bacterial extracellular vesicles

专利摘要:
The present invention relates to methods and compositions related to EVs useful as therapeutic agents.
公开号:BR112020004264A2
申请号:R112020004264-1
申请日:2018-09-10
公开日:2020-10-06
发明作者:Brian Goodman；Baundauna Bose；Christopher J. H. Davitt
申请人:Evelo Biosciences, Inc.；
IPC主号:

专利说明:

[001] [001] This application claims priority benefit from provisional patent applications numbers US 62 / 556,015, filed on September 8, 2017, and US 62 / 669,151, filed on May 9, 2018, the contents of which are incorporated into this document as a reference in its entirety. SUMMARY
[002] [002] In certain respects, pharmaceutical compositions comprising bacterial extracellular vesicles (IV) useful for the treatment and / or prevention of disease (e.g., cancer, autoimmune disease, inflammatory disease, metabolic disease) are provided herein as well as methods for producing and / or identifying such EVs and methods for using such pharmaceutical compositions (for example, for the treatment of cancers, autoimmune diseases, inflammatory diseases, metabolic diseases, alone or in combination with other therapeutics). In some embodiments, the pharmaceutical compositions comprise both EV and total bacteria (for example, live bacteria, dead bacteria, weakened bacteria). In certain embodiments, pharmaceutical compositions comprising bacteria in the absence of EV are provided herein. In some embodiments, the pharmaceutical compositions comprise EV in the absence of bacteria. In some embodiments, the pharmaceutical compositions comprise EV and / or bacteria from one or more of the strains or species of bacteria listed in Table 1 and / or Table 2.
[003] [003] In certain embodiments, the pharmaceutical composition comprises a specific ratio between bacteria and EV particles. For example, in some embodiments, the pharmaceutical composition comprises at least 1 bacterium for each 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 , 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3 , 2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4 , 5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6, 2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7, 5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8, 8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1x103, 2x103, 3x103, 4x103, 5x103, 6x103, 7x103, 8x103, 9x103, 1x104, 2x104, 3x104, 4x104, 5x104, 6x104, 7x104, 8x104, 9x104, 1x105, 2x105, 2x105, 3x105, 4x105, 5x105, 6x105, 7x105, 8x105, 9x105, 1x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 1x107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107, 8x10 7, 9x107, 1x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1x109, 2x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109, 9x109, 1x1010, 2x1010, 3x1010, 4x1010, 5x10, 5x10, 5x10 6x1010, 7x1010, 8x1010, 9x1010, 1x1011, 2x1011, 3x1011, 4x1011, 5x1011, 6x1011, 7x1011, 8x1011, 9x1011, and / or 1x1012 EV particles.
[004] [004] In certain respects, EVs come from a genetically modified bacterium that is modified to enhance certain desirable properties. For example, in some modalities, genetically modified bacteria are modified to increase the production of EV. In some embodiments, genetically modified bacteria are modified to produce EV with enhanced oral administration (for example, improving resistance to acid, mucus adhesion and / or penetration and / or resistance to bile acids, resistance to antimicrobial peptides and / or antibody neutralization), to target desired cell types (for example, M cells, goblet cells, enterocytes, dendritic cells, macrophages) to improve bioavailability systemically or in a suitable niche (for example, mesenteric lymph nodes, Peyer's patches, lamina propria, tumor drainage lymph nodes and / or blood), to enhance the immunomodulatory and / or therapeutic effect of the EV they produce (for example, alone or in combination with another therapeutic agent), to enhance the immune activation by the EVs they produce and / or to improve bacterial and / or EV manufacturing (for example, greater stability, better tolerance to freeze-drying freezing,
[005] [005] In certain embodiments, methods are provided in this document to treat a subject who has cancer which comprise administering to the subject a pharmaceutical composition described in this document. In certain embodiments, methods are provided herein to treat a subject who has an immune disorder (for example, an autoimmune disease, an inflammatory disease, an allergy) which comprises administering to the subject a pharmaceutical composition described in this document. In certain embodiments, methods are provided herein to treat a subject who has a metabolic disease which comprises administering to the subject a pharmaceutical composition described herein.
[006] [006] In some modalities, the method also includes administering an antibiotic to the subject. In some embodiments, the method also comprises administering one or more other cancer therapies to the subject (for example, surgical removal of a tumor, administration of a chemotherapeutic agent, administration of radiotherapy and / or administration of a cancer immunotherapy, such as an immune checkpoint inhibitor, a cancer specific antibody, a cancer vaccine, a prepared antigen presenting cell, a cancer specific T cell, a chimeric antigen receptor (CAR) T cell specific for cancer, an immune-activating protein and / or an adjuvant). In some embodiments, the method also comprises the administration of another bacterium and / or therapeutic IV. In some embodiments, the method further comprises the administration of an immune suppressor and / or an anti-inflammatory agent. In some embodiments, the method further comprises the administration of a therapeutic agent against metabolic disease. BRIEF DESCRIPTION OF THE FIGURES
[007] [007] Figure 1 shows the efficacy of Blautia massiliensis administered by iv compared to that anti-PD-1 administered intraperitoneally (i.p.) in a mouse model with colorectal carcinoma.
[008] [008] Figure 2 shows the inhibition of tumor growth (by volume) by iv administration of Blautia massiliensis compared to anti-PD-1 administered intraperitoneally (i.p.) in a mouse model with colorectal carcinoma.
[009] [009] Figure 3A shows the efficacy of Prevotella histicola and IV derivatives of P. histicola administered orally or intraperitoneally in reducing antigen-specific ear swelling (ear thickness) 24 hours after antigen challenge in a mouse model with delayed KLH-type hypersensitivity. Efficacy was observed in groups of both oral administration and i.p.
[0010] [0010] Figure 3B shows the efficacy of Prevotella histicola and IV derived from P. histicola administered orally or intraperitoneally in reducing antigen-specific ear swelling (ear thickness) 48 hours after antigen challenge in a mouse model with delayed KLH-type hypersensitivity.
[0011] [0011] Figure 3C shows the efficacy of P. histicola-derived IV administered intraperitoneally at the indicated dose (10 µg, 3 µg, 1 µg and 0.1 µg) in reducing the swelling of the antigen-specific ear (thickness of the ear) 48 hours after antigen challenge in a mouse model with delayed KLH-type hypersensitivity.
[0012] [0012] Figure 3D shows the EV capacity derived from
[0013] [0013] Figure 3E shows the ability of IV derived from Prevotella histicola administered intraperitoneally to increase the accumulation of Tregs in cervical lymph nodes 48 hours after antigen challenge in a mouse model with delayed KLH-type hypersensitivity.
[0014] [0014] Figure 4 shows that P. histicola was effective in reducing the NASH activity score (NAS) in mice that received a deficient methionine and choline (MCD) diet, which induces NASH symptoms.
[0015] [0015] Figure 5A shows that P. histicola reduced steatosis in mice that were fed an MCD diet.
[0016] [0016] Figure 5B and Figure 5C show that P. histicola reduced inflammation in mice that were fed an MCD diet.
[0017] [0017] Figure 5D shows that P. histicola reduced ballooning in mice that were fed an MCD diet.
[0018] [0018] Figure 6 shows that P. histicola reduced total liver cholesterol in mice that were fed an MCD diet.
[0019] [0019] Figure 7A and Figure 7B show that P. histicola reduced the fibrosis score in mice that were fed an MCD diet.
[0020] [0020] Figure 8A shows the effectiveness of the administration of Prevotella histicola, P. melanogenica, EV derived from P. histicola, EV derived from P. melanogenica in reducing antigen-specific ear swelling (ear thickness) 24 hours after challenge with antigen in a mouse model with delayed KLH-type hypersensitivity. EVs derived from P. melanogenica are more effective than P. melanogenica.
[0021] [0021] Figure 8B shows the effectiveness of the administration of Prevotella histicola, P. melanogenica, EV derived from P. histicola, EV derived from P. melanogenica in reducing antigen-specific ear swelling (ear thickness) 48 hours after challenge with antigen in a mouse model with delayed KLH-type hypersensitivity. EVs derived from P. melanogenica are more effective than P. melanogenica.
[0022] [0022] Figure 9A shows the effect of P. histicola on free liver fatty acids in mice that were fed an MCD diet.
[0023] [0023] Figure 9B shows the effect of P. histicola on total liver cholesterol in mice that were fed an MCD diet.
[0024] [0024] Figure 9C shows the effect of P. histicola on liver triglycerides in mice that were fed an MCD diet.
[0025] [0025] Figure 9D shows the effect of P. histicola and P. melanogenica on alanine aminotransferase in mice that were fed an MCD diet.
[0026] [0026] Figure 9E shows the effect of P. histicola and P. melanogenica on aspartate aminotransferase in mice that were fed an MCD diet.
[0027] [0027] Figure 10A shows that P. histicola is effective in reducing the NASH activity score (NAS) in mice that received a deficient methionine and choline (MCD) diet, which induces NASH symptoms.
[0028] [0028] Figure 10B shows that Prevotella histicola, P.
[0029] [0029] Figure 11 shows the effectiveness of Veillonella tobetsuensis and Veillonella parvula strains administered orally in reducing antigen-specific ear swelling (ear thickness) in 24 hours compared to anti-inflammatory vehicle (negative control) dexamethasone (positive control) and Bifidobacterium animalis lactis in a mouse model with delayed KLH-type hypersensitivity.
[0030] [0030] Figure 12 shows the efficacy of EV from two strains of Veillonella tobetsuensis and Veillonella parvula compared to anti-PD-1 injected intraperitoneally (i.p.) or vehicle in a mouse model with colorectal carcinoma.
[0031] [0031] Figure 13 shows the efficacy of EV from strains of Veillonella tobetsuensis and Veillonella parvula compared to anti-PD-1 injected intraperitoneally (i.p.) or vehicle in a mouse model with colorectal carcinoma on day 11.
[0032] [0032] Figure 14 shows the dose-dependent efficacy and route of IV administration from Veillonella tobetsuensis and Veillonella parvula strains compared to anti-PD-1 injected intraperitoneally (ip) or vehicle in a mouse model with colorectal carcinoma.
[0033] [0033] Figure 15 shows the dose-dependent efficacy and route of administration of EV from strains of Veillonella tobetsuensis and Veillonella parvula compared to anti-PD-1 injected intraperitoneally (ip) or vehicle in a mouse model with colorectal carcinoma on day 11.
[0034] [0034] Figure 16 shows that Veillonella parvula was effective in reducing the NASH activity score (NAS) in mice that received a deficient methionine and choline (MCD) diet, which induces NASH symptoms.
[0035] [0035] Figure 17 shows that Veillonella parvula reduced fibrosis in mice that were fed an MCD diet.
[0036] [0036] Figure 18 shows that Veillonella parvula reduced total liver cholesterol in mice that were fed an MCD diet.
[0037] [0037] Figure 19 shows that Veillonella parvula reduced liver triglycerides in mice that were fed an MCD diet
[0038] [0038] Figure 20 shows the EV efficacy of Burkholderia pseudomallei compared to anti-PD-1 administered intravenously (i.v.) or vehicle in a mouse model with colorectal carcinoma.
[0039] [0039] Figure 21 shows the efficacy of EV of Burkholderia pseudomallei compared to anti-PD-1 administered intravenously (iv) or vehicle in a mouse model with colorectal carcinoma on day 11. ** The difference in tumor volume in the group treated with EV of Burkholderia pseudomallei compared to the control group treated with vehicle on day 11 it was highly significant, with a P value of 0.0011, as determined by the T test (two-tailed, unpaired, corrected by Welch) calculated in GraphPad.
[0040] [0040] Figure 22 shows the EV efficacy of Neisseria Meningitidis compared to that of anti-PD-1 administered intraperitoneally (i.p.) or vehicle in a mouse model with colorectal carcinoma.
[0041] [0041] Figure 23 shows the EV efficacy of Neisseria Meningitidis compared to that of anti-PD-1 administered intraperitoneally (i.p.) or vehicle in a mouse model with colorectal carcinoma on day 11.
[0042] [0042] "Adjuvant" or "adjuvant therapy" refers broadly to an agent that affects an immune or physiological response in a patient or subject. For example, an adjuvant can increase the presence of an antigen over time or in an area of interest such as a tumor, help absorb an antigen-presenting cell, activate macrophages and lymphocytes and support cytokine production. By altering an immune response, an adjuvant may allow a lower dose of an immune interaction agent to increase the effectiveness or safety of a specific dose of the immune interaction agent. For example, an adjuvant can prevent the exhaustion of T cells and thus increase the effectiveness or safety of a specific immune interaction agent.
[0043] [0043] "Administration" refers broadly to a route of administration of a composition to a subject. Examples of routes of administration include oral administration, rectal administration, topical administration, inhalation (nasal) or injection. Administration by injection includes intravenous (IV), intramuscular (IM), intratumor (IT) and subcutaneous (SC) administration. The pharmaceutical compositions described in this document can be administered in any form by any effective route, including, but not limited to, intratumoral, oral, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal (for example, using any standard patch) ), intradermal, ophthalmic, (intra) nasal, local, non-oral, such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, (trans) rectal, vaginal, intraarterial and intrathecal, transmucosal (for example, sublingual, lingual, (trans) buccal, (trans) urethral, vaginal (for example, trans and perivaginally), implanted, intravesical, intrapulmonary, intraduodenal, intragastric and intrabronchial. In preferred embodiments, the pharmaceutical compositions described in this document are administered by oral, rectal, intratumoral, topical, intravesical, by injection in or adjacent to the drainage lymph node, intravenously, by inhalation or aerosol or subc utaneous.
[0044] [0044] As used herein, the term "antibody" can refer to both an intact antibody and an antigen-binding fragment thereof. Intact antibodies are glycoproteins that include at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determination regions (CDR), interspersed with regions that are more conserved, called framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino termination to the carboxy termination in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The term "antibody" includes, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (for example, bispecific antibodies), single chain antibodies and antigen binding antibody fragments.
[0045] [0045] The terms "antigen binding fragment" and "antigen binding portion" of an antibody, as used herein, refer to one or more fragments of an antibody that retain the ability to bind to an antigen. Examples of binding fragments covered by the term "antigen binding fragment" of an antibody include Fab, Fab ', F (ab') 2, Fv, scFv, disulfide-bound 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 way as intact antibodies.
[0046] [0046] "Cancer" refers largely to an uncontrolled abnormal growth of cells in a host, leading to invasion of the surrounding tissue and potentially the distal tissue to the initial site of abnormal cell growth in the host. The main classes include carcinomas that are cancers of the epithelial tissue (eg, skin, squamous cells); sarcomas that are cancers of connective tissue (for example, bone, cartilage, fat, muscle, blood vessels, etc.); leukemias that are cancers of blood-forming tissue (for example, bone marrow tissue); lymphomas and myelomas that are cancers of immune cells; and cancers of the central nervous system that include cancers of the brain and spinal tissue. "Cancer (or cancers)", "neoplasia (or neoplasms)" and "tumor (or tumors)" are used in this document interchangeably. As used herein, "cancer" refers to all types of cancer or neoplasia or malignant tumors, including leukemias, carcinomas and sarcomas, new or recurring. Specific examples of cancers are: carcinomas,
[0047] [0047] "Cell augmentation" refers largely 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 are not present in the composition itself. Cells that enhance the environment include immune cells, stromal cells, bacterial and fungal cells. The environments of specific interest are the microenvironments in which cancer cells reside or are located. In some cases, the microenvironment is a tumor microenvironment or a tumor drainage lymph node. In other cases, the microenvironment is a precancerous tissue site or the site of local administration of a composition or a site where the composition will accumulate after remote administration.
[0048] [0048] "Clado" refers to 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 which is a distinctive monophyletic evolutionary unit and which shares, to some extent, sequence similarity. "Operational taxonomic units", "OTU" (or plural, "OTUs") refer to a terminal leaf in a phylogenetic tree and are defined by a nucleic acid sequence, for example, the entire genome, or a specific genetic sequence and all sequences that share sequence identity with that species-level nucleic acid sequence. In some embodiments, the specific genetic sequence can be the 16S sequence or a portion of the 16S sequence.
[0049] [0049] A "combination" of EVs from two or more microbial strains includes the physical coexistence of the two EVs, in the same material or product or in physically connected products, as well as the co-administration or temporal co-administration of the EVs from the two strains.
[0050] [0050] The term "decrease" or "deplete" means a change, so that the difference is, depending on the circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1/100, 1 / 1,000, 1 / 10,000, 1 / 100,000, 1 / 1,000,000 or undetectable after treatment compared to a pre-treatment state.
[0051] [0051] As used in this document, the term "dibiosis" refers to a state in which the synergy between microbes and the tumor is disrupted, as the microbes no longer support nucleation, maintenance, progression or spreading or metastasis of a tumor.
[0052] [0052] The term "epitope" means a protein determinant with the ability to specifically bind to an antibody or T cell receptor. Epitopes typically consist of groups of chemically active surface molecules, such as amino acids or sugar side chains. Certain epitopes can be defined by a specific amino acid sequence to which an antibody is able to bind.
[0053] [0053] As used herein, "genetically modified bacteria" are any bacteria that have been genetically altered from their natural state by human intervention and the progeny of any such bacteria. Genetically modified bacteria include, for example, targeted genetic modification products, products of random mutagenic screening and products of directed evolution.
[0054] [0054] The term "gene" is used widely 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 mRNA encoded by that genomic sequence.
[0055] [0055] "Identity" 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, standard parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. EUA 85: 2,444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (I): 387 (1984)), BLASTP, BLASTN, FASTA Atschul, SF, et al ., J Molec Biol 215: 403 (1990); Guide to Huge Computers, Mrtin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al. (1988) SIAM J Applied Math 48: 1,073). For example, the BLAST function in the database of the National Center for Biotechnology Information can be used to determine identity. Other commercially or publicly available programs include DNAStar's "MegAlign" program (Madison, Wis.) And the University of Wisconsin Genetics (UWG) computer group's "Gap" program (Madison Wis.)).
[0056] [0056] As used herein, the term "immune disorder" refers to any disease, disorder or symptom of disease caused by an activity of the immune system, including autoimmune diseases, inflammatory diseases and allergies. Immune disorders include, but are not limited to, autoimmune diseases (eg, lupus, scleroderma, hemolytic anemia, vasculitis, type one diabetes, Grave's disease, rheumatoid arthritis, multiple sclerosis, Goodpasture's syndrome, pernicious anemia and / or myopathy), diseases inflammatory (for example, acne vulgaris, asthma, celiac disease, chronic prostatitis, glomerulonephritis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis,
[0057] [0057] "Immunotherapy" is the treatment that uses a subject's immune system to treat the disease (for example, immune disease, inflammatory disease, metabolic disease, cancer) and includes, for example, checkpoint inhibitors, vaccines against cancer, cytokines, cell therapy, CAR-T cells and dendritic cell therapy.
[0058] [0058] The term "increase" means a change, so that the difference is, depending on the circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 %, 2 times, 4 times, 10 times, 100 times, 10 ^ 3 times, 10 ^ 4 times, 10 ^ 5 times, 10 ^ 6 times and / or 10 ^ 7 times higher after treatment compared to a state of pre-treatment. Properties that can be increased include immune cells, bacterial cells, stromal cells, myeloid-derived suppressor cells, fibroblasts, metabolites and cytokines.
