive impairment, confusion, conjunctivitis, constipation, coughing, cramping, cystitis, deep vein thrombosis, dehydration, depression, diarrhea, dizziness, dry mouth, dry skin, dyspepsia, dysp
  • 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, 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
  • Cancers treated in some embodiments include non-cancerous or benign tumors, e.g., of endodermal, ectodermal or mesenchymal origin, including, but not limited to
  • cholangioma cholangioma, colonic polyp, adenoma, papilloma, cystadenoma, liver cell adenoma,
  • hydatidiform mole renal tubular adenoma, squamous cell papilloma, gastric polyp, hemangioma, osteoma, chondroma, lipoma, fibroma, lymphangioma, leiomyoma, rhabdomyoma, astrocytoma, nevus, meningioma, and ganglioneuroma.
  • the methods and compositions described herein relate to the treatment of Nonalcoholic Fatty Liver Disease (NAFLD) and Nonalcoholic Steatohepatitis (NASH).
  • NAFLD Nonalcoholic Fatty Liver Disease
  • NASH Nonalcoholic Steatohepatitis
  • the engineered Burkholderia bacteria are modified to enhance certain desirable properties.
  • the engineered Burkholderia bacteria are modified to increase production of EVs by the Burkholderia bacteria.
  • the engineered Burkholderia bacteria are modified to increase production of EVs by the Burkholderia bacteria.
  • Burkholderia bacteria are modified to produce EVs with enhanced oral delivery (e.g., by improving acid resistance and/or resistance to bile acids), to enhance the therapeutic effect of the EVs they produce (e.g., either alone or in combination with another therapeutic agent), to enhance immune activation by the EVs they produce and/or to improve Burkholderia bacterial and/or EV manufacturing (e.g., higher oxygen tolerance, improved freeze-thaw tolerance, shorter generation times).
  • the engineered Burkholderia bacteria may be produced using any technique known in the art, including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, CRISPR/Cas9, or any combination thereof.
  • the Burkholderia bacteria is modified by directed evolution.
  • the directed evolution comprises exposure of the Burkholderia to an environmental condition and selection of Burkholderia with improved survival and/or growth under the environmental condition.
  • the method comprises a screen of mutagenized Burkholderia bacteria using an assay that identifies enhanced Burkholderia bacteria.
  • the method further comprises mutagenizing the Burkholderia bacteria (e.g., by exposure to chemical mutagens and/or UV radiation) followed by an assay to detect Burkholderia bacteria having the desired phenotype (e.g., an in vivo assay, an ex vivo assay, or an in vitro assay).
  • an assay to detect Burkholderia bacteria having the desired phenotype e.g., an in vivo assay, an ex vivo assay, or an in vitro assay.
  • the Burkholderia bacteria provided herein are modified by exposure to a stress-inducing environment (e.g., an environment that induces envelope stress).
  • a stress-inducing environment e.g., an environment that induces envelope stress.
  • growth under such growth conditions increase production of EVs by the Burkholderia bacteria.
  • the. Burkholderia bacteria is grown in the presence of subinhibitory concentrations of an antibiotic described herein (e.g., 0.1-1 pg/mL chloramphenicol, or 0.1-0.3 pg/mL gentamicin).
  • an antibiotic described herein e.g., 0.1-1 pg/mL chloramphenicol, or 0.1-0.3 pg/mL gentamicin.
  • host e.g., 0.1-1 pg/mL chloramphenicol, or 0.1-0.3 pg/mL gentamicin.
  • antimicrobial peptides e.g., lysozyme, defensins, and Reg proteins
  • Burkholderia bacterially-produced antimicrobial peptides e.g., Burkholderia bacteriocins and microcins
  • the stress is temperature stress (e.g., growth at 37-50°C).
  • the stress is carbon limitation stress (e.g., growth in a media comprising limited carbon sources, such as media with carbon source restricted below 1% (w/v)).
  • the stress is salt stress (e.g., growth in a medium containing 0.5M NaCl).
  • the stress is UV stress (e.g., growth under a UV lamp, either throughout the entire cultivation period or only during a portion of the cultivation period).
  • the stress is reactive oxygen stress (e.g., growth in media containing subinhibitory concentrations of hydrogen peroxide, such as 250-1,000 pM hydrogen peroxide).
  • a combination of the stresses disclosed herein are applied to the Burkholderia bacteria.
  • Extracellular vesicles are prepared from Burkholderia bacterial cultures using methods known to those skilled in the art (S. Bin Park, et al. PLoS ONE. 6(3):el7629 (2011)).
  • Burkholderia 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.
  • using a filtration technique using an Amicon Ultra spin filter or by tangential flow filtration, supernatants are filtered so as to retain species of molecular weight >
  • EVs are obtained from Burkholderia 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 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. 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 Burkholderia 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
  • EVs are labeled as previously described (N. Kesty, et al. EMBO Journal. 23: 4538-4549 (2004)).
  • purified EVs are incubated with fluorescein isothiocyanate (FITC) (Sigma- Aldrich, USA), Cy7, or any other fluorochrome suitable for flow cytometry, 1 : 1 for 1 hour at 25°C. The incubation step may be extended overnight at 4°C.
  • FITC fluorescein isothiocyanate
  • EVs are then either (1) pelleted by centrifugation at 200,000 x g for 3 hr- overnight, washed, and resuspended in PBS or another appropriate buffer for downstream applications; or (2) buffer exchanged into PBS or another appropriate buffer for downstream applications by dialysis or by filtration (e.g, using an Amicon Ultra column).
  • EVs will be obtained from Burkholderia bacteria cultured in medium containing 0.8 mM 3-azido-D-alanine or HADA. EVs will be resuspended or buffer exchanged into PBS and a portion will be further labeled with lOuM Dibenzo-aza-cyclooctyne (DIBAC)-fluorescent dye in l%BSA/PBS (dyes include Cy5, TAMRA, Rhodamine-green, and Cy7) if grown with 3-azido-D-alanine. Unincorporated dye will be removed as described above, by (1) ultracentrifugation, washing, and resuspension; or (2) buffer exchange by dialysis or filtration.
  • DIBAC Dibenzo-aza-cyclooctyne
  • Labeled EVs may also be generated from Burkholderia bacteria expressing green- fluorescent protein (GFP), or any other fluorescent protein.
  • GFP green- fluorescent protein
  • Quantum dots may be used to label EVs for non-invasive in vivo imaging studies (K. Kikushima, et al. Scientific Reports. 3(1913) (2013)). Quantum dots are conjugated to an antibody confirmed to be present in the EV membrane. Isolated EVs are incubated with quantum dot conjugates, and extra conjugates are removed as described above, by (1) ultracentrifugation, washing, and resuspension; or (2) buffer exchange by dialysis or filtration.
  • Fluorescently labeled EVs are detected in in vitro and ex vivo samples by confocal microscopy, nanoparticle tracking analysis, and/or flow cytometry. Additionally, fluorescently labeled EVs will be detected in whole animals and/or dissected organs and tissues using an instrument such as the IVIS spectrum CT (Perkin Elmer), as in H-I. Choi, et al. Experimental & Molecular Medicine . 49: e330 (2017).
  • EVs may be radiolabeled as previously described (Z. Varga et al., Cancer Biother Radiopharm. 2016 Jun;3l(5): l68-73).