[0059] [0059] "Inborn immunological agonists" or "immunoadjuvates" are small molecules, proteins or other agents that specifically target innate immune receptors, including Toll type receptors (TLR), NOD receptors, RLRs, type C lectin receptors, pathway components STING-cGAS, inflammasome complexes. For example, LPS is a TLR-4 agonist that is derived or synthesized by bacteria, and aluminum can be used as an adjunct to immune stimulation. Immunoadjuvants are a specific class of adjuvant or broader adjuvant therapy. Examples of STING agonists include, but are not limited to, 2'3'- cGAMP, 3'3'- cGAMP, c-di-AMP, c-di-GMP, 2'2'-cGAMP and 2'3'-cGAM (PS) 2 (Rp / Sp) (Rp, Sp isomers of the 2'3'-cGAMP bis-phosphorothioate analogue). Examples of TLR agonists include, but are not limited to, TLR1,
[0060] [0060] The "internal transcript spacer" or "ITS" is a part of non-functional RNA located between structural ribosomal RNAs (rRNA) in a common precursor transcript, often used to identify eukaryotic species in specific fungi. The fungi rRNA that forms the nucleus of the ribosome is transcribed as a signal gene and consists of regions 8S, 5.8S and 28S with ITS4 and 5 between regions 8S and 5.8S and 5.8S and 28S, respectively. These two intercistronic segments between regions 18S and 5.8S and 5.8S and 28S are removed by splicing and contain significant variations between species for barcode purposes as previously described (Schoch et al., Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi (PNAS 109: 6,241 to 6,246. 2012). 18S rDNA is traditionally used for phylogenetic reconstruction, however, ITS can perform this function, since it is generally highly conserved, but it contains hypervariable regions that harbor enough nucleotide diversity to differentiate genera and species from most fungi.
[0061] [0061] The term "isolated" or "enriched" encompasses a microbe, an EV or other entity or substance that has been (1) separated from at least part of the components to which it was associated when initially produced (in nature or in an environment) experimental) and / or (2) produced, prepared, purified and / or manufactured by the hand of man. Isolated microbes can be separated by 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 to which they were initially associated.
[0062] [0062] "Metabolite", as used herein, refers to any and all molecular compounds, compositions, molecules, ions, cofactors, catalysts or nutrients used as substrates in any cellular or microbial or resulting metabolic reaction as compounds of product, compositions, molecules, ions, cofactors, catalysts or nutrients from any cellular or microbial metabolic reaction.
[0063] [0063] "Microbe" refers to any natural or genetically modified organism characterized as bacteria, fungi, microscopic algae, protozoa, and developmental stages or life-cycle stages (eg vegetative, spore (including sporulation, dormancy and germination), latent, biofilm) associated with the organism. Examples of microbes from the gastrointestinal tract include: Actinomyces graevenitzii, Actinomyces odontolyticus, Akkermansia muciniphila, Bacteroides caccae, Bacteroides fragilis, Bacteroides putredinis, Bacteroides thetaiotaomicron, Bacteroides vultagus, Bifectobyl, Bifidobacter, Bifidobacterium, Bifidobacter, III, Clostridia cluster IV, Clostridia cluster IX (Acidaminococcaceae group), Clostridia cluster XI, Clostridia cluster XIII (Peptostreptococcus group), Clostridia cluster XIV, Clostridia cluster XV, Collinsella aerofaciens, Coprococcus, Corynebacterium sunsvicea, Dorulfomena, Dorulfomener, , Escherichia coli, Eubacterium hadrum, Eubacterium rectale, Faecalibacteria prausnitzii, Gemella, Lactococcus, Lanchnospira, Mollicutes cluster XVI, Mollicutes cluster XVIII, Prevotella, Rotia mucilaginosa, Ruminococcus callidus, Ruminococcus gnavus and Ruminococcus gnavus, Ruminococcus gnavus treptococcus.
[0064] [0064] "Microbioma" refers broadly to microbes that reside on or in the body site of a subject or patient. Microbes in a microbiome can include bacteria, viruses, eukaryotic microorganisms and / or viruses. Individual microbes in a microbiome can be metabolically active, dormant, dormant or exist as spores, they can exist planktonically or in biofilms, or they can be present in the microbiome in a sustainable or transient manner. The microbiome can be a commensal or healthy microbiome or a microbiome in a disease state. The microbiome can be native to the subject or patient, or components of the microbiome can be modulated, introduced or depleted due to changes in health status (eg, pre-cancerous or cancerous) or treatment conditions (eg, antibiotic treatment , exposure to different microbes). In some ways, the microbiome occurs on a mucosal surface. In some respects, the microbiome is a microbiome of the gastrointestinal tract. In some ways, the microbiome is a tumor microbiome.
[0065] [0065] A "microbiome profile" or "microbiome signature" of a tissue or sample refers to an at least partial characterization of the bacterial constitution of a microbiome. In some embodiments, 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. In some embodiments, 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 associated with cancer are present in a sample. In some modalities, the microbiome profile indicates the relative or absolute amount of each bacterial strain detected in the sample. In some modalities, the microbiome profile is a microbiome profile associated with cancer. A cancer-associated microbiome profile is a microbiome profile that occurs more frequently in a subject who has cancer than in the general population. In some embodiments, the cancer-associated microbiome profile comprises a greater number of cancer-associated bacteria than is normally present in a tissue or sample microbiome, otherwise an equivalent harvested from an individual who does not have cancer .
[0066] [0066] "Modified", in reference to a bacterium, refers largely to a bacterium that has undergone a change in its wild-type shape. Examples of 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, for example, attenuation, auxotrophy, anchoring or antigenicity. Phenotype modification can include, by way of example, the development of bacteria in media that modify the phenotype of a bacterium, which increase or decrease virulence.
[0067] [0067] As used in this document, a gene is "overexpressed" in a bacterium if it is expressed at a higher level in a genetically modified bacterium under at least some conditions than is expressed by a wild type bacterium of the same species under the same conditions. Similarly, a gene is "underexpressed" in a bacterium if it is expressed at a lower level in a genetically modified bacterium under at least some conditions than is expressed by a wild type bacterium of the same species under the same conditions.
[0068] [0068] The terms "polynucleotide" and "nucleic acid" are used interchangeably. They refer to a polymeric form of nucleotides of any length,
[0069] [0069] An "oncobioma", as used in this document, comprises a pathogenic, tumorigenic and / or cancer-associated microbiota, the microbiota comprising one or more of a virus, a bacterium, a fungus, a protist, a parasite or another microbe.
[0070] [0070] "Oncotrophic" or "oncophilic" bacteria and microbes are highly associated microbes or present in a cancer microenvironment. They can preferably be selected into the environment, preferably grow in a cancer microenvironment or perfect a said environment.
[0071] [0071] "Operational taxonomic units" and "OTU (or OTUs)"
[0072] [0072] As used herein, the term "extracellular vesicle" or "EV" refers to a composition derived from a bacterium comprising bacterial lipids and bacterial proteins and / or bacterial nucleic acids and / or chemical carbohydrate portions contained in a nanoparticle. These EVs may contain 1, 2, 3, 4, 5, 10 or more than 10 different lipid species. EV can contain 1, 2, 3, 4, 5, 10 or more than 10 different protein species. EV can contain 1, 2, 3, 4, 5, 10 or more than 10 different nucleic acid species. EV can contain 1, 2, 3, 4, 5, 10 or more than 10 different carbohydrate species.
[0073] [0073] As used herein, a substance is "pure" if it is substantially free of other components. The terms "purify", "purifying" and "purified" refer to an EV or other material that has been separated from at least part of the components with which it was associated when initially produced or generated (for example, in nature or in experimental environment), or any time after its initial production. An EV can be considered purified if it is isolated in or after production, such as one or more other bacterial components, and a purified microbe or microbial population can contain up to about 10%, about 20%, about 30% , about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or above about 90% of other materials and still be considered "isolated". In some embodiments, 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 their microbial components are, for example, purified from habitat waste products.
[0074] [0074] As used herein, the term "purified EV composition" or "EV composition" refers to a preparation that includes EVs that have been separated from at least one associated substance found in a source material (for example , separated from at least one other bacterial component) or any material associated with EV in any process used to produce the preparation. It also refers to a composition that has been significantly enriched or concentrated. In some modalities, EVs are concentrated in 2 times, 3 times, 4 times, 5 times, 10 times, 100 times, 1,000 times, 10,000 times or more than
[0075] [0075] "Habitat waste products" refer to material derived from the habitat for microbiota within or on a subject. For example, microbes live in feces in the gastrointestinal tract, in the skin itself, in saliva, in mucus in the respiratory tract or in secretions from the genitourinary tract (ie biological matter associated with the microbial community). Substantially free of habitat waste 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 products from the habitat may include abiotic materials (including undigested food) or may include unwanted microorganisms. Substantially free of waste products from the habitat can also mean that the microbial composition does not contain detectable cells from a human or animal and that only microbial cells are detectable. In one embodiment, substantially free of waste products from the habitat can also mean that the microbial composition does not contain viral contaminants (including microbial viruses (eg, phage)), fungal, detectable mycoplasmatics. In another embodiment, this means that less than 1x10 to 2%, 1x10 to 3%, 1x10 to 4%, 1x10 to 5%, 1x10 to 6%, 1x10 to 7%, 1x10 to 8% of viable cells in the microbial composition are human or animal, compared to microbial cells. There are multiple ways to achieve this degree of purity, none of which is limiting. Thus, contamination can be reduced by isolating the desired constituents through multiple streaking stages on single colonies in solid media until replicated streaks (such as, but not limited to, two) from single colonies in series show only a single morphology of Cologne. Alternatively, contamination reduction can be achieved by multiple rounds of serial dilutions for single desired cells (for example, a 10 to 8 or 10 to 9 dilution), such as by multiple 10-fold serial dilutions. This can also 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 (for example, PCR, DNA sequencing), serology and antigen analysis, enzyme and metabolic analysis, and methods that use instrumentation, such as flow cytometry with reagents that distinguish desired constituents from contaminants.
[0076] [0076] As used herein, "specific binding" refers to the ability of an antibody to bind to a predetermined antigen or the ability of a polypeptide to bind to its predetermined binding partner. Typically, an antibody or polypeptide specifically binds to its predetermined antigen or binding partner with an affinity that corresponds 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) which is at least 10 times less, at least 100 times less or at least 1,000 times less than its affinity for binding to a non-specific and unrelated antigen / binding partner (eg, BSA, casein). Alternatively, the specific bond applies more widely to a two-component system, where one component is a protein, lipid or carbohydrate, or a combination thereof, and engages with the second component which is a protein, lipid, carbohydrate or combination of themselves in a specific way.
[0077] [0077] The terms "subject" or "patient" refer to any animal. A subject or a patient described as "in need of it" refers to someone in need of treatment for an illness. Mammals (i.e., mammalian animals) include humans, laboratory animals (for example, primates, rats, mice), cattle (for example, cows, sheep, goats, pigs) and laboratory animals (for example, dogs, cattle , rodents).
[0078] [0078] "Cepa" refers to a member of a bacterial species with a genetic signature so that it can be distinguished from closely related members of the same bacterial species. The genetic signature can be the absence of all or part of at least one gene, the absence of all or part of at least one regulatory region (for example, a promoter, a terminator, a riboswitch, a ribosome binding site), the absence ("cure") 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 of at least one mutated regulatory region (for example, 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 resistance cassette antibiotic, or a combination thereof. Genetic signatures between different strains can be identified by PCR amplification optionally followed by DNA sequencing of the genomic region (or genomic regions) of interest or of the entire genome. In the event that a strain (compared to another of the same species) has gained or lost antibiotic resistance or has gained or lost a biosynthetic capacity (such as an auxotrophic strain), strains can be differentiated by selection or counter-selection with the use of an antibiotic or nutrient / metabolite, respectively.
[0079] [0079] As used herein, the term "treating" a disease in a subject or "treating" a subject suspected of having a disease refers to the subject's submission to a pharmaceutical treatment, for example, the administration of a or more agents, so that at least one symptom of the disease is diminished or prevented from worsening. Thus, in one embodiment, "treatment" refers, among others, to delaying progression, speeding up remission, inducing remission, increasing remission, accelerating recovery, increasing efficiency or decreasing resistance to alternative therapies, or a combination of them. Bacteria
[0080] [0080] In certain respects, pharmaceutical compositions comprising bacteria and / or EV produced from bacteria are provided herein.
[0081] [0081] In some embodiments, the bacteria from which EVs are obtained are modified to intensify the production of EVs, to intensify the oral administration of the produced EVs (for example, improving resistance to acid, mucus adhesion and / or penetration and / or resistance to bile acids, digestive enzymes, resistance to antimicrobial peptides and / or antibody neutralization), to target desired cell types (for example, M cells, goblet cells, enterocytes, dendritic cells, macrophages), to enhance its immunomodulatory and / or therapeutic effect of the produced EVs (for example, alone or in combination with another therapeutic agent) and / or to enhance the immune activation or suppression by the produced EVs (for example, through modified polysaccharide production, pili, fimbriae, adhesins). In some embodiments, the genetically modified bacteria described in this document are modified to improve bacterial and / or EV manufacturing (eg, higher oxygen tolerance, stability, better freeze-thaw tolerance, shorter generation times). For example, in some embodiments, the genetically modified bacteria described include bacteria that harbor one or more genetic changes, the change being an insertion, deletion, translocation or substitution, or any combination thereof, of one or more nucleotides contained in the chromosome bacterial or endogenous plasmid and / or one or more foreign plasmids, the genetic alteration of which may result in the overexpression and / or underexpression of one or more genes. The genetically modified microbe (or genetically modified microbes) can be produced using any technique known in the art, including, but not limited to, sitiodirected mutagenesis, transposon mutagenesis, knockouts, knockins, polymerase chain reaction mutagenesis, chemical mutagenesis , mutagenesis by ultraviolet light, transformation (chemically or by electroporation), phage transduction, directed evolution or any combination thereof.
[0082] [0082] As used herein, the term "bacteria" refers broadly to the domain of prokaryotic organisms, including Gram positive and Gram negative organisms. Examples of species and / or strains of bacteria that can be used to produce the EVs described in this document are provided in Table 1 and / or Table 2 and elsewhere throughout this specification. In some embodiments, the bacterial strain is a bacterial strain that has a genome that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% of sequence identity with a strain listed in Table 1 and / or Table 2. In some embodiments, the EVs come from an oncotrophic bacterium. In some modalities, the EVs come from an immunostimulating bacterium. In some modalities, the EVs come from an immunosuppressive bacterium. In some modalities, the EVs come from an immunomodulatory bacteria. In certain embodiments, EVs are generated from a combination of the bacterial strains provided in this document. In some embodiments, the combination is a combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 bacterial strains. In some embodiments, the combination includes EV from bacterial strains listed in Table 1 and / or Table 2 and / or bacterial strains that have a genome that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6% , 99.7%, 99.8% or 99.9% sequence identity with a strain listed in Table 1 and / or Table 2. Table 1: Exemplary bacterial strains OTU Public DB record Abiotrophia defectiva ACIN02000016 Abiotrophia para_adiacens AB022027 Abiotrophia sp. oral clone P4PA_155 P1 AY207063 Acetanaerobacterium elongatum NR_042930 Acetivibrio cellulolyticus NR_025917 Acetivibrio ethanolgignens FR749897 Acetobacter aceti NR_026121 Acetobacter fabarum NR_042678 Acetobacter lovaniensis NR_040832 Acetobacter malorum NR_025513 Acetobacter orientalis NR_028625 Acetobacter pasteurianus NR_026107 Acetobacter pomorum NR_042112 Acetobacter syzygii NR_040868 Acetobacter tropicalis NR_036881 Acetobacteraceae bacterium A_5844 AGEZ01000040 Acholeplasma laidlawii NR_074448 Achromobacter denitrificans NR_042021 Achromobacter piechaudii ADMS01000149 Achromobacter xylosoxidans ACRC01000072 Acidaminococcus fermentans CP001859 Acidaminococcus intestini CP003058 Acidaminococcus sp.
[0083] [0083] In certain embodiments, the EVs described in this document are obtained from mandatory anaerobic bacteria. Examples of mandatory anaerobic bacteria include gram-negative rods (including the genera of Bacteroides, Prevotella, Porphyromonas, Fusobacterium, Bilophila and Sutterella spp.), Gram-positive cocci (mainly Peptostreptococcus spp.), Gram-positive spore builders (Clostridium spp .), non-spore forming bacilli (Actinomyces, Propionibacterium, Eubacterium, Lactobacillus and Biofobacterium spp.) and gram-negative cocci (mainly Veillonella spp.). In some modalities, the mandatory anaerobic bacteria are of a genus selected from Agathobaculum, Atopobium, Blautia, Burkholderia, Dielma, Longicatena, Paraclostridium, Turicibacter and
[0084] [0084] In some modalities, the EVs described in this document are obtained from bacteria of a genus selected from Escherichia, Klebsiella, Lactobacillus, Shigella and Staphylococcusa.
[0085] [0085] In some embodiments, the bacteria and / or EV described in this document are of a species selected from Blautia massiliensis, Paraclostridium benzoelyticum, Dielma fastidiosa, Longicatena caecimuris, Veillonella tobetsuensis.
[0086] [0086] In some embodiments, the EV and / or bacteria described in this document are modified so that they comprise, are linked and / or are linked by a therapeutic chemical portion. In some embodiments, the therapeutic chemical portion is a cancer-specific chemical portion. In some embodiments, the cancer-specific chemical moiety has binding specificity for a cancer cell (for example, it has binding specificity for a cancer-specific antigen). In some embodiments, the cancer-specific chemical moiety comprises an antibody or antigen-binding fragment thereof. In some embodiments, the cancer-specific chemical moiety comprises a T cell receptor or a chimeric antigen (CAR) receptor. In some embodiments, the cancer-specific chemical moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor binding fragment thereof. In some embodiments, the cancer-specific chemical moiety is a bipartite fusion protein that has two parts: a first part that binds and / or is bound to the bacteria and a second part that is able to bind to a cancer cell (for example, example, because it has binding specificity for a cancer specific antigen).
[0087] [0087] In some embodiments, the EVs described in this document are modified so that they comprise, are linked and / or are linked by a magnetic and / or paramagnetic chemical portion (for example, a magnetic microsphere). In some embodiments, the magnetic and / or paramagnetic chemical portion is comprised of and / or directly linked to bacteria. In some embodiments, the magnetic and / or paramagnetic chemical portion is linked and / or part of an EV-binding chemical portion that binds to the EV. In some embodiments, the chemical EV-binding moiety is a full-length peptidoglycan recognition fragment or protein, such as PGRP. In some embodiments, the chemical EV-binding moiety has binding specificity for EV (for example, because it has binding specificity for a bacterial antigen). In some embodiments, the chemical EV-binding moiety comprises an antibody or antigen-binding fragment thereof. In some embodiments, the chemical EV-binding portion comprises a T cell receptor or a chimeric antigen (CAR) receptor. In some embodiments, the chemical EV-binding portion comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof. In certain embodiments, the co-administration of the magnetic and / or paramagnetic chemical portion with the EVs (together or in separate administrations) can be used to increase the targeting of the EVs in cancer cells and / or in a part of a subject in which the cells carcinogens are present. EV production
[0088] [0088] In certain respects, the EVs described in this document can be prepared using any method known in the art.
[0089] [0089] In some modalities, EVs are prepared without an EV purification step. For example, in some embodiments, the bacteria that comprise the EVs described in this document are exterminated using a method that leaves the bacterial EVs intact, and the resulting bacterial components, including the EVs, are used in the methods and compositions described in this document. In some embodiments, the bacteria are exterminated using an antibiotic (for example, using an antibiotic described in this document). In some embodiments, the bacteria are exterminated with the use of UV irradiation.
[0090] [0090] In some embodiments, the EVs described in this document are purified from one or more other bacterial components. Methods for purifying EV from bacteria are known in the art. In some modalities, EVs are prepared from bacterial cultures using the methods described in S. Bin Park, et al. PLoS ONE. 6 (3): e17629 (2011) or G. Norheim, et al. PLoS ONE. 10 (9): e0134353 (2015), each of which is incorporated herein by reference in its entirety. In some embodiments, the bacteria are grown at high optical density and then centrifuged to agglomerate the bacteria (for example, at 10,000 x g for 30 min at 4 ° C). In some embodiments, the culture supernatants are then passed through the filter to exclude intact bacterial cells (for example, a 022 µm filter). In some embodiments, filtered supernatants are centrifuged to agglomerate bacterial EVs (for example,
[0091] [0091] For example, in some embodiments, the bacterial cultures disclosed in this document can be centrifuged at
[0092] [0092] Alternatively, EVs can be obtained from bacterial cultures continuously during development, or at selected points in time during development, by connecting a bioreactor to an alternating tangential flow (ATF) system (eg, XCell Repligen ATF). The ATF system retains intact cells (> 0.22 µm) in the bioreactor and allows smaller components (eg, EV, free proteins) to pass through a filter for collection. For example, the system can be configured so that the filtrate <0.22 µm is then passed through a second 100 kDa filter, allowing species, such as EV between 0.22 µm and 100 kDa, to be collected and species smaller than 100 kDa are pumped back into the bioreactor. Alternatively, the system can be configured to allow the medium in the bioreactor to be replenished and / or modified during the development of the culture. EVs collected by this method can also be purified and / or concentrated by ultracentrifugation or filtration as described above for filtered supernatants.
[0093] [0093] EVs obtained by methods provided in this document can also be purified by column chromatography based on size, affinity chromatography and by gradient ultracentrifugation, using methods that may include, but are not limited to, use sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, the pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is changed from buffer to 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column. Samples are applied to a 35 to 60% batch sucrose gradient and centrifuged at 200,000 x g for 3 to 24 hours at 4 ° C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation is used to concentrate the filtered supernatants, the pellets are resuspended in 35% Optiprep in PBS. In some embodiments, if filtration is used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final 35% Optiprep concentration. Samples are applied to a 35 to 60% batch sucrose gradient and centrifuged at 200,000 x g for 3 to 24 hours at 4 ° C.
[0094] [0094] In some modalities, to confirm the sterility and isolation of the EV preparations, the EVs are serially diluted in agar medium used for routine culture of the bacteria to be tested and incubated with the use of routine conditions. Non-sterile preparations are passed through a 0.22 µm filter to exclude intact cells. To further increase purity, isolated EVs can be treated with DNase or proteinase K.
[0095] [0095] In some modalities, for the preparation of EVs used for in vivo injections, the purified EVs are processed as previously described (G. Norheim, et al. PLoS ONE. 10 (9): e0134353 (2015)). Briefly, after sucrose gradient centrifugation, bands containing EV are resuspended to a final concentration of 50 µg / ml in a solution containing 3% sucrose or another suitable solution for in vivo injection known to the person skilled in the art. This solution can also contain adjuvant, for example, aluminum hydroxide, at a concentration of 0 to 0.5% (w / v).
[0096] [0096] In certain embodiments, to make samples compatible with additional tests (for example, to remove sucrose prior to TEM imaging or in vitro assays), samples are switched from buffer to PBS or 30 mM Tris, pH 8.0 with the use of filtration (for example, Amicon Ultra columns), dialysis, or ultracentrifugation (200,000 xg, ≥ 3 hours, 4 ° C) and resuspension.
[0097] [0097] In some modalities, the sterility of the EV preparations can be confirmed by placing a portion of the EVs on the agar medium used for the standard culture of the bacteria used in the generation of the EVs and incubating it using standard conditions. .
[0098] [0098] In some modalities, the selected EVs are isolated and enriched by chromatography and chemical portions of the EV binding surface. In other modalities, the selected EVs are isolated and / or enriched by fluorescent cell classification by methods that use affinity reagents, chemical dyes, recombinant proteins or other methods known to the person skilled in the art. Pharmaceutical compositions
[0099] [0099] In certain embodiments, the methods provided in this document are pharmaceutical compositions comprising EV and / or bacteria provided in this document (for example, an EV composition). In some embodiments, the EV composition comprises an EV and / or a combination of EV described herein and a pharmaceutically acceptable carrier.
[00100] [00100] In some embodiments, the pharmaceutical compositions comprise substantially or entirely bacteria-free EV. In some embodiments, the pharmaceutical compositions comprise both EV and total bacteria (for example, live bacteria, dead bacteria, weakened bacteria). In certain embodiments, the pharmaceutical compositions comprise bacteria that are substantially or entirely free of EV. In some embodiments, the pharmaceutical compositions comprise EV and / or bacteria from one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) among the strains or species of bacteria listed in Table 1 and / or Table 2.
[00101] [00101] In some embodiments, the pharmaceutical composition comprises at least 1 bacterium for each 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 . 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9,9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1x103, 2x103, 3x103, 4x103, 5x103, 6x103, 7x103, 8x103, 9x103, 1x104, 2x104, 3x104, 4x104, 5x104, 6x104, 7x104, 8x104, 9x104, 1x105, 2x105, 3x105, 4x105, 5x105, 6x105, 7x105, 8x105, 9x105, 1x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 1x107, 2x107, 2x107, 2x107
[00102] [00102] In some embodiments, the pharmaceutical composition comprises about 1 bacterium for each 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 . 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9,9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1x103, 2x103, 3x103, 4x103, 5x103, 6x103, 7x103, 8x103, 9x103, 1x104, 2x104, 3x104, 4x104, 5x104, 6x104, 7x104, 8x104, 9x104, 1x105, 2x105, 3x105, 4x105, 5x105, 6x105, 7x105, 8x105, 9x105, 1x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 1x107, 2x107, 3x107, 2x107, 3x107 5x107, 6x107, 7x107, 8x107, 9x107, 1x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1x109, 2x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109, 9x109, 1x10, 10x10 3x1010, 4x1010, 5x1010, 6x1010, 7x1010, 8x1010, 9x1010, 1x1011, 2x1011, 3x1011, 4x1011, 5x1011, 6x1011, 7x1011, 8x1011, 9x1011, and / or 1x1012 particles of EV.
[00103] [00103] In certain embodiments, the pharmaceutical composition comprises a certain ratio between particles of bacteria and particles of EV. The number of bacteria particles can be based on the actual number of particles or (if the bacteria are alive) the number of CFUs. The number of particles can be established by combining a defined number of purified EVs with a defined number of purified bacteria, changing the development conditions under which the bacteria are grown or modifying the bacteria themselves to produce more or less EV.
[00104] [00104] In some modalities, to quantify the numbers of EV and / or bacteria present in a bacterial sample, electron microscopy (for example, EM from ultrafine frozen sections) can be used to visualize the vesicles and bacteria and count their relative numbers. Alternatively, combinations of nanoparticle tracking analysis (NTA), particle counter and dynamic light scattering (DLS) or a combination of these techniques can be used. NTA and the particle counter count particles and show their sizes. DLS provides the particle size distribution, but not the concentration. Bacteria are often 1 to 2 µm in diameter. The total range is 0.2 to 20 µm. Combined results from the particle counter and ATN can reveal the numbers of bacteria in a given sample. The particle counter reveals the numbers of particles with diameters from 0.7 to 10 µm. NTA reveals the numbers of particles with diameters from 50 to 1,400 nm. For most bacterial samples, the particle counter alone can reveal the number of bacteria in a sample. EV have a diameter of 20 to 250 nm. NTA will allow counting the numbers of particles that have a diameter of 50 to 250 nm. DLS reveals the distribution of particles of different diameters in an approximate range of 1 nm to 3 um.
[00105] [00105] In some embodiments, the pharmaceutical composition comprises a maximum of 1 bacteria for each 1, 1.1, 1.2, 1.3, 1.4, 1.5,
[00106] [00106] In some embodiments, the pharmaceutical composition comprises at least 1 particle of EV for each 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1 , 8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9,9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62,
[00107] [00107] In some embodiments, the pharmaceutical composition comprises about 1 particle of EV for each 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1 , 8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9,9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1x103, 2x103, 3x103, 4x103, 5x103, 6x103, 7x103, 8x103, 9x103, 1x104, 2x104, 3x104, 4x104, 5x104, 6x104, 7x104, 8x104, 9x104, 1x105, 2x105, 3x105, 4x105, 5x105, 6x105, 7x105, 8x105, 9x105, 1x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 1x107, 2x107, 3x107, 2x107, 3x107 5x107, 6x107, 7x107, 8x107, 9x107, 1x108, 2x108, 3x108,
[00108] [00108] In some modalities, 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%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,
[00109] [00109] In some modalities, 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%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% , 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the particles in the pharmaceutical composition are bacteria.
[00110] [00110] In some modalities, at most 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%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% , 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the particles in the pharmaceutical composition are EV.
[00111] [00111] In some modalities, at most 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%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,
[00112] [00112] In some modalities, about 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%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% , 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the particles in the pharmaceutical composition are EV.
[00113] [00113] In some modalities, about 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%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% , 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the particles in the pharmaceutical composition are bacteria.
[00114] [00114] In some modalities, 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%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,
[00115] [00115] In some modalities, 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%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% , 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the protein in the pharmaceutical composition is bacterial protein.
[00116] [00116] In some modalities, at most 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%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% , 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the protein in the pharmaceutical composition is EV protein.
[00117] [00117] In some modalities, at most 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%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,
[00118] [00118] In some modalities, about 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%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% , 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the protein in the pharmaceutical composition is EV protein.
[00119] [00119] In some modalities, about 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%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% , 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the protein in the pharmaceutical composition is bacterial protein.
[00120] [00120] In some modalities, 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%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,
[00121] [00121] In some modalities, 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%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% , 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceutical composition are lipids from bacteria.
[00122] [00122] In some modalities, at most 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%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% , 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceutical composition are EV lipids.
[00123] [00123] In some modalities, at most 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%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,
[00124] [00124] In some modalities, about 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%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% , 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceutical composition are EV lipids.
[00125] [00125] In some modalities, about 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%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% , 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceutical composition are lipids from bacteria.
[00126] [00126] In some embodiments, the EVs in the pharmaceutical composition are purified from one or more other bacterial components. In some embodiments, the pharmaceutical composition further comprises other bacterial components.
[00127] [00127] In certain respects, pharmaceutical compositions are provided for administration to subjects. In some embodiments, the pharmaceutical compositions are combined with additional active and / or inactive materials in order to produce a final product, which can be in a single dosage unit or in a multiple dose format. In some embodiments, the pharmaceutical compositions are combined with an adjuvant, such as an immunoadjuvant (for example, STING agonists, TLR agonists, NOD agonists).
[00128] [00128] In some embodiments, the composition comprises at least one carbohydrate. A "carbohydrate" refers to a sugar or sugar polymer. The terms "saccharide", "polysaccharide", "carbohydrate" and "oligosaccharide" can be used interchangeably. Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one on each carbon atom in the molecule. Carbohydrates generally have the molecular formula CnH2nOn. A carbohydrate can be a monosaccharide, a disaccharide, trisaccharide, oligosaccharide or polysaccharide. Most basic carbohydrates are monosaccharides, such as glucose, sucrose, galactose, mannose, ribose, arabinose, xylose and fructose. Disaccharides are two monosaccharides joined together. Exemplary disaccharides include sucrose, maltose, cellobiose and lactose. Typically, an oligosaccharide includes between three and six monosaccharide units (for example, raffinose, stachyose), and polysaccharides include six or more monosaccharide units. Exemplary polysaccharides include starch, glycogen and cellulose. Carbohydrates can contain modified saccharide units, such as 2'-deoxyribose, with a hydroxyl group being removed, 2'-
[00129] [00129] In some embodiments, the composition comprises at least one lipid. As used herein, 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). In some embodiments, 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), margárico 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 tetracosanoic acid (24: 0). In some embodiments, the composition comprises at least one modified lipid, for example, a lipid that has been modified by cooking.
[00130] [00130] In some embodiments, the composition comprises at least one supplementary mineral or mineral source. Examples of minerals include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium and selenium. Suitable forms of any of the aforementioned 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.
[00131] [00131] In some embodiments, the composition comprises at least one supplementary vitamin. 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 above are vitamin salts, derivatives of the vitamin, compounds that have the same or similar activity as the vitamin and metabolites of the vitamin.
[00132] [00132] In some embodiments, the composition comprises an excipient. Non-limiting examples of suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compacting agent, a lubricant, a dispersion enhancer, a disintegrating agent, a flavoring agent, a sweetener and a coloring agent.
[00133] [00133] In some embodiments, the excipient is a buffering agent. Non-limiting examples of suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate and calcium bicarbonate.
[00134] [00134] In some embodiments, the excipient comprises a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol and phenol.
[00135] [00135] In some embodiments, the composition comprises a binder as an excipient. Non-limiting examples of suitable binders include starches, pregelatinized starches,
[00136] [00136] In some embodiments, the composition comprises a lubricant as an excipient. Non-limiting examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, esterotex, polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate and light mineral oil .
[00137] [00137] In some embodiments, the composition comprises a dispersion enhancer as an excipient. Non-limiting examples of suitable dispersants include starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoarmorphic silicate and microcrystalline cellulose as high HLB emulsifiers.
[00138] [00138] In some embodiments, the composition comprises a disintegrant as an excipient. In some embodiments, the disintegrant is a non-effervescent disintegrant. Non-limiting examples of non-effervescent disintegrants include starches, such as corn starch, potato starch, pregelatinized and modified starches, sweeteners, clays, such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, gums, such as water, guar, carob, caraia, pectin and tragacanth. In some embodiments, the disintegrant is an effervescent disintegrant. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.
[00139] [00139] In some embodiments, the composition is a food product (for example, a food or drink), such as a healthy food or drink, a baby food or drink, a food or drink for pregnant women, athletes, the elderly or other specified group, a functional food, drink, food or drink for specified health use, dietary supplement, patient food or drink or animal feed. Specific examples of food and drink include various drinks, such as juices, refreshing drinks, tea drinks, drink preparations, soft drinks and functional drinks; alcoholic beverages, such as beers; carbohydrate-containing foods, such as rice, pasta, bread and pasta food products; pasta products such as fish hams, sausages, seafood pasta products; retort packaging products, such as curries, food covered with thick starchy sauces and Chinese soups; soups; dairy products, such as milk, dairy drinks, ice cream, cheese and yogurt; fermented products, such as fermented soy pastes, yoghurts, fermented drinks and pickles; bean products; various confectionery products, including cookies, crackers and the like, sweets, chewing gums, gums, cold desserts, including jellies, cream caramels and frozen desserts; instant foods, such as instant soups and instant soy soups; microwave food; and the like In addition, the examples also include healthy foods and drinks in the form of powders, granules, tablets, capsules, liquids, pastes and jellies.
[00140] [00140] In some embodiments, the composition is a food product for animals, including humans. Animals, in addition to humans, are not particularly limited, and the composition can be used by various livestock, birds, pets, experimental animals and the like. Specific examples of animals include pigs, cattle, horses, sheep, goats, chickens, wild ducks, ostriches, domestic ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys and the like, but animals are not limited to these. Therapeutic agents
[00141] [00141] In certain respects, the methods provided in this document include administering to a subject a pharmaceutical composition described in this document alone or in combination with an additional therapeutic. In some modalities, the additional treatment is an immunosuppressant, a steroid, a cancer treatment.
[00142] [00142] In some embodiments, EV is administered to the subject before the therapy is administered (for example, 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). In some embodiments, EV is administered to the subject after the therapy is administered (for example, 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 later 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 later). In some modalities, IV and therapeutic are administered to the subject simultaneously or almost simultaneously (for example, the administrations occur within one hour of each other). In some embodiments, the subject is administered an antibiotic before EV is administered to the subject (for example, 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,
[00143] [00143] In some modalities, the additional treatment is a cancer treatment. In some modalities, cancer therapy is a chemotherapeutic agent. Examples of such chemotherapeutic agents include, but are not limited to, alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan and piposulfan; aziridines, such as benzodopa, carbocone, meturedopa and uredopa; ethylenimines and methylamelamines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethylolomelamine; acetogenins (especially bulatacin and bulatacinone); a camptothecin (including synthetic analogue topotecan); briostatin; calistatin; CC-1065 (including their synthetic analogues adozelesin, carzelesin and bizelesin); cryptoficina (particularly cryptoficina 1 and criptoficina 8); dolastatin; duocarmycin (including the synthetic analogs KW-2189 and CB1-TM1); eleuterobin; pancratistatin; sarcodictine; spongistatin; nitrogen mustards, such as chlorambucil, chlornafazine, colophosphamide, estramustine, ifosfamide, mecloretamine, meclorethamine oxide hydrochloride, melphalan, novembiquine, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosureas, such as carmustine, chlorozotocin, photemustine,
[00144] [00144] In some modalities, the cancer therapy is an immunotherapeutic agent against cancer. Immunotherapy refers to a treatment that uses a subject's immune system to treat cancer, for example, checkpoint inhibitors, cancer vaccines, cytokines, cell therapy, CAR-T cells and dendritic cell therapy. Non-limiting examples of immunotherapies are checkpoint inhibitors, including, nivolumab (BMS, anti-PD-1), pembrolizumab (Merck, anti-PD-1), ipilimumab (BMS, anti-CTLA-4), MEDI4736 (AstraZeneca , anti-PD-L1) and MPDL3280A (Roche, anti-PD-L1). Other immunotherapies may be tumor vaccines, such as Gardail, Cervarix, BCG, sipulencel-T, Gp100: 209-217, AGS-003, DCVax-L, Algenpantucel-L, Tergenpantucel-L, TG4010, ProstAtak, Prostvac-V / R-TRICOM, Rindopepimul, peptide acetate E75, IMA901,
[00145] [00145] In some embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor. Immune checkpoint inhibition largely refers to the inhibition of checkpoints that cancer cells can produce to prevent or downwardly regulate an immune response. Examples of immune checkpoint proteins include, but are not limited to, CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3 or VISTA. Immune checkpoint inhibitors can be antibodies or antigen-binding fragments thereof that bind to and inhibit an immune checkpoint protein. Examples of 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.
[00146] [00146] In some embodiments, the methods provided in this document include administering a pharmaceutical composition described herein in combination with one or more additional therapies. In some embodiments, the methods disclosed in this document include the administration of two additional immunotherapeutic agents (for example, immune checkpoint inhibitor). For example, the methods provided herein include administering a pharmaceutical composition described herein in combination with a PD-1 inhibitor and a CLTA-4 inhibitor or a PD-L1 inhibitor and a CTLA-4 inhibitor .