  • purified EVs are radiolabeled with the 99m Tc-tricarbonyl complex [ 99m Tc(C0)3(H20)3] + using a commercial kit (Isolink®; Mallinckrodt Medical B.V.), according to the manufacturer's instructions.
  • Example 3 Transmission electron microscopy to visualize bacterial production of EVs and purified bacterial EVs
  • TEM Transmission electron microscopy
  • EMs are prepared from bacteria batch culture as described in Example 1. EVs are mounted onto 300- or 400-mesh-size carbon-coated copper grids (Electron Microscopy Sciences, ETSA) for 2 min and washed with deionized water. EVs are negatively stained using 2% (w/v) uranyl acetate for 20 sec - 1 min. Copper grids are washed with sterile water and dried. Images are acquired using a transmission electron microscope with 100-120 kV acceleration voltage. Stained EVs appear between 20-250 nm in diameter and are electron dense. 10-50 fields on each grid are screened.
  • Example 4 Profiling EV composition and content
  • EVs may be characterized by any one of various methods including, but not limited to, NanoSight characterization, SDS-PAGE gel electrophoresis, Western blot, ELISA, liquid chromatography-mass spectrometry and mass spectrometry, dynamic light scattering, lipid levels, total protein, lipid to protein ratios, and zeta potential.
  • Nanoparticle tracking analysis is used to characterize the size distribution of purified bacterial EVs. Purified EV preps are run on a NanoSight machine (Malvern
  • Samples are boiled in lx SDS sample buffer for 10 min, cooled to 4°C, and then centrifuged at 16,000 x g for 1 min. Samples are then run on a SDS-PAGE gel and stained using one of several standard techniques (e.g., Silver staining, Coomassie Blue, Gel Code Blue ) for visualization of bands.
  • Silver staining e.g., Coomassie Blue, Gel Code Blue
  • a LC-MS system includes a 4000 QTRAP triple quadrupole mass spectrometer (AB SCIEX) combined with 1100 Series pump (Agilent) and an HTS PAL autosampler (Leap Technologies).
  • the column is eluted by flowing a 5% mobile phase [lOmM ammonium formate, 0.1% formic acid in water] for 1 min at a rate of 250uL/min followed by a linear gradient over 10 min to a solution of 40% mobile phase [acetonitrile with 0.1% formic acid].
  • the ion spray voltage is set to 4.5 kV and the source temperature is 450 °C.
  • DLS Dynamic light scattering
  • Lipid levels are quantified using FM4-64 (Life Technologies), by methods similar to those described by A.J. McBroom et al. J Bacteriol 188:5385-5392. and A. Frias, et al.
  • Protein levels are quantified by standard assays such as the Bradford and BCA assays.
  • the Bradford assays are run using Quick Start Bradford lx Dye Reagent (Bio-Rad), according to manufacturer’s protocols.
  • BCA assays are run using the Pierce BCA Protein Assay Kit (Thermo-Fisher Scientific). Absolute concentrations are determined by comparison to a standard curve generated from BSA of known concentrations.
  • Lipid:protein ratios are generated by dividing lipid concentrations by protein concentrations. These provide a measure of the purity of vesicles as compared to free protein in each preparation.
  • the zeta potential of different preparations will be measured using instruments such as the Zetasizer ZS (Malvern Instruments).
  • Example 5 Manipulating bacteria through stress to produce various amounts of EVs and/or to vary content of EVs
  • EV production will be quantified (1) in complex samples of bacteria and EVs by nanoparticle tracking analysis (NTA) or transmission electron microscopy (TEM); or (2) following EV purification by NTA, lipid quantification, or protein quantification. EV content will be assessed following purification by methods described above.
  • NTA nanoparticle tracking analysis
  • TEM transmission electron microscopy
  • Bacteria are cultivated under standard growth conditions with the addition of subinhibitory concentrations of antibiotics. This may include 0.1-1 pg/mL chloramphenicol, or 0.1 -0.3 pg/mL gentamicin, or similar concentrations of other antibiotics (i.e. ampicillin, polymyxin B). Host antimicrobial peptides such as lysozyme, defensins, and Reg proteins may be used in place of antibiotics. Bacterially-produced antimicrobial peptides, including bacteriocins and microcins may also be used.
  • Bacteria are cultivated under standard growth conditions, but at higher or lower temperatures than are typical for their growth. Alternatively, bacteria are grown under standard conditions, and then subjected to cold shock or heat shock by incubation for a short period of time at low or high temperatures respectively. For example, bacteria grown at 37°C are incubated for 1 hour at 4°C-l8°C for cold shock or 42°C-50°C for heat shock.
  • bacteria are cultivated under conditions where one or more nutrients are limited. Bacteria may be subjected to nutritional stress throughout growth or shifted from a rich medium to a poor medium.
  • Some examples of media components that are limited are carbon, nitrogen, iron, and sulfur.
  • An example medium is M9 minimal medium (Sigma- Aldrich), which contains low glucose as the sole carbon source. Particularly for
  • Burkholderia spp. iron availability will be varied by altering the concentration of haemin in media and/or by varying the type of porphyrin or other iron carrier present in the media, as cells grown in low haemin conditions were found to produce greater numbers of EVs (S. Stubbs et al. Letters in Applied Microbiology. 29:31-36 (1999) [234] Saturation
  • Bacteria will be grown to saturation and incubated past the saturation point for various periods of time. Alternatively, conditioned media will be used to mimic saturating environments during exponential growth. Conditioned media will be prepared by removing intact cells from saturated cultures by centrifugation and filtration, as described in Example 1, and conditioned media may be further treated to concentrate or remove specific components.
  • Bacteria are cultivated in or exposed for brief periods to medium containing NaCl, bile salts, or other salts.
  • UV stress is achieved by cultivating bacteria under a UV lamp or by exposing bacteria to UV using an instrument such as a Stratalinker (Agilent). UV may be administered throughout the entire cultivation period, in short bursts, or for a single defined period following growth.
  • Stratalinker Stratalinker
  • Bacteria are cultivated in the presence of subinhibitory concentrations of hydrogen peroxide (250-1,000 mM) to induce stress in the form of reactive oxygen species. Anaerobic bacteria will be cultivated in or exposed to concentrations of oxygen that are toxic to them.
  • Bacteria will be cultivated in or exposed to detergent, such as sodium dodecyl sulfate (SDS) or deoxycholate.
  • detergent such as sodium dodecyl sulfate (SDS) or deoxycholate.
  • Bacteria will be cultivated in or exposed for limited times to media of different pH.
  • Bacterial samples containing minimal amounts of EVs are prepared. EV production are quantified (1) in complex samples of bacteria and extracellular components by NTA or TEM; or (2) following EV purification from bacterial samples, by NTA, lipid quantification, or protein quantification.
  • a. Centrifugation and washing Bacterial cultures are centrifuged at 11,000 x g to separate intact cells from supernatant (including free proteins and vesicles). The pellet is washed with buffer, such as PBS, and stored in a stable way (e.g., mixed with glycerol, flash frozen, and stored at -80°C).