[00147] [00147] In some embodiments, the immunotherapeutic agent is an antibody or antigen-binding fragment that, for example, binds to an antigen associated with cancer. Examples of cancer-associated antigens include, but are not limited to, adipophyllin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein ("AFP"), ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen ("CEA"), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, stretching factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen ("ETA"), ETV6-AML1, EZH2, FGF5, FLT3-ITD, FN1, G250 / MN / CAIX, GAGE-1,2,8, GAGE-3,4,5 fusion protein , 6,7, GAS7, glypican-3, GnTV, gp100 / Pmel17, GPNMB, HAUS3, hepsin, HER-2 / neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2 , IDO1, IGF2B3, IL13Ralfa2, intestinal carboxyl esterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1, LDLR-fucosyltransferase fusion protein AS, lengsine, M-CSF, MAGE-A1, MA GE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mamaglobin-A, MART2, MATN, MC1R, MCSP , mdm-2, ME1, melan-A / MART-1, Meloe, midquina, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, myosin, myosin class I , N-crude, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1 / LAGE-2, OA1, OGT, OS-9, polypeptide P, p53, PAP, PAX5, PBF, pml-RARalfa fusion protein, polymorphic epithelial mucin ("PEM"), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secernina 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17,
[00148] [00148] In some embodiments, the immunotherapeutic agent is a cancer vaccine and / or a component of a cancer vaccine (for example, an antigenic peptide and / or protein). The cancer vaccine can be a protein vaccine, a nucleic acid vaccine, or a combination thereof. For example, in some embodiments, the cancer vaccine comprises a polypeptide that comprises an epitope of a cancer-associated antigen. In some embodiments, the cancer vaccine comprises a nucleic acid (for example, DNA or RNA, such as mRNA) that encodes an epitope of a cancer-associated antigen. Examples of cancer-associated antigens include, but are not limited to, adipophyllin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein ("AFP"), ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen ("CEA"), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, stretching factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen ("ETA"), ETV6-AML1, EZH2, FGF5, FLT3-ITD, FN1, G250 / MN / CAIX, GAGE-1,2,8, GAGE-3,4,5 fusion protein , 6,7, GAS7, glypican-3, GnTV, gp100 / Pmel17, GPNMB, HAUS3, hepsin, HER-2 / neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2 , IDO1, IGF2B3, IL13Ralfa2, intestinal carboxyl esterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1, LDLR- fusion protein
[00149] [00149] In some embodiments, the immunotherapeutic agent is an immunomodulatory protein for the subject. In some embodiments, the immunomodulatory protein is a cytokine or chemokine. Examples of immunomodulatory proteins include, but are not limited to, B lymphocyte chemo attractant ("BLC"), C-C motif chemokine 11
[00150] [00150] In some modalities, the therapeutic agent against cancer is an anticancer compound. Exemplary anticancer compounds include, but are not limited to, alentuzumab (Campath®), alitretinoin (Panretin®), anastrozole (Arimidex®), bevacizumab (Avastin®), bexarotene (Targretin®), bortezomibe (Velcade®), bosutinib (Bosulif®) , brentuximab vedotine (Adcetris®), cabozantinib (Cometriq ™), carfilzomib (Kyprolis ™), cetuximab (Erbitux®), crizotinib (Xalkori®), dasatinib (Sprycel®), denileucine diphthitox (Ontak®), hydrochloride, hydrochloride and hydrochloride (Tarceva®), everolimus (Afinitor®), exemestane (Aromasin®), fulvestrant (Faslodex®), gefitinib (Iressa®), ibritumomab tiuxetan (Zevalin®), imatinib mesylate (Gleevec®), ipilimumabe (Yervoy ™) , lapatinib ditosylate (Tykerb®), letrozole (Femara®), nilotinib (Tasigna®), ofatumumab (Arzerra®), panitumumab (Vectibix®), pazopanib hydrochloride (Votrient®), pertuzumab (Perjeta ™), pralatrexate (Folotynx ®), regorafenib (Stivarga®), rituximab (Rituxan®), romidepsin (Istodax®), sorafenib tosylate (Nexavar®), sun malate itinib (Sutent®), tamoxifen, tensirolimus (Torisel®), toremifene (Fareston®), tositumomab and 131I-tositumomab (Bexxar®), trastuzumab (Herceptin®), tretinoin (Vesanoid®), vandetanib (Caprelsa®) Zelboraf®), vorinostat
[00151] [00151] Exemplary anticancer compounds that modify the function of proteins that regulate gene expression and other cellular functions (for example, HDAC inhibitors, retinoid receptor ligands) are vorinostat (Zolinza®), bexarotene (Targretin®) and romidepsin (Istodax ®), alitretinoin (Panretin®) and tretinoin (Vesanoid®).
[00152] [00152] Exemplary anticancer compounds that induce apoptosis (for example, proteasome inhibitors, antifolates) are bortezomib (Velcade®), carfilzomib (Kyprolis ™) and pralatrexate (Folotyn®).
[00153] [00153] Exemplary anticancer compounds that increase the anti-tumor immune response (eg, anti CD20, anti CD52; antigen-4 associated with anti-cytotoxic T-lymphocyte) are rituximab (Rituxan®), alentuzumab (Campath®), ofatumumab (Arzerra®) and ipilimumab (Yervoy ™).
[00154] [00154] Exemplary anticancer compounds that deliver toxic agents to cancer cells (for example, anti-CD20-radionuclide fusions; IL-2-diphtheria toxin fusions; anti-CD30-monomethylauristatin E (MMAE) fusions) are tositumomab and 131I-tositumomab (Bexxar®) and ibritumomab tiuxetan (Zevalin®), denileucin diphthitox (Ontak®) and brentuximab vedotine (Adcetris®).
[00155] [00155] Other exemplary anticancer compounds are small cell inhibitors and conjugates thereof, for example, Janus kinase, ALK, Bcl-2, PARP, PI3K, VEGF receptor, Braf, MEK, CDK and HSP90.
[00156] Exemplary platinum-based anticancer 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.
[00157] [00157] In some embodiments, the cancer therapy is a radioactive chemical moiety that comprises a radionuclide. Exemplary radionuclides include, but are not limited to, Cr-51, Cs-131, Ce-134, Se-75, Ru-97, I-125, Eu-149, Os-189m, Sb-119, I-123, Ho- 161, Sb-117, Ce-139, In-111, Rh-103m, Ga-67, T1-201, Pd-103, Au-195, Hg-197, Sr-87m, Pt-191, P-33, Er-169, Ru-103, Yb-169, Au-199, Sn-121, Tm-167, Yb-175, In-113m, Sn-113, Lu-177, Rh-105, Sn-117m, Cu- 67, Sc-47, Pt-195m, Ce-141, I-131, Tb-161, As-77, Pt-197, Sm-153, Gd-159, Tm-173, Pr-143, Au-198, Tm-170, Re-186, Ag-111, Pd-109, Ga-73, Dy-165, Pm-149, Sn-123, Sr-89, Ho-166, P-32, Re-188, Pr- 142, Ir-194, In-114m / In-114 and Y-90.
[00158] [00158] In some modalities, the cancer therapy is an antibiotic. For example, if the presence of a cancer-associated bacteria and / or a cancer-associated microbiome profile is detected according to the methods provided in this document, antibiotics can be administered to eliminate the bacteria associated with the subject's cancer. "Antibiotics" refers broadly to compounds capable of inhibiting or preventing bacterial infection. Antibiotics can be classified in a number of ways, including their use for specific infections, their mechanism of action, their bioavailability or their target microbe spectrum (e.g. Gram-negative vs. Gram-positive bacteria, aerobic vs. anaerobic bacteria, etc.), and these can be used to kill specific bacteria in specific areas of the host ("niches") (Leekha, et al 2011. General Principles of Antimicrobial Therapy. Mayo Clin Proc. 86 (2): 156 to 167). In certain embodiments, antibiotics can be used to selectively target bacteria in a specific niche. In some embodiments, antibiotics known to treat a specific infection that includes a cancerous niche can be used to target cancer-associated microbes, including cancer-associated bacteria in that niche. In other modalities, antibiotics are administered after bacterial treatment. In some embodiments, antibiotics are administered after bacterial treatment to remove the graft.
[00159] [00159] In some respects, antibiotics can be selected based on their bactericidal or bacteriostatic properties. Bactericidal antibiotics include mechanisms of action that disrupt the cell wall (eg, β-lactams), the cell membrane (eg, daptomycin) or bacterial DNA (eg, fluoroquinolones). Bacteriostatic agents inhibit bacterial replication and include sulfonamides, tetracyclines and macrolides, and work by inhibiting protein synthesis. In addition, although some drugs may be bactericidal in certain organisms and bacteriostatic in others, knowing the target organism allows the person skilled in the art to select an antibiotic with the appropriate properties. Under certain treatment conditions, bacteriostatic antibiotics inhibit the activity of bactericidal antibiotics. Thus, in certain embodiments, bactericidal and bacteriostatic antibiotics are not combined.
[00160] [00160] Antibiotics include, but are not limited to, aminoglycosides, ansamycins, carbacefemas, carbapenemas, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptide fluids, tincanones, fluoroquinolones, quinolones and combinations thereof.
[00161] [00161] Aminoglycosides include, but are not limited to, amikacin,
[00162] [00162] 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.
[00163] [00163] Carbacefemas include, but are not limited to, loracarbef. Carbacephs are believed to inhibit bacterial cell wall synthesis.
[00164] [00164] Carbapenemas include, but are not limited to, ertapenema, doripenema, imipenema / cilastatin and meropenema. Carbapenemas are bactericidal for both Gram-positive bacteria and Gram-negative bacteria such as broad-spectrum antibiotics. Carbapenemas are believed to inhibit bacterial cell wall synthesis.
[00165] [00165] Cephalosporins include, but are not limited to, cefadroxil, cefazolin, cephalothin, cephalothin, cephalexin, cefaclor, cefamandole, cefoxitin, cefprozila, cefuroxime, cefixime, cefdinir, cefditorhem, cefizime, cefoperoxime, cefoperoxime, cefoperoxime, cefoperzone, cefepime, ceftaroline fosamyl and ceftobiprol. Selected cephalosporins are effective, for example, 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 interrupting the peptidoglycan layer synthesis of bacterial cell walls.
[00166] [00166] Glycopeptides include, but are not limited to, teicoplanin, vancomycin and telavancin. Glycopeptides are effective, for example, against aerobic and anaerobic Gram-positive bacteria, including MRSA and Clostridium difficile. Glycopeptides are believed to inhibit bacterial cell wall synthesis by interrupting the peptidoglycan layer synthesis of bacterial cell walls.
[00167] [00167] Lincosamides include, but are not limited to, clindamycin and lincomycin. Lincosamides are effective, for example, against anaerobic bacteria, as well as Staphylococcus and Streptococcus. Lincosamides are believed to bind to the 50S bacterial ribosomal subunit, thereby inhibiting bacterial protein synthesis.
[00168] [00168] Lipopeptides include, but are not limited to, daptomycin. Lipopeptides are effective, for example, against Gram-positive bacteria. Lipopeptides are believed to bind to the bacterial membrane and cause rapid depolarization.
[00169] [00169] Macrolides include, but are not limited to, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin and spiramycin. Macrolides are effective, for example, against Streptococcus and Mycoplasma. Macrolides are believed to bind to the 50S bacterial or ribosomal subunit, thereby inhibiting bacterial protein synthesis.
[00170] [00170] Monobactams include, but are not limited to, aztreonam. Monobactams are effective, for example, against Gram-negative bacteria. Monobactams are believed to inhibit bacterial cell wall synthesis by interrupting the peptidoglycan layer synthesis of bacterial cell walls.
[00171] [00171] Nitrofurans include, but are not limited to, furazolidone and nitrofurantoin.
[00172] [00172] Oxazolidonones include, but are not limited to, linezolid, posizolid, radezolid and torezolid. Oxazolidonones are believed to be inhibitors of protein synthesis.
[00173] [00173] Penicillins include, but are not limited to, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, naphthylin, oxacillin, penicillin G, penicillin V, ticacillin and piperacillin. Penicillins are effective, for example, against Gram-positive bacteria, facultative anaerobes, for example, Streptococcus, Borrelia and Treponema. Penicillins are believed to inhibit bacterial cell wall synthesis by interrupting the peptidoglycan layer synthesis of bacterial cell walls.
[00174] [00174] Penicillin combinations include, but are not limited to, amoxicillin / clavulanate, ampicillin / sulbactam, piperacillin / tazobactam and ticarcillin / clavulanate.
[00175] [00175] Polypeptide antibiotics include, but are not limited to, bacitracin, colistin and polymyxin B and E. Polypeptide antibiotics are effective, for example, against Gram-negative bacteria. Certain polypeptide antibiotics are believed to inhibit the isoprenyl pyrophosphate involved in the synthesis of the peptidoglycan layer of bacterial cell walls, while others destabilize the outer bacterial membrane by displacing bacterial counterions.
[00176] [00176] Quinolones and fluoroquinolone include, but are not limited to, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grezpafloxacin, grema. Quinolones / fluoroquinolone are effective, for example, against Streptococcus and Neisseria. Quinolones / fluoroquinolone are believed to inhibit bacterial DNA gyrase or topoisomerase IV, thereby inhibiting DNA replication and transcription.
[00177] [00177] Sulfonamides include, but are not limited to, mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole (co-trimoxazidine) and sulfonamide. Sulphonamides are believed to inhibit folate synthesis by competitive inhibition of dihydropterate synthetase, thereby inhibiting nucleic acid synthesis.
[00178] [00178] Tetracyclines include, but are not limited to, demeclocycline, doxycycline, minocycline, oxytetracycline and tetracycline. Tetracyclines are effective, for example, against Gram-negative bacteria. Tetracyclines are believed to bind to the bacterial ribosomal subunit 30S, thereby inhibiting bacterial protein synthesis.
[00179] [00179] Antimycobacterial compounds include, but are not limited to, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine and streptomycin.
[00180] [00180] Suitable antibiotics also include arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin / dalfopristin, tigecycline, tinidazole, trimethoprim amoxicillin / clavulanicine, amycotin, amycin, amycin, amycin, amycin, amycin, amycin, amycin, amycin, amycin, amycin, amine , Pl cecropine, clarithromycin, erythromycin, furazolidone, fusidic acid, Na fusidate, gramicidin, imipenem, indolicidin, josamycin, magainan II, metronidazole, nitroimidazoles, mycumin, B-Ny266 mutacin, B-JHl 140 mutacin, Tacin nisin, nisin A, novobiocin, oleandomycin, ostreogricine, piperacillin / tazobactam, pristinamycin, ramosplanin, ranalexin, reuterine, rifaximin, rosamycin, rosamycin, spectinomycin,
[00181] [00181] In some embodiments, the additional therapy 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. Representative agents include, but are not limited to, cyclosporine, retinoids, corticosteroids, propionic acid derivative, acetic acid derivative, enolic acid derivatives, phenamic acid derivatives, Cox-2 inhibitors, lumiracoxib, ibuprofen, magnesium choline salicylate, fenprofen , salsalate, difunisal, tolmetin, ketoprofen, flurbiprofen, oxaprozine, indomethacin, sulindac, etodolac, ketorolac, nabumetone, naproxen, valdecoxib, etoricoxib, MK0966; rofecoxib, acetominophen, celecoxib, diclofenac, tramadol, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefanamic acid, meclofenamic acid, flufenamic acid, tolfenamic, valdecoxib, parecoxib, metodol, aspirin, metodol, ), antimalarial drugs (for example, hydroxychloroquine and chloroquine), sulfasalazine, leflunomide, azathioprine, cyclosporine, gold salts, minocycline, cyclophosphamide, D-penicillamine, minocycline, auranofin, tacrolimus, for example, myocrisine, , TNF alpha antagonists or TNF alpha receptor antagonists), for example, ADALIMUMABE (Humira®), ETANERCEPT (Enbrel®), INFLIXIMABE (Remicade®; TA-650), CERTOLIZUMABE PEGOL (Cimzia®; CDP870), GOLIMUMABE ( Simpom®; CNTO 148), ANAKINRA (Kineret®), RITUXIMABE (Rituxan®; MabThera®), ABATACEPT (Orencia®), TOCILIZUMABE (RoActemra / Actemra®), integrin antagonists (TYSABRI® (natalizumab)), IL antagonists - 1 (ACZ885 (Ilaris)), Anakinra (Kineret®)), CD4 antagonists, IL-23 antagonists, IL-20 antagonists, IL-6 antagonists, BLyS antagonists (for example, Atacicept, Benlysta® / LymphoStat -B® (belimumab)), p38 inhibitors, CD20 antagonists (ocrelizumab, ofatumumab (Arzerra®)), gamma interferon antagonists (fontolizumab), prednisolone, prednisone, dexamethasone, cortisol, cortisone, hydrocortisone, methylprednisolone, triamcinolone, betamcinolone, betamethasone , beclomethasoma, fludrocortisone, deoxycorticosterone, aldosterone, doxycycline, vancomycin, pioglitazone, SBI-087, SCIO- 469, Cura-100, oncoxin + viusida, TwHF, methoxsalene, vitamin D - ergocalciferol, tnacritol, tyrolazole, taclazolone, ®), RADOOl, rapamune, rapamycin, fostamatinib, fentanyl, XOMA 052, disodium fostamatinib, rosigtazone, curcumin (Longvida ™), rosuvastatin, maraviroc, ramipnl, milnacipran, cobiprostone, somatropin, gene therapy vector, MK069994 esome prazole, everolimus, trastuzumab, JAK1 and JAK2 inhibitors, JAK pan inhibitors, for example, tetracyclic pyridone 6 (P6), 325, PF-956980, denosumab, IL-6 antagonists, CD20 antagonists, CTLA4 antagonists, antagonists IL-8 antagonists, IL-21 antagonists, IL-22 antagonists, integrin antagonists (Tysarbri® (natalizumab)), VGEF antagonists, CXCL antagonists, MMP antagonists, defensin antagonists (IL-1 antagonists ( including IL-1 beta antagonists) and IL-23 antagonists (e.g., receptor decoys, antagonist antibodies, etc.).
[00182] [00182] In some embodiments, the agent is an immunosuppressive agent. Examples of immunosuppressive agents include, but are not limited to, corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporine A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine, epinephrine, epinephrine, epinephrine, epinephrine, epinephrine , anticholinergic drugs for rhinitis, TLR antagonists, inflammasome inhibitors, anticholinergic decongestants, mast cell stabilizers, anti-IgE monoclonal antibodies, vaccines (for example, vaccines used for vaccination in which the amount of an allergen is gradually increased), inhibitors of cytokines, such as anti-IL-6 antibodies, TNF inhibitors, such as infliximab, adalimumab, certolizumab pegol, golimumab or etanercept, and combinations thereof. Administration
[00183] [00183] In certain respects, a method for administering a pharmaceutical composition described in this document to a subject is provided herein. In some embodiments of the methods provided herein, the pharmaceutical composition is administered in conjunction with the administration of an additional therapeutic. In some embodiments, the pharmaceutical composition comprises EV and / or bacteria co-formulated with the additional therapeutic. In some embodiments, the pharmaceutical composition is co-administered with additional therapy. In some embodiments, additional therapy is administered to the subject prior to administration of the pharmaceutical composition (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 , 35, 40, 45, 50 or 55 minutes before, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22 or 23 hours before or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days before). In some embodiments, additional therapy is administered to the subject after administration of the pharmaceutical composition (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 , 35, 40, 45, 50 or 55 minutes later, 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 later or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days later). In some embodiments, the same mode of delivery is used to administer both the pharmaceutical composition and the additional therapeutic. In some embodiments, different modes of delivery are used to administer the pharmaceutical composition and additional therapy. For example, in some embodiments, the pharmaceutical composition is administered orally, while additional therapy is administered by injection (for example, an intravenous, intramuscular and / or intratumoral injection).
[00184] [00184] In certain embodiments, the pharmaceutical compositions, dosage forms and described in this document can be administered in conjunction with any other conventional anticancer treatment, such as, for example, radiotherapy and surgical removal of the tumor. These treatments can be applied as needed and / or as indicated and can occur before, concomitantly or after administration of the pharmaceutical compositions, dosage forms and kits described in this document.
[00185] [00185] The dosage regimen can be any one of a variety of methods and amounts and can be determined by the person skilled in the art according to known clinical factors. As is known in medical techniques, dosages for any patient can depend on many factors, including the subject's species, size, body surface area, age, sex, immunocompetence and health in general, the specific microorganism to be administered, the duration and route of administration, the type and stage of the disease, for example, tumor size, and other compounds, such as drugs being administered concomitantly. 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 the person skilled in the art. In the present methods, adequate minimum dosage levels of microorganisms can be sufficient levels for the microorganism to survive, develop and replicate. The dose of the pharmaceutical compositions described in this document can be suitably defined or adjusted according to the dosage form, the route of administration, the degree or stage of a target disease and the like. For example, the overall effective dose of the agents can vary between 0.01 mg / kg body weight / day and 1,000 mg / kg body weight / day, between 0.1 mg / kg body weight / day and
[00186] [00186] In some modalities, the dose administered to a subject is sufficient to prevent diseases (for example, autoimmune disease, inflammatory disease, metabolic disease, cancer), delay its onset or slow or stop its progression. A person skilled in the art will recognize that the dosage will depend on a variety of factors, including the strength of the specific compound employed, as well as the subject's age, species, condition and body weight. The dose size 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 may accompany the administration of a specific compound and the desired physiological effect.
[00187] [00187] Adequate doses and dosage regimes can be determined by conventional range detection techniques known to those of ordinary skill in the art. In general, treatment is started at lower dosages, which are less than the ideal dose of the compound. Thereafter, the dosage is increased by small increments until the ideal effect under the circumstances is achieved. An effective dosing and treatment protocol can be determined by routine and conventional means, starting, for example, with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosing regimen as well. . Animal studies are commonly used to determine the maximum tolerable dose ("BAT") of bioactive agent per kilogram of weight. Those skilled in the art regularly extrapolate doses for effectiveness, while avoiding toxicity, in other species, including humans.
[00188] [00188] According to the above, in therapeutic applications, the dosages of the active agents used according to 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 doctor or professional who administers the therapy, among other factors that affect the selected dosage. In general, the dose should be sufficient to result in retardation and, preferably, regression of tumor growth and, most preferably, to cause complete regression of the cancer.
[00189] [00189] Separate administrations may include any number among two or more administrations, including two, three, four, five or six administrations. A person skilled in the art can readily determine the number of administrations to be performed or the desire to perform one or more additional administrations in accordance with methods known in the art for monitoring therapeutic methods and other monitoring methods provided herein. Accordingly, the methods provided in this document include methods for providing the subject with one or more administrations of a pharmaceutical composition, in which the number of administrations can be determined by monitoring the subject and, based on the results of the monitoring, determine whether or not to or do not provide one or more additional administrations. Deciding whether or not to provide one or more additional administrations can be based on a variety of monitoring results.
[00190] [00190] The time period between administrations can be any one of a variety of time periods. The length of time between administrations can be a function of any one of a variety of factors, including monitoring steps, as described in relation to the number of administrations, the length of time for a subject to show an immune response and / or the period of time for a subject to eliminate EV from normal tissue. In one example, the length of time may be a function of the length of time for a subject to have an immune response; for example, the length of time may be longer than the length of time for a subject to have an immune response, such as more than about a week, more than about ten days, more than about two weeks or more than about a month; in another example, the time period may be less than the time for a subject to show an immune response, such as less than about a week, less than about ten days, less than about two weeks or less that about a month. In another example, the length of time may be a function of the length of time for a subject to eliminate EV from normal tissue; for example, the time period can be longer than the time for a subject to clear 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.
[00191] [00191] In some embodiments, the delivery of an additional therapeutic in combination with the pharmaceutical composition described in this document reduces adverse effects and / or improves the effectiveness of the additional therapeutic.
[00192] [00192] The effective dose of an additional therapeutic described in this document is the amount of the therapeutic agent that is effective in achieving the desired therapeutic response for a specific patient, the composition and the mode of administration, with the least toxicity for the patient. The effective dosage level can be identified using the methods described in this document and will depend on a variety of pharmacokinetic factors, including the activity of the specific compositions administered, the route of administration, the time of administration, the rate of excretion of the compound. specific to be employed, duration of treatment, other drugs, compounds and / or materials used in combination with the specific compositions employed, age, sex, weight, condition, general health and previous medical history of the patient to be treated and similar factors well known in the medical arts. In general, an effective dose of an additional therapy will be the amount of the therapeutic agent that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above.
[00193] [00193] The toxicity of an additional therapy is the level of adverse effects experienced by the subject during and after treatment. Adverse events associated with additional therapy toxicity include, but are not limited to, abdominal pain, acid indigestion, acid reflux, allergic reactions, alopecia, anaphylaxis, anemia, anxiety, lack of appetite, arthralgia, asthenia, ataxia, azotemia, loss of balance , bone pain, bleeding, blood clots, low blood pressure, high blood pressure, difficulty breathing, bronchitis, bruises, 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, cataract, central neurotoxicity, cognitive impairment, confusion, conjunctivitis, constipation, cough, cramp, cystitis, deep vein thrombosis, dehydration, depression, diarrhea, dizziness, dry mouth, dry skin, dyspepsia, dyspnoea, edema, electrolyte imbalance, esophagitis, fatigue, loss of fertility, fever, flatulence, flushing, gastric reflux, gastroesophageal reflux disease, genital pain, granulocytopenia, gynecomastia, glaucoma, hair loss, palmar-plantar syndrome, headache, hearing loss, heart failure, heart palpitations, heartburn, hematoma, hemorrhagic cystitis, hepatotoxicity, hyperamylasemia, hypercalcemia, hyperchloremia, hyperglycemia, hyperkalemia, hyperlipasemia, hypermagnesemia, hypernatremia, hyperphosphatemia, hyperpigmentation, hypertriglyceridemia, hyperuricemia, hypoalbuminemia, hypocalcaemia, hypochloremia, hypoglycemia, hypokalemia, hypnotic, infection, hypomagnesemia, hypomagnesemia, hypotonia iron deficiency, itching, joint pain, kidney failure, leukopenia, liver dysfunction, memory loss, menopause, mouth sores, mucositis, muscle pain, myalgia, myelosuppression, myocarditis, neutropenic fever, nausea, nephrotoxicity, neutropenia, nosebleeds , numbness, ototoxicity, pain, erythrodysesthesia pa lmar-plantar,
[00194] [00194] In some embodiments, the methods and compositions described in this document refer to the treatment or prevention of a disease or disorder associated with a pathological immune response, such as an autoimmune disease, an allergic reaction and / or an inflammatory disease. In some embodiments, the disease or disorder is an inflammatory bowel disease (for example, Crohn's disease or ulcerative colitis).
[00195] [00195] The methods described in this document can be used to treat any subject in need of it. As used herein, a "subject in need" includes any subject who has a disease or disorder associated with a pathological immune response (for example, an inflammatory bowel disease), as well as any subject who is more likely to acquire such disease or disorder.
[00196] [00196] The compositions described in this document can be used, for example, as a pharmaceutical composition to prevent or treat (reduce, partially or completely, the adverse effects of) an autoimmune disease, such as chronic inflammatory bowel disease, systemic lupus erythematosus , psoriasis,
[00197] [00197] In some embodiments, the methods provided in this document are useful for the treatment of inflammation. In certain embodiments, inflammation of any tissue and organs in the body, including musculoskeletal inflammation, vascular inflammation, neural inflammation, inflammation of the digestive system, eye inflammation, inflammation of the reproductive system and other inflammation, as discussed below.
[00198] [00198] Immune disorders of the musculoskeletal system include, but are not limited to, those conditions that affect skeletal joints, including joints of the hands, wrist, elbow, shoulder, jaw, spine, neck, hip, knee, ankle and feet, and conditions that affect the tissues that connect muscles to bones, such as tendons. Examples of such immunological disorders, which can be treated with the methods and compositions described herein, include, but are not limited to, arthritis (including, for example, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acute and chronic infectious arthritis,
[00199] [00199] Eye immunological disorders refer to an immune disorder that affects any structure of the eye, including the eyelids. Examples of ocular immune disorders that can be treated with the methods and compositions described in this document include, but are not limited to, blepharitis, blepharocalase, conjunctivitis, dacrioadenitis, keratitis, dry queeratoconjunctivitis (dry eye), scleritis, trichiasis and uveitis
[00200] [00200] Examples of immune disorders of the nervous system that can be treated with the methods and compositions described in this document include, but are not limited to, encephalitis, Guillain-Barre syndrome, meningitis, neuromyotonia, narcolepsy, multiple sclerosis, myelitis and schizophrenia. Examples of inflammation of the vasculature or lymphatic system that can be treated with the methods and compositions described in this document include, but are not limited to, atherosclerosis, arthritis, phlebitis, vasculitis and lymphangitis. Examples of immune disorders of the digestive system that can be treated with the methods and compositions described in this document include, but are not limited to, cholangitis, cholecystitis, enteritis, enterocolitis, gastritis, gastroenteritis, inflammatory bowel disease, ileitis and proctitis. Inflammatory bowel diseases include, for example, certain forms recognized in the art for a group of related conditions. Several major forms of inflammatory bowel disease are known, with Crohn's disease (regional intestinal disease, for example, inactive and active forms) and ulcerative colitis (for example, inactive and active forms) being the most common of these disorders. In addition, inflammatory bowel disease includes irritable bowel syndrome, microscopic colitis, lymphocytic-plasmacytic enteritis, celiac disease, collagenous colitis, lymphocytic colitis and eosinophilic enterocolie. Other less common forms of IBD include indeterminate colitis, pseudomembranous colitis (necrotizing colitis), ischemic inflammatory bowel disease, Behcet's disease, sarcoidosis, scleroderma, IBD-associated dysplasia, masses or lesions associated with dysplasia and primary sclerosing cholangitis.
[00201] [00201] Examples of immunological disorders of the reproductive system that can be treated with the methods and compositions described in this document include, but are not limited to, cervicitis, chorioamnionitis, endometritis, epididymitis, omphalitis, oophoritis, orchitis, salpingitis, abscess in the ovarian tube, urethritis, vaginitis, vulvitis and vulvodynia.
[00202] [00202] The methods and compositions described in this document can be used to treat immune disorders that have an inflammatory component. Such conditions include, but are not limited to, acute universal disseminated alopecia, Behcet's disease, Chagas' disease, chronic fatigue syndrome, dysautonomia, encephalomyelitis, ankylosing spondylitis, aplastic anemia, suppurative hidradenitis, autoimmune hepatitis, autoimmune oophoritis, celiac disease, disease of Crohn's, type 1 diabetes mellitus, giant cell arteritis, Goodpasture's syndrome, Grave's disease, Guillain-Barre's syndrome, Hashimoto's disease, Henoch-Schonlein's purpura, Kawasaki's disease, lupus erythematosus, microscopic colitis, microscopic polyarteritis, disease mixed connective tissue, Muckle-Wells syndrome, multiple sclerosis, myasthenia gravis, opsoclonus-myoclonus syndrome, optic neuritis, Ord thyroiditis, pemphigus, polyarteritis nodosa, polymyalgia, rheumatoid arthritis, Reiter's syndrome, Sjogren's syndrome, temporal arteritis , Wegener's granulomatosis, hot autoimmune hemolytic anemia, interstitial cystitis, Lyme disease,
[00203] [00203] The methods and compositions described in this document can be used to treat T-cell-mediated hypersensitivity diseases that have an inflammatory component. Such conditions include, but are not limited to, hypersensitivity to contact, contact dermatitis (including that caused by poison ivy), hives, skin allergies, respiratory allergies (hay fever, allergic rhinitis, allergy to residential mites) and gluten sensitive enteropathy ( celiac disease).
[00204] [00204] Other immunological disorders that can be treated with the methods and compositions include, for example, appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, hepatitis, suppurative hidradenitis, iritis, laryngitis, mastitis, myocarditis, nephritis, otitis , pancreatitis, parotitis, percarditis, peritonitis, pharyngitis, pleuritis, pneumonitis, prostatitis, pyelonephritis and stomatitis, transplant rejection (involving organs such as kidney, liver, heart, lung, pancreas (for example, islet cells), bone marrow , cornea, small intestine, cutaneous allografts, cutaneous homografts and heart valve xenografts, serum disease and graft vs. host disease), acute pancreatitis, chronic pancreatitis, acute respiratory distress syndrome, Sexary syndrome, congenital adrenal hyperplasia, thyroiditis non-suppurative, cancer-associated hypercalcemia, pemphigus, herpetiform bullous dermatitis, severe erythema multiforme, exfoliative dermatitis, dermatitis seborrheic, seasonal or perennial allergic rhinitis, bronchial asthma, contact dermatitis, atopic dermatitis, drug hypersensitivity reactions, allergic conjunctivitis, keratitis, ophthalmic herpes zoster, iritis and oiridocyclitis, chorioretinitis, optic neuritis, pulmonary sarcoidosis, symptomatic sarcoidosis fulminant or disseminated, idiopathic thrombocytopenic purpura in adults, secondary thrombocytopenia in adults, acquired hemolytic anemia (autoimmune), leukemia and lymphomas in adults, acute childhood leukemia, regional enteritis, autoimmune vasculitis, multiple sclerosis, chronic obstructive pulmonary disease, transplantation rejection of solid organs, sepsis. Preferred treatments include transplant rejection treatment, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, type 1 diabetes, asthma, inflammatory bowel disease, systemic lupus erythematosus, psoriasis, chronic obstructive pulmonary disease and inflammation accompanied by infectious diseases (for example, sepsis) . Metabolic disorders
[00205] [00205] The methods and compositions described in this document can be used to treat metabolic disorders and metabolic syndromes. Such conditions include, but are not limited to, type II diabetes, encephalopathy, Tay-Sachs disease, Krabbe disease, galactosemia, phenylketonuria (PKU) and maple syrup urine disease. Accordingly, in certain embodiments, methods are provided herein to treat metabolic disorders which comprise administering to a subject a composition provided herein. In certain embodiments, the metabolic disease is type II diabetes, encephalopathy, Tay-Sachs disease, Krabbe disease, galactosemia, phenylketonuria (PKU) or maple syrup urine disease. Cancer
[00206] [00206] In some embodiments, the methods and compositions described in this document refer to the treatment of cancer. In some modalities, any cancer can be treated using the methods described in this document. Examples of cancers that can be treated by the methods and compositions described in this document include, but are not limited to, bladder cancer cells, blood, bone, bone marrow, brain,
[00207] [00207] In some embodiments, the methods and compositions provided in this document refer to the treatment of a leukemia. The term "leukemia" means largely 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 non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, leukemia leukemia, leukemia leukemia, basophilic leukemia, basophilic leukemia blastic, bovine leukemia, chronic myelocytic leukemia, cutaneous leukemia, embryonic leukemia, eosinophilic leukemia, Gross leukemia, Rieder cell leukemia, Schilling leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hair cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphoid leukemia, lymphocytic leukemia, lymphocytic leukemia micr leukemia omieloblastic, monocytic leukemia,
[00208] [00208] In some embodiments, the methods and compositions provided in this document refer to the treatment of a carcinoma. The term "carcinoma" refers to a malignant growth made up of epithelial cells that tend to infiltrate the surrounding tissues and / or resist the signs of physiological and non-physiological cell death and give rise to metastases. Exemplary non-limiting types of carcinoma include, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adenomatous carcinoma, adrenal cortex carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basal cell carcinoma, basal cell carcinoma, basal cell carcinoma, basal cell carcinoma basosquamous, bronchialalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, body carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma carcinoma, cutaneous carcinoma duct, hard carcinoma, embryonic carcinoma, encephaloid carcinoma, squamous cell carcinoma, adenoid epithelial carcinoma, exophytic carcinoma, ex ulcere carcinoma, fibrous carcinoma, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, ring cell carcinoma, sinus ring carcinoma simplex arcinoma, small cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, spongy carcinoma, squamous carcinoma, squamous cell carcinoma, string carcinoma, telangiectatic carcinoma, telangiectoid carcinoma, transitional cell carcinoma, tubular carcinoma , tuberous carcinoma, verrucous carcinoma,
[00209] [00209] In some embodiments, the methods and compositions provided in this document refer to the treatment of a sarcoma. The term "sarcoma" usually refers to a tumor that consists of a substance similar to embryonic connective tissue and is usually composed of tightly 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, large cell sarcoma, liposomal sarcoma, Abemeth sarcoma alveolar soft tissue, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonic sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymphoblastic B cell lymphoma, lymphoblastic sarcoma , Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymal sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma and telangiectal sarcoma.
[00210] [00210] Additional exemplary neoplasms that can be treated using the methods and compositions described in this document include Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, thrombocytosis primary, 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 , cancer of the genitourinary tract, malignant hypercalcemia, cervical cancer, endometrial cancer, plasmacytoma, colorectal cancer, rectal cancer and adrenal cortical cancer.
[00211] [00211] In some modalities, the cancer treated is a melanoma. The term "melanoma" is considered to be a tumor that originates from the melanocytic system of the skin and other organs. Non-limiting examples of melanomas are Harding-Passey melanoma, juvenile melanoma, malignant lentigo melanoma, malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, nodular melanoma, subangual melanoma superficial.
[00212] [00212] Specific categories of tumors that can be treated using the methods and compositions described in this document include lymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, thyroid cancer, head and neck cancer, central nervous system cancer, peripheral nervous system cancer, skin cancer, kidney cancer, as well as metastases from all those mentioned above.
[00213] [00213] Cancers treated in certain modalities also include precancerous lesions, for example, actinic keratosis (solar keratosis), moles (dysplastic nevi), actinic celite (farmer's lips), cutaneous horns, Barrett's esophagus, atrophic gastritis, dyskeratosis congenital, sideropenic dysphagia, lichen planus, oral submucosal fibrosis, actinic (solar) elastosis and cervical dysplasia.
[00214] [00214] Cancers treated in some modalities include non-cancerous or benign tumors, for example, of endodermal, ectodermal or mesenchymal origin, including, but not limited to, cholangioma, colonic polyp, adenoma, papilloma, cystadenoma, adenoma of liver cells, hydatidiform mole , renal tubular adenoma, squamous cell papilloma, gastric polyp, hemangioma, osteoma, chondroma, lipoma, fibroma, lymphangioma, leiomyoma, rhabdomyoma, astrocytoma, nevus, meningioma and ganglioneuroma. Other diseases and disorders
[00215] [00215] In some embodiments, the methods and compositions described in this document refer to the treatment of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).