  • ATF Bacteria and EVs are separated by connection of a bioreactor to an ATF system. EV-free bacteria is retained within the bioreactor, and may be further separated from residual EVs by centrifugation and washing, as described above.
  • Bacteria are grown under conditions that are found to limit production of EVs. Conditions that may be varied include those listed for Example 5.
  • Example 7 In vitro screening of EVs for enhanced activation of dendritic cells
  • Vibrio cholerae EVs to activate dendritic cells indirectly through epithelial cells is one nonlimiting mechanism by which they stimulate an immune response in mammalian hosts (D. Chatterjee, K. Chadhuri. J Biol Chem. 288(6):4299-309. (2013)).
  • this EV activity is likely shared with other bacteria that stimulate pro-inflammatory cascades in vivo
  • in vitro methods to assay DC activation by bacterial EVs are disclosed herein. Briefly, PBMCs are isolated from heparinized venous blood from CMs by gradient centrifugation using
  • Lymphoprep (Nycomed, Oslo, Norway) or from mouse spleens or bone marrow using the magnetic bead-based Human Blood Dendritic cell isolation kit (Miltenyi Biotech, Cambridge, MA).
  • the monocytes are purified by Moflo and cultured in cRPMI at a cell density of 5e5 cells/ml in a 96-well plate (Costar Corp) for 7 days at 37°C.
  • the culture is stimulated with 0.2 ng/mL IL-4 and 1000 U/ml GM- CSF at 37°C for one week.
  • Mouse DCs can be harvested directly from spleens using bead enrichment or differentiated from haematopheotic stem cells. Briefly, bone marrow is obtained from the femurs of mice. Cells are recovered and red blood cells lysed. Stem cells are cultured in cell culture medium together with 20ng/ml mouse GMCSF for 4 days. Additional medium containing 20ng/ml mouse GM-CSF is added. On day 6 the medium and non-adherent cells are removed and replaced with fresh cell culture medium containing 20ng/ml GMCSF. A final addition of cell culture medium with 20ng/ml GM-CSF is added on day 7. On day 10 non adherent cells are harvested and seeded into cell culture plates overnight and stimulated as required. Dendritic cells are then treated with 25-75 ug/mL EVs for 24 hours with antibiotics.
  • EV compositions tested may include a EVs from a single bacterial species or strain. EV compositions tested may also include a mixture of EVs from bacterial genera, species within a genus, or strains within a species. PBS and EVs from Lactobacillus are included as negative controls and LPS, anti-CD40 antibodies, and EVs from Burkholderia spp. are used as positive controls. Following incubation, DCs are stained with anti CDl lb, CDl lc, CD103, CD8a, CD40, CD80, CD83, CD86, MHCI and MHCII, and analyzed by flow cytometry. DCs that are significantly increased in CD40, CD80, CD83, and CD86 as compared to negative controls are considered to be activated by the associated bacterial EV composition. These experiments are repeated three times at minimum.
  • Epithelial cell lines may include Int407, HEL293, HT29, T84 and CAC02.
  • the beads are then washed twice with 200 m ⁇ wash buffer. 100 m ⁇ of IX biotinylated detector antibody is added and the suspension is incubated for 1 hr with shaking in the dark. Two, 200 m ⁇ washes are then performed with wash buffer. 100 m ⁇ of lx SAV-RPE reagent is added to each well and is incubated for 30 min at RT in the dark. Three 200 m ⁇ washes are performed and 125 m ⁇ of wash buffer is added with 2-3 min shaking occurs. The wells are then submitted for analysis in the Luminex xMAP system.
  • cytokines including GM-CSF, IFN- g, IFN-a, IFN-B, IL-la, IL-1B, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-12 (p40/p70), IL- 17A, IL-17F, IL-21, IL-22 IL-23, IL-25, IP-10, KC, MCP-l, MIG, MIPla, TNFa, and VEGF.
  • cytokines are assessed in samples of both mouse and human origin. Increases in these cytokines in the bacterial treated samples indicate enhanced production of proteins and cytokines from the host.
  • cytokine mRNA is also assessed to address cytokine release in response to a EV composition.
  • This DC stimulation protocol may be repeated using combinations of purified EVs and live bacterial strains to maximize immune stimulation potential.
  • Example 8 In vitro screening of EVs for enhanced activation of CD8+ T cell killing when incubated with tumor cells
  • DCs are isolated from human PBMCs or mouse spleens and incubated with single-strain EVs, mixtures of EVs, and appropriate controls.
  • CD8+ CD8+
  • T cells are obtained from human PBMCs or mouse spleens using the magnetic bead-based Mouse CD8a+ T Cell Isolation Kit and the magnetic bead-based Human CD8+ T Cell Isolation Kit (both from Miltenyi Biotech, Cambridge, MA).
  • EVs are removed from cells with PBS washes, lOOul of fresh media with antibiotics is added to each well, and 200,000 T cells are added to each experimental well in the 96-well plate.
  • Anti-CD3 antibody is added at a final concentration of 2ug/ml. Co-cultures are then allowed to incubate at 37°C for 96 hours under normal oxygen conditions.
  • mice tumor cell lines used include B16.F10, SIY+ B16.F10, and others.
  • Human tumor cell lines are HLA-matched to donor, and can include PANC-l, ETNKPC960/961, UNKC, and HELA cell lines.
  • 100 m ⁇ of the CD8+ T cell and DC mixture is transferred to wells containing tumor cells. Plates are incubated for 24 hours at 37°C under normal oxygen conditions. Staurospaurine is used as negative control to account for cell death.
  • flow cytometry is used to measure tumor cell death and characterize immune cell phenotype. Briefly, tumor cells are stained with viability dye. FACS analysis is used to gate specifically on tumor cells and measure the percentage of dead (killed) tumor cells. Data are also displayed as the absolute number of dead tumor cells per well.
  • Cytotoxic CD8+ T cell phenotype may be characterized by the following methods: a) concentration of supernatant granzyme B, IFNy and TNFa in the culture supernatant as described below, b) CD8+ T cell surface expression of activation markers such as DC69, CD25, CD 154, PD-l, gamma/delta TCR, Foxp3, T-bet, granzyme B, c) intracellular cytokine staining of IFNy, granzyme B, TNFa in CD8+ T cells.
  • CD4+ T cell phenotype may also be assessed by
  • intracellular cytokine staining in addition to supernatant cytokine concentration including INFy, TNFa, IL-12, IL-4, IL-5, IL-17, IL-10, chemokines etc.
  • the beads are then washed twice with 200 m ⁇ wash buffer. 100 m ⁇ of IX biotinylated detector antibody is added and the suspension is incubated for 1 hr with shaking in the dark. Two, 200 m ⁇ washes are then performed with wash buffer. 100 m ⁇ of lx SAV-RPE reagent is added to each well and is incubated for 30 min at RT in the dark. Three 200 m ⁇ washes are performed and 125 m ⁇ of wash buffer is added with 2-3 min shaking occurs. The wells are then submitted for analysis in the Luminex xMAP system.
  • cytokines including GM-CSF, IFN- g, IFN-a, IFN-B IL-la, IL-1B, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-12 (p40/p70), IL-17, IL-23, IP-10, KC, MCP-l, MIG, MIPla, TNFa, and VEGF.
  • cytokines are assessed in samples of both mouse and human origin. Increases in these cytokines in the bacterial treated samples indicate enhanced production of proteins and cytokines from the host.
  • cytokine mRNA is also assessed to address cytokine release in response to an EV composition.
  • This CD8+ T cell stimulation protocol may be repeated using combinations of purified EVs and live bacterial strains to maximize immune stimulation potential.
  • Example 9 In vitro screening of EVs for enhanced tumor cell killing by PBMCs