[00216] [00216] In some embodiments, the methods and compositions described in this document refer to the treatment of liver diseases. Such diseases include, but are not limited to, Alagille syndrome, alcohol-related liver disease, alpha-1 antitrypsin deficiency, autoimmune hepatitis, benign liver tumors, biliary atresia, cirrhosis, galactosemia, Gilbert's syndrome, hemochromatosis, hepatitis A, hepatitis B , hepatitis C, hepatic encephalopathy, intrahepatic cholestasis of pregnancy (PCI), lysosomal acid lipase deficiency (LAL-D), hepatic cysts,
[00217] [00217] The methods and compositions described in this document can be used to treat neurodegenerative and neurological diseases. In certain embodiments, the neurodegenerative and / or neurological disease is Parkinson's disease, Alzheimer's disease, prion disease, Huntington's disease, motor neuron disease (MND), spinocerebellar ataxia, spinal muscular atrophy, dystonia, intracranial idiopathic hypertension, epilepsy, nervous system disease, central nervous system disease, movement disorders, multiple sclerosis, encephalopathy, peripheral neuropathy or postoperative cognitive dysfunction. Methods to produce intensified bacteria
[00218] [00218] In certain respects, methods for producing genetically modified bacteria for the production of the EVs described in this document are provided in this document. In some embodiments, genetically modified bacteria are modified to enhance certain desirable properties. For example, in some embodiments, genetically modified bacteria are modified to increase the production of EV by the bacteria. In some embodiments, genetically modified bacteria are modified to produce EV with enhanced oral administration (for example, improving acid resistance and / or resistance to bile acids), to enhance the immunomodulatory and / or therapeutic effect of the EVs they produce. (eg, alone or in combination with another therapeutic agent), to enhance immune activation by the EVs they produce and / or to improve bacterial and / or EV manufacturing (eg, higher tolerance to oxygen, better tolerance to freeze-thaw, shorter generation times). Genetically modified bacteria can be produced using any technique known in the art, including, but not limited to, sitiodirected mutagenesis, transposon mutagenesis, knockouts, knockins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, CRISPR / Cas9 or any combination thereof.
[00219] [00219] In some modalities of the methods provided in this document, the bacterium is modified by directed evolution. In some modalities, directed evolution comprises the exposure of the bacteria to an environmental condition and the selection of bacteria with improved survival and / or development under the environmental condition. In some modalities, the method comprises screening mutagenized bacteria using an assay that identifies the intensified bacteria. In some embodiments, the method further comprises mutagenizing the bacteria (for example, by exposure to chemical mutagenesis and / or UV radiation) followed by an assay to detect bacteria that have the desired phenotype (for example, an in vivo assay, a ex vivo assay or an in vitro assay).
[00220] [00220] In some embodiments, the bacteria provided in this document is modified by exposure to a stress-inducing environment (for example, an environment that induces stress in the envelope). In some embodiments, development under such developmental conditions increases the production of EV by the bacterium. For example, in some embodiments, the bacterium is developed in the presence of subinhibitory concentrations of an antibiotic described in this document (for example, 0.1 to 1 µg / ml chloramphenicol or 0.1 to 0.3 µg / ml gentamicin ). In some embodiments, host antimicrobial peptides (for example, lysozyme, defensins and Reg proteins) are used instead of antibiotics, or in combination with them. In some embodiments, antimicrobial peptides produced by bacteria (for example, bacteriocins and microcins) are used. In some modalities, stress is temperature stress (for example, development at 37 to 50 ° C). In some embodiments, stress is carbon limiting stress (for example, development in a medium that comprises limited carbon sources, such as medium with a carbon source restricted to below 1% (w / v)). In some modalities, stress is salt stress (for example, development in a medium containing 0.5 M NaCl). In some modalities, stress is UV stress (for example, development under a UV lamp, over the entire growing period or only during a portion of the growing period). In some embodiments, stress is reactive oxygen stress (eg, development in media containing subinhibitory concentrations of hydrogen peroxide, such as 250 to 1,000 µM of hydrogen peroxide). In some embodiments, a combination of these stresses revealed in this document is applied to the bacteria.
[00221] [00221] Extracellular vesicles (EV) are prepared from bacterial cultures using the methods known to those skilled in the art (S. Bin Park, et al. PLoS ONE. 6 (3): e17629 (2011)).
[00222] [00222] For example, bacterial cultures are centrifuged at
[00223] [00223] Alternatively, EVs are obtained from bacterial cultures continuously during development, or at selected points in time during development, by connecting a bioreactor to an alternating tangential flow (ATF) system (eg XCell ATF Repligen) according to the manufacturer's instructions. The ATF system retains intact cells (> 0.22 µm) in the bioreactor and allows smaller components (eg, EV, free proteins) to pass through a filter for collection. For example, the system can be configured so that the filtrate <0.22 µm is then passed through a second 100 kDa filter, allowing species, such as EV between 0.22 µm and 100 kDa, to be collected and species smaller than 100 kDa are pumped back into the bioreactor. Alternatively, the system can be configured to allow the medium in the bioreactor to be replenished and / or modified during the development of the culture. EVs collected by this method can also be purified and / or concentrated by ultracentrifugation or filtration as described above for filtered supernatants.
[00224] [00224] The EVs obtained by the methods described above can also be purified by gradient ultracentrifugation, using methods that may include, but are not limited to, the 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, the pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is changed from buffer to 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column. Samples are applied to a 35 to 60% batch sucrose gradient and centrifuged at 200,000 x g for 3 to 24 hours at 4 ° C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation is used to concentrate the filtered supernatants, the pellets are resuspended in 35% Optiprep in PBS. If filtration is used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep. Samples are applied to a 35 to 60% batch sucrose gradient and centrifuged at
[00225] [00225] In order to confirm the sterility and isolation of the EV preparations, the EVs are serially diluted in agar medium used for routine culture of the bacteria to be tested and incubated with the use of routine conditions. Non-sterile preparations are passed through a 0.22 µm filter to exclude intact cells. To further increase purity, isolated EVs can be treated with DNase or proteinase K.
[00226] [00226] Alternatively, for the preparation of EVs used for in vivo injections, the purified EVs are processed as previously described (G. Norheim, et al. PLoS ONE. 10 (9): e0134353 (2015)). Briefly, after sucrose gradient centrifugation, bands containing EV are resuspended to a final concentration of 50 µg / ml in a solution containing 3% sucrose or another suitable solution for in vivo injection known to the person skilled in the art. This solution can also contain adjuvant, for example, aluminum hydroxide, at a concentration of 0 to 0.5% (w / v).
[00227] [00227] To make the samples compatible with additional tests (for example, to remove sucrose before TEM imaging or in vitro assays), the samples are changed from buffer to PBS or 30 mM Tris, pH 8.0 with the use of filtration (eg Amicon Ultra columns), dialysis, or ultracentrifugation (200,000 xg, ≥ 3 hours, 4 ° C) and resuspension. Example 2: Identification of bacterial EV
[00228] [00228] In order to track their biodistribution in vivo and quantify them localize in vitro in various preparations and in tests conducted with mammalian cells, EVs are identified as previously described (N. Kesty, et al. EMBO Journal. 23: 4,538 a
[00229] [00229] For example, 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 can be extended overnight at 4 ° C. To remove excess fluorochrome, the EVs are then (1) agglomerated by centrifugation at 200,000 x g for 3 h - overnight, washed and resuspended in PBS or another buffer suitable for downstream applications; or (2) switched from buffer to PBS or another buffer suitable for downstream dialysis or filtration applications (for example, using an Amicon Ultra column).
[00230] [00230] Alternatively, EVs are obtained from bacteria grown in medium containing 0.8 mM 3-azido-D-alanine or HADA. The EVs are resuspended or changed from buffer to PBS, and a portion is additionally identified with fluorescent dye of 10 µM dibenzozaza-cyclooctin (DIBAC) in 1% BSA / PBS (dyes include Cy5, TAMRA, Rhodamine-green and Cy7) if developed with 3-azido-D-alanine. The unincorporated dye is removed as described above, by (1) ultracentrifugation, washing and resuspension; or (2) changing the buffer by dialysis or filtration.
[00231] [00231] The identified EVs can also be generated from bacteria that express green fluorescent protein (GFP), or any other fluorescent protein. For Gram negative bacteria, periplasmic targeting sequences are attached to the fluorescent proteins, so that they are adequately located to be internalized by the EV as they form.
[00232] [00232] Quantum dots can be used to identify EV for non-invasive imaging studies in vivo (K. Kikushima, et al. Scientific Reports. 3 (1913) (2013)). Quantum dots are conjugated to an antibody confirmed to be present on the EV membrane. Isolated EVs are incubated with quantum dot conjugates, and the excess conjugates are removed as described above, by (1) ultracentrifugation, washing and resuspension; or (2) changing the buffer by dialysis or filtration.
[00233] [00233] EVs identified with fluorescence are detected in in vitro and ex vivo samples by confocal microscopy, nanoparticle tracking analysis and / or flow cytometry. Additionally, the EVs identified with fluorescence are detected in whole animals and / or organs and tissues dissected using an instrument, such as the IVIS CT spectrum (Perkin Elmer), as in H-I. Choi, et al. Experimental & Molecular Medicine. 49: e330 (2017).
[00234] [00234] Additionally, EVs can be radioidentified as previously described (Z. Varga et al., Cancer Biother Radiopharm. June 2016; 31 (5): 168 to 173).
[00235] [00235] For example, the purified EVs are radioidentified with 99m Tc-tricarbonyl complex [99mTc (CO) 3 (H2O) 3] + using a commercial kit (Isolink®; Mallinckrodt Medical BV), according to the manufacturer's instructions. Example 3: Transmission electron microscopy to view bacterial production of purified bacterial EV and EV
[00236] [00236] Transmission electron microscopy (TEM) is used to visualize bacteria as they produce purified bacterial EV or EV (S. Bin Park, et al. PLoS ONE. 6 (3): e17629 (2011). EV are prepared from batch culture of bacteria as described in Example 1. The EVs are mounted on carbon-coated copper gratings with a 300 or 400 weft size (Electron Microscopy Sciences, USA) for 2 min and washed with deionized water. EV are stained negatively with the use of 2% uranyl acetate (w / v) for 20 s to 1 min. The copper gratings are washed with sterile water and dried. The images are captured using a transmission electron microscope with acceleration voltage from 100 to 120 kV. The spotted EVs appear between 20 and 250 nm in diameter and are electrodense 10 to 50 fields in each grid are screened Example 4: Composition profiling and EV content
[00237] [00237] EVs can be characterized by any of several methods, including, but not limited to, NanoSight characterization, SDS-PAGE gel electrophoresis, Western blot, ELISA,
[00238] [00238] EV NanoSight Characterization
[00239] [00239] Nanoparticle tracking analysis (NTA) is used to characterize the size distribution of purified bacterial EVs. Purified EV preparations are performed on a NanoSight machine (Malvern Instruments) to assess the size and concentration of EV.
[00240] [00240] SDS-PAGE Gel electrophoresis gel
[00241] [00241] To identify the purified EV protein components (Example 1), the samples are run on a 12% Bolt Bis-Tris Plus 4 gel (Thermo-Fisher Scientific) using standard techniques. The samples are boiled in 1x SDS sample buffer for 10 min, cooled to 4 ° C and then centrifuged at 16,000 x g for 1 min. The samples are then run on an SDS-PAGE gel and stained using one of several standard techniques (for example, Silver staining, Coomassie Blue, Gel Code Blue) for viewing bands.
[00242] [00242] Western blot analysis
[00243] [00243] To identify and quantify specific purified EV protein components, EV proteins are separated by SDS-PAGE as described above and subjected to Western blot analysis (Cvjetkovic et al., Sci. Rep. 6, 36338 (2016 )) and are quantified by ELISA.
[00244] [00244] Liquid chromatography mass spectrometry (LC-MS / MS) and Mass spectrometry (MS)
[00245] [00245] The proteins present in EV are identified and quantified by mass spectrometry techniques.
[00246] [00246] The data are analyzed using commercially available software such as AB SCIEX Multiquant 1.2 for peak mass spectrum integration. Peaks of interest must be manually selected and compared to standards to confirm the peak identity. Quantification with suitable standards is performed to determine the number of metabolites present in the initial media, after bacterial conditioning and after the growth of tumor cells.
[00247] [00247] Dynamic light scattering (DLS)
[00248] [00248] DLS measurements, including the distribution of particles of different sizes in different EV preparations, are taken using instruments, such as DynaPro NanoStar (Wyatt Technology) and Zetasizer Nano ZS (Malvern Instruments).
[00249] [00249] Lipid levels
[00250] [00250] Lipid levels are quantified using FM4-64 (Life Technologies), by methods similar to those described by A.J. McBroom et al. J Bacteriol 188: 5,385 to 5,392 and A. Frias, et al. Microb Ecol. 59: 476 to 486 (2010). The samples are incubated with FM4-64 (3.3 µg / ml in PBS for 10 min at 37 ° C in the dark). After excitation at 515 nm, the emission at 635 nm is measured using a Spectramax M5 plate reader (Molecular Devices). Absolute concentrations are determined by comparing unknown samples with standards (such as palmitoyloleoylphosphatidylglycerol (POPG) vesicles) of known concentrations.
[00251] [00251] Total protein
[00252] [00252] Protein levels are quantified by standard assays, such as Bradford and BCA assays. Bradford assays are performed using a Quick Start Bradford 1x dye reagent (Bio-Rad), according to the manufacturer's protocols. BCA assays are performed using the Pierce BCA assay kit (Thermo-Fisher Scientific). Absolute concentrations are determined by comparison with a standard curve generated from BSA of known concentrations.
[00253] [00253] Reasons lipid: protein
[00254] [00254] The lipid: protein ratios are generated by dividing the lipid concentrations by the protein concentrations. These provide a measure of the purity of vesicles compared to free protein in each preparation.
[00255] [00255] Nucleic acid analysis
[00256] [00256] Nucleic acids are extracted from EV and quantified using a Qubit fluorometer. The size distribution is assessed using a bioanalyzer, and the material is sequenced.
[00257] [00257] Zeta potential
[00258] [00258] The zeta potential of different preparations is measured using instruments, such as the Zetasizer ZS (Malvern Instruments). Example 5: Manipulation of bacteria through stress to produce various amounts of EV and / or to vary the EV content
[00259] [00259] Stress, and in particular envelope stress, has been shown to increase the production of EV in some bacterial strains (I. MacDonald, M. Kuehn. J Bacteriol 195 (13): doi: 10/1128 / JB .02267-12). In order to vary the production of EV by bacteria, bacteria are stressed using various methods.
[00260] [00260] Bacteria can be subjected to single stressors or stressors in combination. The effects of different stressors on different bacteria are determined empirically by varying the stress condition and determining the IC50 value (the conditions necessary to inhibit cell growth by 50%). EV purification, quantification and characterization occur as detailed in Examples 1 to 4. EV production is quantified (1) in complex bacterial and EV samples by nanoparticle tracking analysis (NTA) or transmission electron microscopy (TEM) ; or (2) after purification of EV by NTA, lipid quantification or protein quantification. The EV content is assessed after purification by the methods described above.
[00261] [00261] Stress with antibiotics
[00262] [00262] Bacteria are grown under standard development conditions with the addition of subinhibitory concentrations of antibiotics. This can include 0.1 to 1 µg / ml of chloramphenicol or 0.1 to 0.3 µg / ml of gentamicin, or similar concentrations of other antibiotics (for example, ampicillin, polymyxin B). Host antimicrobial products, such as lysozyme, defensins and Reg proteins, can be used instead of antibiotics. Antimicrobial peptides produced by bacteria, including bacteriocins and microcins, can also be used.
[00263] [00263] Temperature stress
[00264] [00264] Bacteria are grown under standard development conditions, but at temperatures higher or lower than the typical temperatures for their development. Alternatively, the bacteria are grown under standard conditions and then subjected to cold shock or hot 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 to 18 ° C for cold shock or 42 ° C at 50 ° C for hot shock.
[00265] [00265] Starvation and limitation of nutrients
[00266] [00266] To induce nutritional stress, bacteria are grown under conditions in which one or more nutrients are limited. Bacteria can be subjected to nutritional stress throughout development or transferred from a rich medium to a poor medium. Some examples of media components that are limited are carbon, nitrogen, iron and sulfur. An exemplary medium is the M9 minimal medium (Sigma-Aldrich), which contains low glucose as the only carbon source. Particularly for Prevotella spp., The availability of iron is varied by changing the concentration of hemin in the media and / or by varying the type of porphyrin or other iron carrier present in the media, since it was found that cells developed in conditions of low hemin content produce higher amounts of EV (S. Stubbs et al. Letters in Applied Microbiology. 29:31 to 36 (1999). Media components are also manipulated by the addition of chelators, such as EDTA and deferoxamine.
[00267] [00267] Saturation
[00268] [00268] The bacteria are grown to saturation and incubated after the saturation point for several periods of time. Alternatively, conditioned media is used to mimic saturation environments during exponential growth. The conditioned medium is prepared by removing intact cells from the saturated cultures by centrifugation and filtration, as described in Example 1, and the conditioned medium can be further treated to concentrate or remove specific components.
[00269] [00269] Stress with salt
[00270] [00270] The bacteria are cultivated or exposed for brief periods to the medium containing NaCl, bile salts or other salts.
[00271] [00271] UV stress
[00272] [00272] UV stress is achieved by growing bacteria under a UV lamp or exposing the bacteria to UV with the use of an instrument, such as a Stratalinker (Agilent). UV can be administered over the entire growing period, in short bursts, or for a single defined period after growth.
[00273] [00273] Stress with reactive oxygen
[00274] [00274] Bacteria are grown in the presence of subinhibitory concentrations of hydrogen peroxide (250 to 1,000 µM) to induce stress in the form of reactive oxygen species. Anaerobic bacteria are either grown or exposed to concentrations of oxygen that are toxic to them.
[00275] [00275] Stress with detergent
[00276] [00276] Bacteria are grown or exposed to detergent, such as sodium dodecyl sulfate (SDS) or deoxycholate.
[00277] [00277] pH stress
[00278] [00278] Bacteria are grown or exposed for a limited time to different pH media. Example 6: Preparation of EV-free bacteria
[00279] [00279] Bacterial samples containing minimal amounts of EV are prepared. EV production is quantified (1) in complex samples of bacteria and extracellular components by NTA or TEM; or (2) after purification of EV from bacterial samples, by NTA, lipid quantification or protein quantification.
[00280] [00280] a. Centrifugation and washing: Bacterial cultures are centrifuged at 11,000 x g to separate intact cells from the supernatant (including free proteins and vesicles). The agglomerate is washed with buffer, such as PBS, and stored in a stable manner (for example, mixed with glycerol, quickly frozen and stored at -80 ° C).
[00281] [00281] b. ATF: Bacteria and EV are separated by connecting a bioreactor to an ATF system. EV-free bacteria are retained in the bioreactor and can also be separated from residual EV by centrifugation and washing, as described above.
[00282] [00282] c. Bacteria are developed under conditions that limit the production of EV. Conditions that can be varied include those listed in Example 5. Example 7: In vitro screening for EV for enhanced activation of dendritic cells
[00283] [00283] The ability of Vibrio cholerae to activate dendritic cells indirectly through epithelial cells is a non-limiting mechanism by which an immune response is stimulated in mammalian hosts (D. Chatterjee, K. Chadhuri. J Biol Chem. 288 (6 ): 4,299 to 4,309. (2013)). Since this EV activity is likely to be shared with other bacteria that stimulate pro-inflammatory cascades in vivo, in vitro methods for testing the activation of DC by bacterial EV are disclosed in this document. Briefly, PBMCs are isolated from CM heparinized venous blood by gradient centrifugation using Lymphoprep (Nycomed, Oslo, Norway) or mouse spleen or bone marrow using the human blood dendritic cell isolation kit based in a magnetic microsphere (Miltenyi Biotech, Cambridge, MA). Using anti-human CD14 mAb, 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. For maturation of dendritic cells, the culture is stimulated with 0.2 ng / ml IL-4 and
[00284] [00284] For screening for the ability of EV-activated epithelial cells to stimulate DCs, the above protocol is followed with the addition of an EV coculture of epithelial cells for 24 hours before incubation with DCs. Epithelial cells are washed after incubation with EV and are then co-cultured with DCs in the absence of EV for 24 hours before being processed as stated above. Epithelial cell lines can include Int407, HEL293, HT29, T84 and CACO2.
[00285] [00285] As an additional measure of DC activation, 100 µl of culture supernatant is removed from the wells after 24 hours of incubation of DCs with EV or epithelial cells treated with EV and are analyzed for secreted cytokines, chemokines and growth factors with the use of Luminex Magpix multiplexed. Kit (EMD Millipore, Darmstadt, Germany). Soon, the wells are pre-wetted with buffer, and 25 µl of 1x antibody-coated magnetic microspheres are added and 2x 200 µl of wash buffer are conducted in each well using the magnet. 50 µl of incubation buffer, 50 µl of diluent and 50 µl of samples are added and mixed by shaking for 2 h at room temperature in the dark. The microspheres are then washed twice with 200 µl of wash buffer. 100 µl of biotinylated 1X detector antibody are added, and the suspension is incubated for 1 h with shaking in the dark. Two 200 µl washes are then performed with the wash buffer. 100 µl of 1x SAV-RPE reagent is added to each well and incubated for 30 min at RT in the dark. Three 200 µl washes are performed, and 125 µl of wash buffer is added with 2 to 3 min of shaking. The wells are then subjected to analysis in the Luminex xMAP system.
[00286] [00286] The standards allow for careful quantification of cytokines, including GM-CSF, IFN-g, IFN-a, IFN-B, IL-1a, 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-1, MIG, MIP1a, TNFa and VEGF. These cytokines are evaluated in samples from both mice and humans. Increases in these cytokines in samples treated with bacteria indicate enhanced production of proteins and cytokines from the host. Other variations in that assay that examine the ability of specific cell types to release cytokines are assessed by acquiring these cells through classification methods and are recognized by a person of ordinary skill in the art. In addition, cytokine mRNA is also evaluated to address cytokine release in response to an EV composition. These changes in host cells stimulate an immune response in a similar way to the in vivo response in a cancer microenvironment.
[00287] [00287] This DC stimulation protocol can be repeated using combinations of purified EV and live bacterial strains to maximize the potential for immune stimulation. Example 8: In vitro screening for EV for enhanced activation of CD8 + T cell extermination when incubated with tumor cells
[00288] [00288] In vitro methods for EV screening that can activate CD8 + T cell killing of tumor cells are described. Briefly, DCs are isolated from human PBMCs or mouse spleens and incubated with single strain EV, mixtures of EV and suitable controls as described in Example 12. In addition, CD8 + T cells are obtained from human PBMCs or spleens from mouse using the mouse CD8a + T cell isolation kit based on magnetic microsphere and the human CD8a + T cell isolation kit based on magnetic microsphere (both from Miltenyi Biotech, Cambridge, MA). After incubation for 24 hours of DCs with EVs, or DCs with epithelial cells stimulated by EVs (detailed in Example 12), the EVs are removed from the cells with PBS washes, 100 µl of fresh antibiotic medium is added to each well and 200,000 T cells are added to each experimental well in the 96-well plate. The anti-CD3 antibody is added to a final concentration of 2 µg / ml. The cocultures are then allowed to incubate at 37 ° C for 96 hours under normal oxygen conditions.
[00289] [00289] 72 hours after coculture incubation, 50,000 tumor cells / well are plated per well in new 96 well plates. The mouse tumor cell lines used include B16.F10, SIY + B16.F10 and others. Human tumor cell lines are compatible with donor HLA and can include PANC-1, UNKPC960 / 961, UNKC and HELA cell lines. Upon completion of 96 hours of co-culture, 100 µl of the mixture of CD8 + T cells and DCs are transferred to wells containing tumor cells. The plates are incubated for 24 hours at 37 ° C under normal oxygen conditions. Staurospaurin is used as a negative control to explain cell death.
[00290] [00290] After this incubation, flow cytometry is used to measure tumor cell death and characterize immune cell phenotype. The tumor cells are soon stained with the viability dye. FACS analysis is used to specifically block tumor cells and measure the percentage of dead (exterminated) tumor cells. The data is also displayed as the absolute number of tumor cells killed per well. The cytotoxic CD8 + T cell phenotype can be characterized by the following methods: a) concentration of granzyme B, IFNy and TNFa supernatant in the culture supernatant as described below, b) surface expression of CD8 + T cells of activation markers, such as DC69 , CD25, CD154, PD-1, gamma / delta TCR, Foxp3, T-bet, granzyme B, c) IFNy intracellular cytokine staining, granzyme B, TNFa in CD8 + T cells. The CD4 + T cell phenotype can also be assessed by intracellular cytokine staining in addition to the concentration of cytokine in the supernatant, including INFy, TNFa, IL-12, IL-4, IL-5, IL-17, IL-10, chemokines, etc.
[00291] [00291] As an additional measure of CD8 + T cell activation, 100 µl of culture supernatant is removed from the wells after 96 hours of incubation of T cells with DCs and analyzed for secreted cytokines, chemokines and growth factors with use of Luminex Magpix multiplexed. Kit (EMD Millipore, Darmstadt, Germany). Soon, the wells are pre-wetted with buffer, and 25 µl of magnetic microspheres coated with antibody
[00292] [00292] The standards allow for careful quantification of cytokines, including GM-CSF, IFN-g, IFN-a, IFN-B IL-1a, 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-1, MIG, MIP1a, TNFα and VEGF. These cytokines are evaluated in samples from both mice and humans. Increases in these cytokines in samples treated with bacteria indicate enhanced production of proteins and cytokines from the host. Other variations in that assay that examine the ability of specific cell types to release cytokines are assessed by acquiring these cells through classification methods and are recognized by a person of ordinary skill in the art. In addition, cytokine mRNA is also evaluated to address cytokine release in response to an EV composition. These changes in host cells stimulate an immune response in a similar way to the in vivo response in a cancer microenvironment.
[00293] [00293] This CD8 + T cell stimulation protocol can be repeated using combinations of purified EV and live bacterial strains to maximize the potential for immune stimulation. Example 9: In vitro screening for EV for extermination of tumor cells enhanced by PBMCs
[00294] [00294] Methods for screening for EV for the ability to stimulate PBMCs, which in turn activate CD8 + T cells to exterminate tumor cells, are included. PBMCs are isolated from heparinized venous blood of CMs by centrifugation in a ficoll-paque gradient for mouse or human blood, or with a means of separating lymphocytic cells (Cedarlane Labs, Ontario, Canada) from mouse blood. PBMCs are incubated with single strain EV, mixtures of EV and appropriate controls as described in Example 12. In addition, CD8 + T cells are obtained from human PBMCs or mouse spleens as in Example 12. After incubation for 24 hours of PBMCs with EV, the EV are removed from the cells with PBS washes, 100 µl of fresh antibiotic medium is added to each well and 200,000 T cells are added to each experimental well in the 96-well plate. The anti-CD3 antibody is added to a final concentration of 2 µg / ml. The cocultures are then allowed to incubate at 37 ° C for 96 hours under normal oxygen conditions.
[00295] [00295] 72 hours after coculture incubation, 50,000 tumor cells / well are plated per well in new 96 well plates. The mouse tumor cell lines used include B16.F10, SIY + B16.F10 and others. Human tumor cell lines are compatible with donor HLA and can include PANC-1, UNKPC960 / 961, UNKC and HELA cell lines. After 96 hours of co-culture is completed, 100 µl of the mixture of CD8 + T cells and PBMC are transferred to wells containing tumor cells. The plates are incubated for 24 hours at 37 ° C under normal oxygen conditions. Staurospaurin is used as a negative control to explain cell death.
[00296] [00296] After this incubation, flow cytometry is used to measure tumor cell death and characterize immune cell phenotype. The tumor cells are soon stained with the viability dye. FACS analysis is used to specifically block tumor cells and measure the percentage of dead (exterminated) tumor cells. The data is also displayed as the absolute number of tumor cells killed per well. The cytotoxic CD8 + T cell phenotype can be characterized by the following methods: a) concentration of granzyme B, IFNy and TNFa supernatant in the culture supernatant as described below, b) surface expression of CD8 + T cells of activation markers, such as DC69 , CD25, CD154, PD-1, gamma / delta TCR, Foxp3, T-bet, granzyme B, c) IFNy intracellular cytokine staining, granzyme B, TNFa in CD8 + T cells. The CD4 + T cell phenotype can also be assessed by intracellular cytokine staining in addition to the concentration of cytokine in the supernatant, including INFy, TNFa, IL-12, IL-4, IL-5, IL-17, IL-10, chemokines, etc.
[00297] [00297] As an additional measure of CD8 + T cell activation, 100 µl of culture supernatant is removed from the wells after 96 hours of incubation of T cells with DCs and analyzed for secreted cytokines, chemokines and growth factors with use of Luminex Magpix multiplexed. Kit (EMD Millipore, Darmstadt, Germany). Soon, the wells are pre-wetted with buffer, and 25 µl of 1x antibody-coated magnetic microspheres are added and 2x 200 µl of wash buffer is conducted in each well using the magnet. 50 µl of incubation buffer, 50 µl of diluent and 50 µl of samples are added and mixed by shaking for 2 h at room temperature in the dark.
[00298] [00298] The standards allow for careful quantification of cytokines, including GM-CSF, IFN-g, IFN-a, IFN-B IL-1a, 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-1, MIG, MIP1a, TNFα and VEGF. These cytokines are evaluated in samples from both mice and humans. Increases in these cytokines in samples treated with bacteria indicate enhanced production of proteins and cytokines from the host. Other variations in that assay that examine the ability of specific cell types to release cytokines are assessed by acquiring these cells through classification methods and are recognized by a person of ordinary skill in the art. In addition, cytokine mRNA is also evaluated to address cytokine release in response to an EV composition. These changes in host cells stimulate an immune response in a similar way to the in vivo response in a cancer microenvironment.
[00299] [00299] This PBMC stimulation protocol can be repeated using combinations of purified EV and live bacterial strains to maximize the potential for immune stimulation. Example 10. In vitro detection of EV in antigen presenting cells
[00300] [00300] Dendritic cells in the lamina propria constantly sample live bacteria, dead bacteria and microbial products in the intestinal lumen, extending their dendrites through the intestinal epithelium, which is a way in which the EVs produced by bacteria in the intestinal lumen can directly stimulate dendritic cells. The following methods represent a way of assessing differential EV absorption by antigen presenting cells. Optionally, these methods can be applied to assess the immunomodulatory behavior of IV administered to a patient.
[00301] [00301] Dendritic cells (DCs) are isolated from human or mouse bone marrow, blood or spleens according to standard methods or kit protocols (eg Inaba K, Swiggard WJ, Steinman RM, Romani N, Schuler G, 2001. Isolation of dendritic cells, Current Protocols in Immunology, Chapter 3: Unit 3.7) and as discussed in Example 12.
[00302] [00302] To assess the entry and / or presence of EV in DCs,
[00303] [00303] To demonstrate the ability of the selected EV compositions to elicit the potent cytotoxicity of NK cells to tumor cells when incubated with the tumor cells, the following in vitro assay is used. Soon, heparinized blood mononuclear cells are obtained from healthy human donors. Optionally, an expansion step to increase the number of NK cells is performed as previously described (for example, see Somanschi et al 2011 J Vis Exp.). They are adjusted to a concentration of 1 and 6 cells / ml in RPMI-1640 medium containing 5% human serum. The PMNC cells are then identified with suitable antibodies, and the NK cells are isolated via FACS as CD3- / CD56 + cells and are ready for the subsequent cytotoxicity assay. Alternatively, NK cells are isolated using the autoMACs instrument and NK cell isolation kit following the manufacturer's instructions (Miltenyl Biotec).
[00304] [00304] NK cells are counted and plated in a 96-well format with 5,000 cells per well, and incubated with single strain EV, EV from mixtures of bacterial strains and appropriate controls as described in Example 12. As a additional negative control, this assay is performed with EV from Fusobacterium nucleatum. F. nucleatum is known to inhibit the activity of NK cells (see, for example, Gur et al 2005 Immunity 42: 1 to 12). After 5 to 24 hours of incubation of NK cells with EV, the EVs are removed from the cells with PBS washes, the NK cells are resuspended in 10 fresh media with antibiotics and added to the 96-well plates containing 50,000 target tumor cells / well. The mouse tumor cell lines used include B16.F10, SIY + B16.F10 and others. Human tumor cell lines are compatible with donor HLA and can include PANC-1, UNKPC960 / 961, UNKC and HELA cell lines. The plates are incubated for 24 hours at 37 ° C under normal oxygen conditions. Staurospaurin is used as a negative control to explain cell death.
[00305] [00305] After this incubation, flow cytometry is used to mediate the death of tumor cells. The tumor cells are soon stained with the viability dye. FACS analysis is used to specifically block tumor cells and measure the percentage of dead (exterminated) tumor cells. The data is also displayed as the absolute number of tumor cells killed per well.
[00306] [00306] This NK cell stimulation protocol can be repeated using combinations of purified EV and live bacterial strains to maximize the potential for immune stimulation. Example 12: Use of in vitro immune activation assays to predict the effectiveness of immunotherapy against cancer in vivo of EV compositions
[00307] [00307] In vitro immune activation assays identify EVs that have the capacity to stimulate dendritic cells, which in turn activate the extermination of CD8 + T cells. The work of A. Sivan, et al, Science 350 (6264): 1,084 to 1,089 (2015) suggested that the intensified extermination of tumor cells by CD8 + T cells in response to oral ingestion of Bifidobacterium spp. it is an effective cancer immunotherapy in mice. Therefore, the in vitro assays described above are used as a rapid predictive screening of a large number of EV candidates for potential immunotherapeutic activity. At
[00308] [00308] Wild type mice (for example, C57BL / 6 or BALB / c) are inoculated orally with the EV composition of interest to determine the in vivo biodistribution profile of purified EV (Example 1). EVs are identified as in Example 2 to aid downstream analysis.
[00309] [00309] Mice can receive a single dose of EV (25 to 100 µg) or several doses over a defined time course (25 to 100 µg). The mice are housed under specific pathogen-free conditions following approved protocols. Alternatively, mice can be bred and maintained under sterile germ-free conditions. Blood and stool samples can be taken at appropriate points in time.
[00310] [00310] Mice are humanely sacrificed at various points in time (i.e., hours to days) after inoculation with the EV compositions, and a complete 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 collected and used directly or frozen for testing later. The tissue samples are dissected and homogenized to prepare single cell suspensions following standard protocols known to the person skilled in the art. The number of EVs present in the sample is then quantified using flow cytometry (Example 17). Quantification can also proceed with the use of fluorescence microscopy after adequate processing of all mouse tissue (Vankelecom H., Fixation and paraffin-embedding of mouse tissues for GFP visualization, Cold Spring Harb. Protoc., 2009). Alternatively, the animals can be analyzed using live imaging according to the EV identification technique. Example 14: Administration of EV compositions with enhanced immune activation in vitro to treat mouse models with syngeneic tumor
[00311] [00311] A mouse model with cancer is generated by subcutaneously injecting a tumor cell line or tumor sample derived from the patient and allowing grafting in female C57BL / 6 mice aged 6 to 8 weeks. The methods provided in this document 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 into a 1x106 cells on the right flank (Maletzki et al
[00312] [00312] A syngeneic mouse model with spontaneous melanoma with a very high metastatic frequency is used to test the ability of bacteria to reduce tumor growth and spread of metastases. The EVs chosen from this assay are compositions that exhibit enhanced activation of subsets of immune cells and stimulate the extermination of tumor cells in vitro (Examples 12 to 16). The B16-F10 mouse melanoma cell line 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% penicillin / streptomycin at 37 ° C in a 5% CO2 atmosphere in the air. Exponentially growing tumor cells are collected by trypsinization, washed three times with 1x cold 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 to 8 weeks old and weigh approximately 16 to 20 g. For tumor development, each mouse is injected SC into the flank with 100 μl of the B16-F10 cell suspension. Mice are anesthetized by ketamine and xylazine before cell transplantation. The animals used in the experiment can be started on an antibiotic treatment by instilling a cocktail and kanamycin (0.4 mg / ml), gentamicin, (0.035 mg / ml), colistin (850 U / ml), metronidazole ( 0.215 mg / ml) and vancomycin (0.045 mg / ml) in drinking water from days 2 to 5 and an intraperitoneal injection of clindamycin (10 mg / kg) on day 7 after the tumor injection.
[00313] [00313] The size of the primary tumor on the flank is measured with a calibrator every 2 to 3 days and the tumor volume is calculated using the following formula: tumor volume = tumor width2 × tumor length × 0.5. After the primary tumor reaches approximately 100 mm3, the animals are classified into different groups based on their body weight. The mice are then randomly removed from each group and assigned to a treatment group. EV compositions are prepared as described in Example
[00314] [00314] Cancer cells can be easily visualized in the mouse model B16-F10 with melanoma due to their production of melanin. Following standard protocols, tissue samples from lymph nodes and organs in the head and chest are collected, and the presence of micro and macrometastases is analyzed using the following classification rule. An organ is classified as positive for metastasis if at least two micrometastatic lesions and one macrometastatic lesion by lymph node or organ were found. Micrometastases are detected by staining sections of lymphoid tissue embedded in paraffin with hematoxylin-eosin, following standard protocols known to those skilled in the art. The total number of metastases is correlated with the volume of the primary tumor and it was found that the tumor volume is significantly correlated with the tumor growth time and the number of macro and micrometastases in lymph nodes and visceral organs and also with the sum of all metastases observed . 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; 21 (8): 705 to 708).
[00315] [00315] Tumor tissue samples are also analyzed for tumor infiltrating lymphocytes. Cytotoxic CD8 + T cells can be isolated by FACS (see Example 17) and can then be further analyzed using custom microarrays for MHC class I to reveal their antigen specificity (see, for example, Deviren G. , et al., Detection of antigen-specific T cells on for MHC microarrays, J. Mol. Recognit., January to February 2007; 20 (1): 32 to 38). CD4 + T cells can be analyzed using microarrays customized for MHC class II.
[00316] [00316] The same experiment is also carried out with a mouse model with multiple metastases of pulmonary melanoma. The B16-BL6 mouse melanoma cell line 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 to 8 weeks old and weigh approximately 16 to 20 g. For tumor development, each mouse is injected into the tail vein with 100 μl of a suspension of 2E6 cells / ml of B16-BL6 cells. Tumor cells grafted by IV injection end up in the lungs.