  • PBMCs are isolated from heparinized venous blood from CMs by ficoll-paque gradient centrifugation for mouse or human blood, or with Lympholyte Cell Separation Media (Cedarlane Labs, Ontario, Canada) from mouse blood.
  • PBMCs are incubated with single-strain EVs, mixtures of EVs, and appropriate controls.
  • CD8+ T cells are obtained from human PBMCs or mouse spleens.
  • EVs are removed from cells with PBS washes, lOOul of fresh media with antibiotics is added to each well, and 200,000 T cells are added to each experimental well in the 96-well plate.
  • Anti-CD3 antibody is added at a final concentration of 2ug/ml. Co cultures are then allowed to incubate at 37°C for 96 hours under normal oxygen conditions.
  • mice tumor cell lines used include B16.F10, SIY+ B16.F10, and others.
  • Human tumor cell lines are HLA-matched to donor, and can include PANC-l, ETNKPC960/961, ETNKC, and HELA cell lines.
  • 100 m ⁇ of the CD8+ T cell and PBMC mixture is transferred to wells containing tumor cells. Plates are incubated for 24 hours at 37°C under normal oxygen conditions. Staurospaurine is used as negative control to account for cell death.
  • flow cytometry is used to measure tumor cell death and characterize immune cell phenotype. Briefly, tumor cells are stained with viability dye. FACS analysis is used to gate specifically on tumor cells and measure the percentage of dead (killed) tumor cells. Data are also displayed as the absolute number of dead tumor cells per well.
  • Cytotoxic CD8+ T cell phenotype may be characterized by the following methods: a) concentration of supernatant granzyme B, IFNy and TNFa in the culture supernatant as described below, b) CD8+ T cell surface expression of activation markers such as DC69, CD25, CD 154, PD-l, gamma/delta TCR, Foxp3, T-bet, granzyme B, c) intracellular cytokine staining of IFNy, granzyme B, TNFa in CD8+ T cells.
  • CD4+ T cell phenotype may also be assessed by
  • intracellular cytokine staining in addition to supernatant cytokine concentration including INFy, TNFa, IL-12, IL-4, IL-5, IL-17, IL-10, chemokines etc.
  • the beads are then washed twice with 200 m ⁇ wash buffer. 100 m ⁇ of IX biotinylated detector antibody is added and the suspension is incubated for 1 hr with shaking in the dark. Two, 200 m ⁇ washes are then performed with wash buffer. 100 m ⁇ of lx SAV-RPE reagent is added to each well and is incubated for 30 min at RT in the dark. Three 200 m ⁇ washes are performed and 125 m ⁇ of wash buffer is added with 2-3 min shaking occurs. The wells are then submitted for analysis in the Luminex xMAP system.
  • cytokines including GM-CSF, IFN- g, IFN-a, IFN-B IL-la, IL-1B, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-12 (p40/p70), IL-17, IL-23, IP-10, KC, MCP-l, MIG, MIPla, TNFa, and VEGF.
  • cytokines are assessed in samples of both mouse and human origin. Increases in these cytokines in the bacterial treated samples indicate enhanced production of proteins and cytokines from the host.
  • Other variations on this assay examining specific cell types ability to release cytokines are assessed by acquiring these cells through sorting methods and are recognized by one of ordinary skill in the art.
  • cytokine mRNA is also assessed to address cytokine release in response to an EV composition.
  • This PBMC stimulation protocol may be repeated using combinations of purified EVs and live bacterial strains to maximize immune stimulation potential.
  • Example 10 In vitro detection of EVs in antigen-presenting cells
  • Dendritic cells in the lamina limbalium constantly sample live bacteria, dead bacteria, and microbial products in the gut lumen by extending their dendrites across the gut epithelium, which is one way that EVs produced by bacteria in the intestinal lumen may directly stimulate dendritic cells.
  • the following methods represent a way to assess the differential uptake of EVs by antigen-presenting cells.
  • these methods may be applied to assess immunomodulatory behavior of EVs administered to a patient.
  • DCs Dendritic cells
  • kit protocols e.g., Inaba K, Swiggard WJ, Steinman RM, Romani N, Schuler G, 2001. Isolation of dendritic cells. Current Protocols in Immunology. Chapter 3:CTnit3.7).
  • DCs are seeded on a round cover slip in complete RPMI-1640 medium and are then incubated with EVs from single bacterial strains or combinations EVs at a multiplicity of infection (MOI) between 1 : 1 and 1 : 10.
  • MOI multiplicity of infection
  • Purified EVs have been labeled with fluorochromes or fluorescent proteins as described in Example 2. After 1 hour of incubation, the cells are washed twice with ice-cold PBS, detached from the plate using trypsin. Cells are either allowed to remain intact or are lysed. Samples are then processed for flow cytometry.
  • Total internalized EVs are quantified from lysed samples, and percentage of cells that uptake EVs is measured by counting fluorescent cells.
  • the methods described above may also be performed in substantially the same manner using macrophages or epithelial cell lines (obtained from the ATCC) in place of DCs.
  • Example 11 In vitro screening of EVs with an enhanced ability to activate NK cell killing when incubated with target cells
  • NK cells are isolated using the autoMACs instrument and NK cell isolation kit following manufacturer’s instructions (Miltenyl Biotec).
  • NK cells are counted and plated in a 96 well format with 5000 cells per well, and incubated with single-strain EVs, EVs from mixtures of bacterial strains, and appropriate controls. As an additional negative control, this assay is run with EVs from Fusobacterium nucleatum. F. nucleatum is known to be inhibitory to NK cell activity (see e.g. Gur et al 2005 Immunity 42: 1-12). After 5-24 hours incubation of NK cells with EVs, EVs are removed from cells with PBS washes, NK cells are resuspended inlO fresh media with antibiotics, and are added to 96-well plates containing 50,000 target tumor cells/well.
  • Mouse tumor cell lines used include B16.F10, SIY+ B16.F10, and others.
  • Human tumor cell lines are HLA- matched to donor, and can include PANC-l, ETNKPC960/961, UNKC, and HELA cell lines. Plates are incubated for 24 hours at 37°C under normal oxygen conditions. Staurospaurine is used as negative control to account for cell death.
  • flow cytometry is used to measure tumor cell death. Briefly, tumor cells are stained with viability dye. FACS analysis is used to gate specifically on tumor cells and measure the percentage of dead (killed) tumor cells. Data are also displayed as the absolute number of dead tumor cells per well.
  • This NK cell stimulation protocol may be repeated using combinations of purified EVs and live bacterial strains to maximize immune stimulation potential.
  • Example 12 Using in vitro immune activation assays to predict in vivo cancer
  • EVs that display enhanced stimulation of dendritic cells, enhanced stimulation of CD8+ T cell killing, enhanced stimulation of PBMC killing, and/or enhanced stimulation of NK cell killing are preferentially chosen for in vivo cancer immunotherapy efficacy studies.
  • Example 13 Determining the biodistribution of EVs when delivered orally to mice
  • Wild-type mice e.g C57BL/6 or BALB/c
  • the EV composition of interest to determine the in vivo biodistibution profile of purified EVs (Example 1).
  • EVs are labeled as in Example 2 to aide in downstream analyses.
  • mice can receive a single dose of the EV (25-100 pg) or several doses over a defined time course (25-100 pg). Mice are housed under specific pathogen-free conditions following approved protocols. Alternatively, mice may be bred and maintained under sterile, Germ-free conditions. Blood and stool samples can be taken at appropriate time points.
  • mice are humanely sacrificed at various time points (i.e., hours to days) post inoculation with the EV compositions and a full necropsy under sterile conditions is performed. Following standard protocols, lymph nodes, adrenal glands, liver, colon, small intestine, cecum, stomach, spleen, kidneys, bladder, pancreas, heart, skin, lungs, brain, and other tissue of interest are harvested and are used directly or snap frozen for further testing. The tissue samples are dissected and homogenized to prepare single-cell suspensions following standard protocols known to one skilled in the art. The number of EVs present in the sample is then quantified through flow cytometry. Quantification may also proceed with use of fluorescence microscopy after appropriate processing of whole mouse tissue (Vankelecom H., Fixation and paraffin- embedding of mouse tissues for GFP visualization, Cold Spring Harb. Protoc., 2009).
  • the animals may be analyzed using live-imaging according to the EV labeling technique.
  • Example 14 Administering EV compositions with enhanced immune activation in vitro to treat syngeneic mouse tumor models
  • a mouse model of cancer is generated by subcutaneously injecting a tumor cell line or patient derived tumor sample and allowing it to engraft into C57BL/6, female mice at ages 6-8 weeks old.
  • the methods provided herein are replicated using several tumor cell lines including: B16-F10 or B16-F10-SIY cells as an orthotopic model of melanoma, Panc02 cells as an orthotopic model of pancreatic cancer, injected at a concentration of lxlO 6 cells into the right flank (Maletzki et al 2008. Gut 57:483-491), LLC1 cells as an orthotopic model of lung cancer, CT-26 as an orthotopic model of colorectal cancer, and RM-l as an orthotopic model of prostate cancer.
  • methods for the B16-F10 model are provided in depth herein.
  • a syngeneic mouse model of spontaneous melanoma with a very high metastatic frequency is used to test the ability of bacteria to reduce tumor growth and the spread of metastases.
  • the EVs chosen for this assay are compositions that display enhanced activation of immune cell subsets and stimulate enhanced killing of tumor cells in vitro.
  • the mouse melanoma cell line B16-F10 is obtained from ATCC. The cells are cultured in vitro as a monolayer in RPMI medium, supplemented with 10% heat-inactivated fetal bovine serum and 1%
  • mice penicillin/streptomycin at 37°C in an atmosphere of 5% C02 in air.
  • the exponentially growing tumor cells are harvested by trypsinization, washed three times with cold lx PBS, and a suspension of 5E6 cells/ml is prepared for administration.
  • Female C57BL/6 mice are used for this experiment. The mice are 6-8 weeks old and weigh approximately 16-20 g.
  • each mouse is injected SC into the flank with 100 m ⁇ of the B16-F10 cell suspension.
  • the mice are anesthetized by ketamine and xylazine prior to the cell transplantation.
  • the animals used in the experiment may be started on an antibiotic treatment via instillation of a cocktail of kanamycin (0.4 mg/ml), gentamicin, (0.035 mg/ml), colistin (850 U/ml),
  • metronidazole 0.215 mg/ml
  • vancomycin 0.045 mg/ml
  • the animals are sorted into several groups based on their body weight. The mice are then randomly taken from each group and assigned to a treatment group.
  • EV compositions are prepared as described in Example 1. The mice are orally inoculated by gavage with either 25-100 pg EV to be tested, 25- 100 pg EV from Lactobacillus (negative control), PBS, or 25-100 pg EV from Burkholderia spp. (positive control).
  • mice are orally gavaged with the same amount of EVs daily, weekly, bi weekly, monthly, bi-monthly, or on any other dosing schedule throughout the treatment period.
  • EVs are IV injected into the tail vein, or injected directly into the tumor, subtumorally or peritumorally.
  • Additional groups of mice may receive some ratio of bacterial cells to EVs.
  • the bacterial cells may be live, dead, or weakened.
  • the bacterial cells may be harvested fresh (or frozen) and administered, or they may be irradiated or heat-killed prior to administration.
  • some groups of mice may receive between lxlO 4 and 5xl0 9 bacterial cells in an administration separate from, or comingled with, the EV administration.
  • bacterial cell administration may be varied by route of administration, dose, and schedule. This can include oral gavage, i.v. injection, i.p. injection, or nasal route injection. Mice can be injected with lOng-lug of EVs, bacteria and EVs, or inactivated bacteria and EVs. Mice can be injected weekly, or at various dosing intervals, including once per month. Mice may also receive combinations of purified EVs and live bacteria to maximize tumor-killing potential. All mice are housed under specific pathogen-free conditions following approved protocols.
  • mice are humanely sacrificed 6 weeks after the B16-F10 mouse melanoma cell injection or when the volume of the primary tumor reaches 1000 mm3. Blood draws are taken weekly and a full necropsy under sterile conditions is performed at the termination of the protocol.
  • Cancer cells can be easily visualized in the mouse B16-F10 melanoma model due to their melanin production.
  • tissue samples from lymph nodes and organs from the neck and chest region are collected and the presence of micro- and macro- metastases is analyzed using the following classification rule.
  • An organ is classified as positive for metastasis if at least two micro-metastatic and one macro-metastatic lesion per lymph node or organ are found.
  • Micro-metastases are detected by staining the paraffin-embedded lymphoid tissue sections with hematoxylin-eosin following standard protocols known to one skilled in the art.
  • the total number of metastases is correlated to the volume of the primary tumor and it is found that the tumor volume correlates significantly with tumor growth time and the number of macro- and micro-metastases in lymph nodes and visceral organs and also with the sum of all observed metastases. Twenty-five different metastatic sites are identified as previously described (Bobek V., et al, Syngeneic lymph-node-targeting model of green fluorescent protein-expressing Lewis lung carcinoma, Clin. Exp. Metastasis, 2004;2l(8):705-8).
  • the tumor tissue samples are further analyzed for tumor infiltrating lymphocytes.
  • the CD8+ cytotoxic T cells can be isolated by FACS and can then be further analyzed using customized p/MHC class I microarrays to reveal their antigen specificity (see e.g. Deviren G., et al., Detection of antigen-specific T cells on p/MHC microarrays, J. Mol. Recognit, 2007 Jan- Feb;20(l):32-8).
  • CD4+ T cells can be analyzed using customized p/MHC class II microarrays.
  • the same experiment is also performed with a mouse model of multiple pulmonary melanoma metastases.
  • the mouse melanoma cell line B16-BL6 is obtained from ATCC and the cells are cultured in vitro as described above.
  • Female C57BL/6 mice are used for this experiment. The mice are 6-8 weeks old and weigh approximately 16-20 g.
  • each mouse is injected into the tail vein with 100 m ⁇ of a 2E6 cells/ml suspension of B16-BL6 cells.
  • the tumor cells that engraft upon IV injection end up in the lungs.
  • mice are humanely killed after 9 days.
  • the lungs are weighed and analyzed for the presence of pulmonary nodules on the lung surface.
  • the extracted lungs are bleached with Fekete’s solution, which does not bleach the tumor nodules because of the melanin in the B16 cells though a small fraction of the nodules is amelanotic (i.e. white).
  • Fekete a small fraction of the nodules is amelanotic (i.e. white).
  • the number of tumor nodules is carefully counted to determine the tumor burden in the mice.
  • 200-250 pulmonary nodules are found on the lungs of the control group mice (i.e. PBS gavage).
  • the percentage tumor burden is calculated for the three treatment groups. This measure is defined as the mean number of pulmonary nodules on the lung surfaces of mice that belong to a treatment group divided by the mean number of pulmonary nodules on the lung surfaces of the control group mice.
  • Biological triplicates of media and spent media samples after bacterial conditioning and after growth of the tumor are deproteinized using Sartorius Centrisart I filters (cutoff 10 kDa). Before use, the filter is washed twice by centrifugation of water to remove glycerol and a small volume (20 ul) of 20.2 mM trimethylsilyl-2,2,3,3-tetradeuteropropionic acid (TSP, sodium salt) in D20 is added to 700ul of the ultrafiltrate, providing a chemical shift reference (0.00 ppm) and a deuterium lock signal. 650 ul of the sample is placed in a 5 mm NMR tube.
  • TSP trimethylsilyl-2,2,3,3-tetradeuteropropionic acid
  • Single pulse 1H-NMR spectra (500 MHz) are obtained on a Bruker DMX-500 spectrometer or comparable instrument as described previously (by Engelke et al. 2006 NMR spectroscopic studies on the late onset form of 3-methylutaconic aciduria type I and other defects in leucine metabolism. NMR Biomed. 19: 271-278). Phase and baseline are corrected manually. All spectra are scaled to TSP and metabolite signals are fitted semi-automatically with a Lorentzian line shape. Metabolite concentrations in the spent media are calculated relative to the known concentration in the standard medium and correspondingly expressed in units of mM. The concentration of a particular metabolite was calculated by the area of the corresponding peak to the area of the valine doublet at 1.04 ppm or an appropriate standard.
  • Metabolic content of a sample is ascertained using liquid chromatography techniques combined with mass spectrometry.
  • a LC-MS system includes a 4000 QTRAP triple quadrupole mass spectrometer (AB SCIEX) combined with 1100 Series pump (Agilent) and an HTS PAL autosampler (Leap Technologies). Media samples or other complex metabolic mixtures (-10 pL) are extracted using nine volumes of 74.9:24.9:0.2 (v/v/v) acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (valine-d8, Isotec; and phenylalanine-d8, Cambridge Isotope Laboratories). Standards may be adjusted or modified depending on the metabolites of interest.
  • the samples are centrifuged (10 min, 9,000g, 4 °C), and the supernatants (10 pL) are submitted to LCMS by injecting the solution onto the HILIC column (150 x 2.1 mm, 3 pm particle size).
  • the column is eluted by flowing a 5% mobile phase [lOmM ammonium formate, 0.1% formic acid in water] for 1 min at a rate of 250uL/min followed by a linear gradient over 10 min to a solution of 40% mobile phase [acetonitrile with 0.1% formic acid].
  • the ion spray voltage is set to 4.5 kV and the source temperature is 450 °C.
  • RNA Seq to Determine Mechanism of Action Dendritic cells are purified from tumors, Peyers patches, and mesenteric lymph nodes. RNAseq analysis is carried out and analyzed according to standard techniques known to one skilled in the art (Z. Hou. Scientific Reports. 5(9570): doi: 10.1038/srep09570 (2015)). In the analysis, specific attention is placed on innate inflammatory pathway genes including TLRs, CLRs, NLRs, and STING, cytokines, chemokines, antigen processing and presentation pathways, cross presentation, and T cell co-stimulation.
  • Example 15 Administering EVs with enhanced immune activation in vitro to treat syngeneic mouse tumor models in combination with PD-1 or PD-L1 inhibition
  • CT-26 colorectal cancer
  • CAT# CRL-2638 tumor cells are cultured in vitro as a monolayer in RPMI-1640 or DMEM supplemented with 10% heat- inactivated fetal bovine serum at 37°C in an atmosphere of 5% C02 in air. The exponentially- growing cells are harvested and counted prior to tumor inoculation. 6-8 week old female
  • mice are used for this experiment.
  • each mouse is injected subcutaneously in one or both rear flanks with 5x10 5 CT-26 tumor cells in 0.1 ml of lx PBS.
  • mice may receive antibiotic pre-treatment. Tumor size and mouse weight are monitored at least thrice weekly on nonconsecutive days.
  • EVs are tested for their efficacy in the mouse tumor model, either alone or in combination with whole bacterial cells and with or without anti-PD-l or anti-PD-Ll .
  • EVs, bacterial cells, and/or anti-PD-l or anti-PD-Ll will be administered at varied time points and at varied doses. For example, on day 10 after tumor injection, or after the tumor volume reaches lOOmm 3 , the mice are treated with EVs alone or in combination with anti-PD-l or anti-PD-Ll.
  • mice are intravenously injected with EVs at 15, 20, or 15 ug/mouse. Other mice may receive 25, 50, or 100 mgs of EVs per mouse. While some mice will receive EVs through i.v. injection, other mice may receive EVs through intraperitoneal (i.p.) injection, subcutaneous (s.c.) injection, nasal route administration, oral gavage, or other means of administration. Some mice may receive EVs every day (e.g. starting on day 1), while others may receive EVs at alternative intervals (e.g. every other day, or once every three days). Additional groups of mice may receive some ratio of bacterial cells to EVs. The bacterial cells may be live, dead, or weakened.
  • the bacterial cells may be harvested fresh (or frozen) and administered, or they may be irradiated or heat-killed prior to administration.
  • some groups of mice may receive between lxlO 4 and 5x10 9 bacterial cells in an administration separate from, or comingled with, the EV administration.
  • bacterial cell administration may be varied by route of administration, dose, and schedule. This can include oral gavage, i.v. injection, i.p. injection, or nasal route injection.
  • Some groups of mice are also injected with effective doses of checkpoint inhibitor.
  • mice receive 100 pg anti-PD-Ll mAB (clone l0f.9g2, BioXCell) or another anti-PD-l or anti-PD-Ll mAB in 100 m ⁇ PBS, and some mice receive vehicle and/or other appropriate control (e.g. control antibody).
  • Mice are injected with mABs 3, 6, and 9 days after the initial injection.
  • control mice receiving anti-PD-l or anti-PD- Ll mABs are included to the standard control panel.
  • Primary (tumor size) and secondary (tumor infiltrating lymphocytes and cytokine analysis) endpoints are assessed, and some groups of mice are rechallenged with a subsequent tumor cell inoculation to assess the effect of treatment on memory response.
  • Example 16 Intravenous administeration of Burkholderia pseudomallei inhibits colorectal carcinoma tumor growth
  • mice Female 6-8 week old Balb/c mice were obtained from Taconic (Germantown,
  • 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) Burkholderia pseudomallei ; and 3) anti-PD-l antibody.
  • Antibodies were administered intraperitoneally (i.p.) at 200 pg mouse (100 m ⁇ final volume) every four days, starting on day 1, and Burkholderia pseudomallei EVs (5 pg) were

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des méthodes et des compositions se rapportant à des vésicules extracellulaires, VE, de Burkholderia utiles en tant qu'agents thérapeutiques.
PCT/US2019/021792 2018-03-12 2019-03-12 Vésicules extracellulaires provenant de burkholderia WO2019178055A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201862641694P 2018-03-12 2018-03-12
US62/641,694 2018-03-12
US201862691352P 2018-06-28 2018-06-28
US62/691,352 2018-06-28

Publications (1)

Publication Number Publication Date
WO2019178055A1 true WO2019178055A1 (fr) 2019-09-19

Family

ID=65911282

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2019/021792 WO2019178055A1 (fr) 2018-03-12 2019-03-12 Vésicules extracellulaires provenant de burkholderia

Country Status (1)

Country Link
WO (1) WO2019178055A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111228315A (zh) * 2020-02-27 2020-06-05 上海上药信谊药厂有限公司 抗肿瘤组合物
US10973890B2 (en) 2016-09-13 2021-04-13 Allergan, Inc. Non-protein clostridial toxin compositions
CN113058031A (zh) * 2021-03-18 2021-07-02 沈阳药科大学 高尔基体与基因工程外泌体杂化膜包被的维甲酸原位喷雾水凝胶疫苗及制备方法和应用
WO2022055250A1 (fr) * 2020-09-08 2022-03-17 주식회사 바이오솔루션 Procédé de fabrication de vésicules extracellulaires dérivées de micro-organismes ayant un rendement amélioré, et composition pour améliorer l'état de la peau comprenant des vésicules extracellulaires ainsi fabriquées
CN115252632A (zh) * 2022-06-23 2022-11-01 湛江中心人民医院 一种基于载药囊泡的突破血脑屏障组合物及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2484752A2 (fr) * 2009-09-04 2012-08-08 Aeon Medix Inc. Vésicules extracellulaires issues de bactéries à gram positif et leur utilisation
EP3263119A2 (fr) * 2015-02-23 2018-01-03 MD Healthcare Inc. Composition comprenant des vésicules extracellulaires dérivées de bactéries de bacillus sp. pour le traitement de maladies associées à la grossesse

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2484752A2 (fr) * 2009-09-04 2012-08-08 Aeon Medix Inc. Vésicules extracellulaires issues de bactéries à gram positif et leur utilisation
EP3263119A2 (fr) * 2015-02-23 2018-01-03 MD Healthcare Inc. Composition comprenant des vésicules extracellulaires dérivées de bactéries de bacillus sp. pour le traitement de maladies associées à la grossesse

Non-Patent Citations (54)

* Cited by examiner, † Cited by third party
Title
"Guide to Huge Computers", 1994, ACADEMIC PRESS
A. FRIAS ET AL., MICROB ECOL., vol. 59, 2010, pages 476 - 486
A. SIVAN ET AL., SCIENCE, vol. 350, no. 6264, 2015, pages 1084 - 1089
A.J. MCBROOM ET AL., J BACTERIOL, vol. 188, pages 5385 - 5392
ACHLMAN M; WAGNER M: "Microbial diversity and the genetic nature of microbial species", NAT. REV. MICROBIOL., vol. 6, 2008, pages 431 - 440
ACHTMAN M; WAGNER M.: "Microbial diversity and the genetic nature of microbial species", NAT. REV. MICROBIOL., vol. 6, 2008, pages 431 - 440
ATSCHUL, S. F. ET AL.: "Molec Biol", vol. 215, 1990, pages: 403
BAKER ET AL., VACCINES (BASEL, vol. 5, no. 4, 9 December 2017 (2017-12-09)
BOBEK V. ET AL.: "Syngeneic lymph-node-targeting model of green fluorescent protein-expressing Lewis lung carcinoma", CLIN. EXP. METASTASIS, vol. 21, no. 8, 2004, pages 705 - 8, XP019235796, DOI: doi:10.1007/s10585-004-8118-8
CARILLO ET AL., SIAM J APPLIED MATH, vol. 48, 1988, pages 1073
CLAESSON M J; WANG Q; O'SULLIVAN 0; GREENE-DINIZ R; COLE J R; ROS R P; O'TOOLE P W: "Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable t6S rRNA gene regions", NUCLEIC ACIDS RES, 2010, pages e200, XP055250083, DOI: doi:10.1093/nar/gkq873
CLAESSON MJ; WANG Q; O'SULLIVAN O; GREENE-DINIZ R; COLE JR; ROSS RP; O'TOOLE PW: "Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions", NUCLEIC ACIDS RES, vol. 38, 2010, pages e200, XP055250083, DOI: doi:10.1093/nar/gkq873
CVJETKOVIC ET AL., SCI. REP., vol. 6, 2016, pages 36338
D. CHATTERJEE; K. CHADHURI, J BIOL CHEM., vol. 288, no. 6, 2013, pages 4299 - 309
DETTMER ET AL.: "Mass spectrometry-based metabolomics", MASS SPECTROM REV., vol. 26, no. 1, 2007, pages 51 - 78, XP002485404, DOI: doi:10.1002/mas.20108
DEVEREUX, J. ET AL., NUCLEIC ACIDS RESEARCH, vol. 12, no. I, 1984, pages 387
DEVIREN G. ET AL.: "Detection of antigen-specific T cells on p/MHC microarrays", J. MOL. RECOGNIT., vol. 20, no. 1, January 2007 (2007-01-01), pages 32 - 8, XP002667108, DOI: doi:10.1002/JMR.805
ENGELKE ET AL.: "NMR spectroscopic studies on the late onset form of 3-methylutaconic aciduria type I and other defects in leucine metabolism", NMR BIOMED., vol. 19, 2006, pages 271 - 278
G. NORHEIM ET AL., PLOS ONE, vol. 10, no. 9, 2015, pages e0134353
G. NORHEIM ET AL., PLOS ONE, vol. 10, no. 9, 2015, pages eOl 34353
GUR ET AL., IMMUNITY, vol. 42, 2005, pages 1 - 12
H-I. CHOI ET AL., EXPERIMENTAL & MOLECULAR MEDICINE, vol. 49, 2017, pages e330
HOBB ET AL., EVALUATION OF PROCEDURES FOR OUTER MEMBRANE ISOLATION FROM CAMPYLOBACTER JEJUNI., vol. 155, 2009, pages 979 - 988
INABA K; SWIGGARD WJ; STEINMAN RM; ROMANI N; SCHULER G: "Current Protocols in Immunology", 2001, article "Isolation of dendritic cells"
K. KIKUSHIMA ET AL., SCIENTIFIC REPORTS, vol. 3, 1913
KONSTANTIMDIS K T; RAMETTE A; TIEDJE J M.: "The bacterial species definition in the genomic era", PHILOS TRANS R SOC LOUD B BIOL SCI, vol. 361, 2006, pages 1 929 - 1 940
KONSTANTINIDIS K T; RAMELTE A; TIEDJE J M: "The bacterial species definition in the genomic era", PHILOS TRANS R SOC LOUD B BIOL SCI, vol. 361, 2006, pages 1929 - 1940
KONSTANTINIDIS KT; RAMETTE A; TIEDJE JM.: "The bacterial species definition in the genomic era", PHILOS TRANS R SOC LOND B BIOL SCI, vol. 361, 2006, pages 1929 - 1940
KONSTANTINIDIS KT; RAMETTE A; TIEDJE JM: "The bacterial species definition in the genomic era", PHILOS TRANS R SOC LOND B BIOL SCI, vol. 361, 2006, pages 1929 - 1940
LEEKHA ET AL.: "General Principles of Antimicrobial Therapy", MAYO CLIN PROC., vol. 86, no. 2, 2011, pages 156 - 167, XP055372896, DOI: doi:10.4065/mcp.2010.0639
MALETZKI ET AL., GUT, vol. 57, 2008, pages 483 - 491
N. KESTY ET AL., EMBO JOURNAL, vol. 23, 2004, pages 4538 - 4549
NIEVES ET AL., CLIN VACCINE IMMUNOL., vol. 21, no. 5, May 2014 (2014-05-01), pages 747 - 54
NIEVES ET AL., VACCINE, vol. 29, no. 46, 26 October 2011 (2011-10-26), pages 8381 - 9
NIEVES W ET AL: "Characterization of Antibody and Cell-Mediated Immune Responses to a Protective Burkholderia pseudomallei Outer Membrane Vesicle Vaccine", ABSTRACTS OF THE GENERAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 112, 1 January 2012 (2012-01-01), pages 822, XP009512887, ISSN: 1060-2011 *
NIEVES WILDALIZ ET AL: "A Burkholderia pseudomallei Outer Membrane Vesicle Vaccine Provides Protection against Lethal Sepsis", vol. 21, no. 5, 30 April 2014 (2014-04-30), pages 747 - 754, XP009512886, ISSN: 1556-6811, Retrieved from the Internet <URL:http://cvi.asm.org/cgi/doi/10.1128/CVI.00119-14> DOI: 10.1128/CVI.00119-14 *
PEARSON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 2444
PETERSEN ET AL., PROCEDIA VACCINOL., vol. 8, 1 January 2014 (2014-01-01), pages 38 - 42
PETERSEN HAILEY ET AL: "Evaluation of a Burkholderia pseudomallei Outer Membrane Vesicle Vaccine in Nonhuman Primates", PROCEDIA IN VACCINOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 8, 1 January 2014 (2014-01-01), pages 38 - 42, XP009512885, ISSN: 1877-282X, DOI: 10.1016/J.PROVAC.2014.07.007 *
PROPST ET AL., INFECT IMMUN., vol. 78, no. 7, July 2010 (2010-07-01), pages 3136 - 3143
PROPST KL ET AL., INFECT IMMUN., vol. 78, no. 7, July 2010 (2010-07-01), pages 3136 - 43
ROBERTS ET AL., TARGETED METABOLOMICS. CURR PROTOC MOL BIOL., vol. 30, 2012, pages 1 - 24
ROBERTS ET AL.: "Targeted Metabolomics", CURR PROTOC MOL BIOL., vol. 30, 2012, pages 1 - 24
S. BIN PARK ET AL., PLOS ONE, vol. 6, no. 3, 2011, pages el 7629
S. BIN PARK ET AL., PLOS ONE, vol. 6, no. 3, 2011, pages el7629
S. STUBBS ET AL., LETTERS IN APPLIED MICROBIOLOGY, vol. 29, 1999, pages 31 - 36
SANDRINI ET AL.: "Fractionation by ultracentrifugation of gram negative cytoplasmic and membrane proteins", BIO PROTOCOL., vol. 4, no. 21, 2014
SCHOLLER ET AL.: "Protoplast and cytoplasmic membrane preparations from Streptococcus sanguis and Streptococcus mutans", J GEN MICRO., vol. 129, 1983, pages 3271 - 3279
SILINDIR M. ET AL., FABAD J. PHARM. SCI., vol. 34, 2009, pages 43 - 53
SOMANSCHI ET AL., J VIS EXP., 2011
THEIN ET AL.: "Efficient subfractionation of gram-negative bacteria for proteomics studies", vol. 9, 2010, AM CHEM SOCIETY, pages: 6135 - 6147
VANKELECOM H.: "Fixation and paraffin-embedding of mouse tissues for GFP visualization", COLD SPRING HARB. PROTOC., 2009
WILDALIZ NIEVES ET AL: "A naturally derived outer-membrane vesicle vaccine protects against lethal pulmonaryinfection", VACCINE, ELSEVIER, AMSTERDAM, NL, vol. 29, no. 46, 7 August 2011 (2011-08-07), pages 8381 - 8389, XP028314387, ISSN: 0264-410X, [retrieved on 20110814], DOI: 10.1016/J.VACCINE.2011.08.058 *
Z. VARGA ET AL., CANCER BIOTHER RADIOPHARM., vol. 31, no. 5, June 2016 (2016-06-01), pages 168 - 73

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10973890B2 (en) 2016-09-13 2021-04-13 Allergan, Inc. Non-protein clostridial toxin compositions
CN111228315A (zh) * 2020-02-27 2020-06-05 上海上药信谊药厂有限公司 抗肿瘤组合物
WO2021170137A1 (fr) * 2020-02-27 2021-09-02 上海上药信谊药厂有限公司 Composition antitumorale
EP4115893A4 (fr) * 2020-02-27 2024-04-17 Sph Sine Pharmaceutical Laboratories Co Ltd Composition antitumorale
WO2022055250A1 (fr) * 2020-09-08 2022-03-17 주식회사 바이오솔루션 Procédé de fabrication de vésicules extracellulaires dérivées de micro-organismes ayant un rendement amélioré, et composition pour améliorer l'état de la peau comprenant des vésicules extracellulaires ainsi fabriquées
CN113058031A (zh) * 2021-03-18 2021-07-02 沈阳药科大学 高尔基体与基因工程外泌体杂化膜包被的维甲酸原位喷雾水凝胶疫苗及制备方法和应用
CN113058031B (zh) * 2021-03-18 2022-05-10 沈阳药科大学 高尔基体与基因工程外泌体杂化膜包被的维甲酸原位喷雾水凝胶疫苗及制备方法和应用
CN115252632A (zh) * 2022-06-23 2022-11-01 湛江中心人民医院 一种基于载药囊泡的突破血脑屏障组合物及其应用
CN115252632B (zh) * 2022-06-23 2023-10-20 湛江中心人民医院 一种基于载药囊泡的突破血脑屏障组合物及其应用

Similar Documents

Publication Publication Date Title
US20230405058A1 (en) Extracellular vesicles from prevotella
US10702561B2 (en) Pharmaceutical compositions comprising a Blautia strain
US11090341B2 (en) Compositions and methods for treating cancer and immune disorders using Veillonella bacteria
US20210052669A1 (en) Compositions and methods for treating immune disorders using lachnospiraceae bacteria
AU2020291003A1 (en) Secreted microbial extracellular vesicles
WO2019099682A1 (fr) Compositions et méthodes pour traiter des troubles immunitaires à l&#39;aide de souches bactériennes lactococcus à modulation immunitaire
WO2019178055A1 (fr) Vésicules extracellulaires provenant de burkholderia
WO2019178487A2 (fr) Compositions et méthodes de traitement d&#39;une maladie à l&#39;aide de klebsiella quasipneumoniae subsp. similipneumoniae
WO2019178490A1 (fr) Compositions et procédés pour traiter le cancer et l&#39;inflammation utilisant klebsiella oxytoca
WO2019178494A1 (fr) Compositions et méthodes de traitement du cancer et de l&#39;inflammation à l&#39;aide de tyzzerella nexilis
WO2019169181A1 (fr) Compositions et méthodes de traitement du cancer à l&#39;aide de lactobacillus salivarius
US20230263838A1 (en) Compositions and methods for treating diseases and disorders using oscillospiraceae microbial extracellular vesicles
WO2019169160A1 (fr) Compositions et méthodes de traitement du cancer à l&#39;aide de ruminococcus gnavus
WO2021252861A1 (fr) Compositions et méthodes de traitement de maladies et de troubles à l&#39;aide de mégasphaera sp.
WO2019075452A1 (fr) Identification de bactéries pour la cancérothérapie
WO2019169143A1 (fr) Compositions et méthodes de traitement du cancer à l&#39;aide de t uricibacter sanguinis
WO2019169168A1 (fr) Compositions et méthodes de traitement du cancer à l&#39;aide d&#39;agathobaculum
WO2019169138A1 (fr) Compositions et méthodes de traitement du cancer à l&#39;aide de paraclostridium benzoelyticum
US20230302061A1 (en) Compositions and methods for treating diseases and disorders using fournierella massiliensis
WO2020006216A1 (fr) Compositions et méthodes de traitement du cancer faisant appel à des bactéries neisseria
WO2022221183A1 (fr) Préparations de vésicules extracellulaires de fournierella

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19713637

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19713637

Country of ref document: EP

Kind code of ref document: A1