[00317] [00317] The mice are humanly exterminated 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 whiten the tumor nodules due to the melanin in the B16 cells, although a small fraction of the nodules is amelanotic (that is, white). The number of tumor nodules is carefully contacted to determine the tumor burden in the mice. Typically, 200 to 250 pulmonary nodules are found in the lungs of the mice in the control group (ie, PBS gavage).
[00318] [00318] The percentage of the tumor burden is calculated for the three treatment groups. This measure is defined as the average number of pulmonary nodules on the lung surfaces of mice that belong to a treatment group divided by the average number of pulmonary nodules on the lung surfaces of the mice in the control group.
[00319] [00319] Determination of metabolic content with 1H NMR
[00320] [00320] Biological triplicates of media and samples of media spent after bacterial conditioning and after tumor development are deproteinized using Sartorius Centrisart I filters (10 kDa cut). Before use, the filter is washed twice by centrifuging water to remove glycerol and a small volume (20 µl) of 20.2 mM trimethylsilyl-2,2,3,3-tetradeuteropropionic acid (TSP, sodium salt) in D2O is added to 700 μl of the ultrafiltrate, providing a chemical shift reference (0.00 ppm) and a deuterium blocking signal. 650 µl of the sample is placed in a 5 mm NMR tube. 1H NMR (500 MHz) single pulse spectra are obtained on a Bruker DMX-500 spectrometer or comparable instrument as previously described (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 to 278). The phase and baseline are corrected manually. All spectra are sized for TSP, and metabolite signals are adjusted semiautomatically with a Lorentzian line format. The metabolite concentrations in the spent medium are calculated in relation to the known concentration in the standard medium and correspondingly expressed in units of mM. The concentration of a specific metabolite was calculated by the peak area corresponding to the area of the 1.04 ppm valine doublet or a suitable standard.
[00321] [00321] Determination of metabolic content with LCMS
[00322] [00322] The metabolic content of a sample is ascertained using liquid chromatography techniques combined with mass spectrometry. There are a variety of techniques for determining the metabolic content of various samples, and they are known to the person skilled in the art, which involve solvent extraction, chromatographic separation and a variety of ionization techniques coupled with mass determination (Roberts et al 2012 Targeted Metabolomics. Curr Protoc Mol Biol. 30: 1 to 24; Dettmer et al. 2007, Mass spectrometry-based metabolomics. Mass Spectrom Rev. 26 (1): 51 to 78). As a non-limiting example, an LC-MS system includes a 4000 QTRAP triple quadrupole mass spectrometer (AB SCIEX) combined with an 1100 Series pump (Agilent) and an HTS PAL autosampler (Leap Technologies). Samples of media or other complex metabolic mixtures (~ 10 µl) are extracted using nine volumes of acetonitrile / methanol / formic acid 74.9: 24.9: 0.2 (v / v / v) containing internal standards identified with stable isotopes (valine-d8, Isotec; and phenylalanine-d8, Cambridge Isotope Laboratories). The patterns can be adjusted or modified, depending on the metabolites of interest. The samples are centrifuged (10 min, 9,000 g, 4 ° C), and the supernatants (10 µl) are subjected to LCMS by injecting the solution into the HILIC column (150 × 2.1 mm, 3 µm particle size) . The column is eluted by flowing a 5% mobile phase [10 mM ammonium formate, 0.1% formic acid in water] for 1 min at a rate of 250 ul / min, followed by a linear gradient over 10 min for a 40% mobile phase solution [acetonitrile with 0.1% formic acid]. The ionic spray voltage is set to 4.5 kV, and the source temperature is 450 ° C.
[00323] [00323] The data are analyzed using commercially available software, such as AB SCIEX Multiquant 1.2 for peak mass spectrum integration. Peaks of interest are manually selected and compared to standards to confirm the peak identity. Quantification with appropriate standards is performed to determine the amount of metabolites present in the initial media, after bacterial conditioning and after the growth of tumor cells.
[00324] [00324] Tumor biopsy and blood samples are subjected to metabolic analysis using LCMS techniques described in this document. Differential levels of amino acids, sugars, lactate and other metabolites, between test groups demonstrate the ability of the microbial composition to disrupt the metabolic state of the tumor.
[00325] [00325] RNA sequence to determine mechanism of action
[00326] [00326] Dendritic cells are purified from tumors, Peyers patches and mesenteric lymph nodes as described in Example 12. The RNAseq analysis is performed and analyzed according to standard techniques known to those skilled in the art (Z. Hou. Scientific Reports.5 (9570): doi: 10.1038 / srep09570 (2015)). In the analysis, specific attention is directed to genes in the innate inflammatory pathways, including
[00327] [00327] To determine the efficacy of EV in mouse models with a syngeneic tumor, colorectal cancer (CT-26) was used. Briefly, CT-26 tumor cells (CAT number CRL-2638) were cultured in vitro as a monolayer in RPMI-1640 or DMEM supplemented with 10% heat-inactivated fetal serum at 37 ° C in a 5% CO2 atmosphere up in the air. Exponential growth cells were collected and counted before tumor inoculation. Female BALB / c mice from 6 to 8 weeks of age were used for this experiment. For tumor development, each mouse was injected subcutaneously into one or both posterior flanks with 5x105 tumor cells CT-26 in 0.1 ml of 1x PBS. Some mice may receive pretreatment with antibiotics. Tumor size and mouse weight were monitored at least three times weekly on non-consecutive days.
[00328] [00328] EVs were tested for effectiveness in the mouse model with tumor, alone or in combination with whole bacterial cells and with or without anti-PD-1 or anti-PD-L1. EVs, bacterial and / or anti-PD-1 or anti-PD-L1 cells were administered at varying points in time and in varying doses. For example, on the 10th day after the tumor injection, or after the tumor volume reached 100 mm3, the mice were treated with EV alone or in combination with anti-PD-1 or anti-PD-L1. The dosage amount was administered according to Table 3. The Blautia massiliensis group showed inhibition of tumor growth comparable to that observed in the anti-PD-1 group (Figures 1 and 2). Table 3. Dosage quantity. Treatment Group Dose, route, schedule 1 (n = 10) Vehicle IV (PBS) N / A, IV, Q3Dx4 2 (n = 10) Anti-PD-1 200 ug, IP, Q4Dx3 3 (n = 10) EV ( IV) Blautia 5 ug, IV, Q3Dx4 massiliensis
[00329] [00329] For example, some mice are injected intravenously with IV at 15, 20 or 15 µg / mouse. Other mice may receive 25, 50 or 100 mg of EV per mouse. Although some mice receive IV via i.v. injection, other mice may receive IV via intraperitoneal injection (i.p.), subcutaneous injection (s.c.), nasal administration, oral gavage or other means of administration. Some mice may receive EV every day (for example, starting on day 1), while others may receive EV at alternate intervals (for example, every other day or once every three days). Additional groups of mice may receive some ratio between bacterial cells and EV. Bacterial cells can be alive, dead or weakened. Bacterial cells can be collected fresh (or frozen) and administered, or they can be irradiated or exterminated by heat before administration. For example, some groups of mice can receive between 1x104 and 5x109 bacterial cells in a separate administration or mixed with the administration of EV. As with EV, the administration of bacterial cells can be varied in relation to the route of administration, dose and schedule. This may include oral gavage, i.v. injection, i.p. or nasal injection. Some groups of mice are also injected with effective doses of checkpoint inhibitor. For example, mice receive 100 µg of anti-PD-L1 mAB (clone 10f.9g2, BioXCell) or another anti-PD-1 or anti-PD-L1 mAB in 100 µl of PBS, and some mice receive vehicle and / or other suitable control (for example, control antibody). The mice are injected with mABs 3, 6 and 9 days after the initial injection. To assess whether checkpoint inhibition and EV immunotherapy have an additive antitumor effect, control mice receiving anti-PD-1 or anti-PD-L1 mABs are included in the standard control panel. Primary (tumor size) and secondary (tumor infiltrating lymphocyte and cytokine analysis) endpoints are assessed, and some groups of mice are again challenged with a subsequent tumor cell inoculation to assess the effect of treatment on memory response. Example 16: EV in a mouse model with experimental autoimmune encephalomyelitis (EAE)
[00330] [00330] EAE is a well studied animal model with multiple sclerosis, as reviewed by Constantinescu et al. (Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br J Pharmacol. October 2011; 164 (4): 1,079 to 1,106). It can be induced in a variety of mouse and rat strains with the use of different peptides associated with myelin, by the adoptive transfer of activated encephalitogenic T cells, or the use of transgenic TCR mice susceptible to EAE, as discussed in Mangalam et al . (Two discreet subsets of CD8 + T cells modulate PLP91-110 induced experimental autoimmune encephalomyelitis in HLA-DR3 transgenic mice. J Autoimmun. June 2012; 38 (4): 344 to 353).
[00331] [00331] EVs are tested for effectiveness in the EAE rodent model, alone or in combination with whole bacterial cells, with or without the addition of other anti-inflammatory treatments. For example, female C57Bl / 6 mice 6 to 8 weeks old are obtained from Taconic (Germantown, NY). Groups of mice are administered with two subcutaneous injections (sc) at two sites on the back (upper and lower) of 0.1 ml of myelin oligodentrocyte glycoprotein 35 to 55 (MOG35-55; 100 µg per injection; 200 µg per mouse (total 0.2 ml per mouse)), emulsified in complete Freund's adjuvant (CFA; 2 to 5 mg of mycobacterium tuberculosis H37Ra exterminated / ml of emulsion). Approximately 1 to 2 hours after the above, the mice are injected intraperitoneally (i.p.) with 200 ng of Pertussis toxin (PTx) in 0.1 ml of PBS (2 µg / ml). An additional IP injection of PTx is administered on day 2. Alternatively, an adequate amount of an alternative myelin peptide (for example, proteolipid protein (PLP)) is used to induce EAE. Some animals represent virgin controls. The severity of EAE is assessed, and a disability score is assigned daily starting on the 4th according to methods known in the art (Mangalam et al. 2012).
[00332] [00332] Treatment with EV starts at some point, either close to the time of immunization or after immunization against EAE. For example, EVs can be administered at the same time as immunization (day 1), or they can be administered after the first signs of disability (eg, soft tail), or during severe EAE. EVs are administered in varying doses and at defined intervals. For example, some mice are injected intravenously with IV at 15, 20 or 15 µg / mouse. Other mice may receive 25, 50 or 100 mg of EV per mouse. Although some mice receive IV via i.v. injection, other mice may receive IV via intraperitoneal injection (i.p.), subcutaneous injection (s.c.), nasal administration, oral gavage or other means of administration. Some mice may receive EV every day (for example, starting on day 1), while others may receive EV at alternate intervals (for example, every other day or once every three days). Additional groups of mice may receive some ratio between bacterial cells and EV. Bacterial cells can be alive, dead or weakened. Bacterial cells can be collected fresh (or frozen) and administered, or they can be irradiated or exterminated by heat before administration.
[00333] [00333] For example, some groups of mice can receive between 1x104 and 5x109 bacterial cells in a separate administration or mixed with the administration of EV. As with EV, the administration of bacterial cells can be varied in relation to the route of administration, dose and schedule. This can include oral gavage, i.v. injection, i.p. injection, subcutaneous injection (s.c.) or nasal administration.
[00334] [00334] Some groups of mice can be treated with additional anti-inflammatory agent (or additional anti-inflammatory agents) or therapeutic for EAE (or therapeutic for EAE) (for example, anti-CD154, blocking of TNF family members, vitamin D or other treatment), and / or an appropriate control (e.g., vehicle or antibody control) at various points in time and in effective doses.
[00335] [00335] In addition, some mice are treated with antibiotics before treatment. For example, vancomycin (0.5 g / l), ampicillin (1.0 g / l), gentamicin (1.0 g / l) and amphotericin B (0.2 g / l) are added to drinking water, and antibiotic treatment is stopped at the time of treatment or a few days before treatment.
[00336] [00336] At various points in time, mice are sacrificed, and sites of inflammation (eg, brain and spinal cord), lymph nodes or other tissues can be removed by histological, cytokine and / or flow cytometric analysis ex vivo using methods known in the art. For example, tissues are dissociated using dissociation enzymes according to the manufacturer's instructions. The cells are stained for flow cytometric analysis using techniques known in the art. Staining antibodies can include anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4 and anti-CD103. Other markers that can be analyzed include immune cell pan marker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzima B, CD69, PD-1, CTLA-4) and macrophage / myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4 / 80). In addition to immunophenotyping, serum cytokines are analyzed, including, but not limited to, TNFα, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2 , IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES and MCP-1. The cytokine analysis can be performed on immune cells obtained from lymph nodes or other tissue, and / or on immune cells infiltrated into the purified CD45 + central nervous system (CNS) obtained ex vivo. Finally, immunohistochemistry is performed on several sections of tissue to measure T cells, macrophages, dendritic cells and protein expression in the checkpoint molecule.
[00337] [00337] In order to examine the impact and longevity of protection against disease, instead of being sacrificed, some mice can be re-challenged with a disease activator (eg, activated encephalitogenic T cells or reinjection of EAE-inducing peptides ). The mice are analyzed for susceptibility to disease and severity of EAE after the new challenge. Example 17: EV in a mouse model with collagen-induced arthritis (ASD)
[00338] [00338] Collagen-induced arthritis (ASD) is an animal model commonly used to study rheumatoid arthritis (RA), as described by Caplazi et al. (Mouse models of rheumatoid arthritis. Veterinary Pathology. September 1, 2015. 52 (5): 819 to 826) (see also Brand et al. Collagen-induced arthritis. Nature Protocols. 2007. 2: 1,269 to 1,275; Pietrosimone et al. Collagen-induced arthritis: a model for murine autoimmune arthritis. Bio Protoc. October 20, 2015; 5 (20): e1626).
[00339] [00339] Among other versions of the CIA rodent model, one model involves immunizing HLA-DQ8 Tg mice with chicken type II collagen as described by Taneja et al. (J. Immunology. 2007. 56: 69 to 78; see also Taneja et al. J. Immunology 2008. 181: 2,869 to 2,877; and Taneja et al. Arthritis Rheum.,
[00340] [00340] The mice are immunized for CIA induction and separated into various treatment groups. EVs are tested for their effectiveness in CIA, alone or in combination with whole bacterial cells, with or without the addition of other anti-inflammatory treatments.
[00341] [00341] Treatment with EV is started near the moment of immunization with collagen or after immunization. For example, in some groups, EVs can be given at the same time as immunization (day 1), or EVs can be given after the first signs of illness, or after the onset of severe symptoms. EVs are administered in varying doses and at defined intervals.
[00342] [00342] For example, some mice are injected intravenously with IV at 15, 20 or 15 µg / mouse. Other mice may receive 25, 50 or 100 mg of EV per mouse. Although some mice receive IV via i.v. injection, other groups of mice may receive IV via intraperitoneal injection (i.p.), subcutaneous injection (s.c.), nasal administration, oral gavage or other means of administration. Some mice may receive EV every day (for example, starting on day 1), while others may receive EV at alternate intervals (for example, every other day or once every three days). Additional groups of mice may receive some ratio between bacterial cells and EV. Bacterial cells can be alive, dead or weakened. Bacterial cells can be collected fresh (or frozen) and administered, or they can be irradiated or exterminated by heat before administration.
[00343] [00343] For example, some groups of mice can receive between 1x104 and 5x109 bacterial cells in a separate administration or mixed with the administration of EV. As with EV, the administration of bacterial cells can be varied in relation to the route of administration, dose and schedule. This may include oral gavage, i.v. injection, i.p. injection, subcutaneous injection (s.c.), intradermal injection (i.d.) or nasal administration.
[00344] [00344] Some groups of mice can be treated with additional anti-inflammatory agent (or additional anti-inflammatory agents) or therapeutic for CIA (or therapeutic for CIA) (for example, anti-CD154, blocking of TNF family members, vitamin D or other treatment), and / or an appropriate control (e.g., vehicle or antibody control) at various points in time and in effective doses.
[00345] [00345] In addition, some mice are treated with antibiotics before treatment. For example, vancomycin (0.5 g / l), ampicillin (1.0 g / l), gentamicin (1.0 g / l) and amphotericin B (0.2 g / l) are added to drinking water, and antibiotic treatment is stopped at the time of treatment or a few days before treatment. Some immunized mice are treated without receiving antibiotics.
[00346] [00346] At various points in time, serum samples are obtained to assess levels of anti-row and anti-mouse CII IgG antibodies using a standard ELISA (Batsalova et al. Comparative analysis of collagen type II-specific immune responses during development of collagen -induced arthritis in two B10 mouse strains. Arthritis Res Ther. 2012. 14 (6): R237). In addition, some mice are sacrificed, and the sites of inflammation (eg, synovium), lymph nodes or other tissues can be removed by histological, cytokine and / or flow cytometric analysis ex vivo using the methods known in the art. The synovium and synovial fluid are analyzed for the infiltration of plasma cells and the presence of antibodies using techniques known in the art. In addition, tissues are dissociated using dissociation enzymes according to the manufacturer's instructions to examine the profiles of cellular infiltrates. The cells are stained for flow cytometric analysis using techniques known in the art. Staining antibodies can include anti-CD11c
[00347] [00347] In order to examine the impact and longevity of protection against disease, instead of being sacrificed, some mice can be re-challenged with a disease activator (for example, reinjection activated with CIA-inducing peptides). The mice are analyzed for susceptibility to disease and severity of ASD after the new challenge. Example 18: EV in a mouse model with colitis
[00348] [00348] Colitis induced by sodium dextran sulfate (DSS) is a well-studied animal model of colitis, as reviewed by Randhawa et al. (A review on chemical-induced inflammmatory bowel disease models in rodents. Korean J Physiol Pharmacol. 2014. 18 (4): 279 to 288; see also Chassaing et al. Dextran sulfate sodium (DSS) -induced colitis in mice. Curr Protoc Immunol. February 4, 2014; 104: Unit 15.25).
[00349] [00349] EVs are tested for effectiveness in a mouse model with DSS-induced colitis, alone or in combination with whole bacterial cells, with or without the addition of other anti-inflammatory agents.
[00350] [00350] Groups of mice are treated with DSS to induce colitis as known in the art (Randhawa et al. 2014; Chassaing et al. 2014; see also Kim et al. Investigating intestinal inflammation in DSS-induced model of IBD. J Vis Exp. 2012. 60: 3,678). For example, 6 to 8 week old male C57Bl / 6 mice are obtained from Charles River Labs, Taconic, or another supplier. Colitis is induced by adding 3% DSS (MP Biomedicals, Cat. Number 0260110) to drinking water. Some mice do not receive DSS in drinking water and represent virgin controls. Some mice receive water for five (5) days. Some mice may receive DSS for a period shorter or longer than five (5) days. Mice are monitored and scored using a disability activity index known in the art based on weight loss (eg, no weight loss (score 0); 1 to 5% weight loss (score 1); 5 to 10% weight loss (score 2)); stool consistency (eg, normal (score 0); loose stools (score 2); diarrhea (score 4)); and bleeding (eg, no blood (score 0), positive blood culture (score 1); positive blood culture and visual cluster bleeding (score 2); bleeding around the anus, gross bleeding (score 4).
[00351] [00351] EV treatment starts sometime, on day 1 of DSS administration or sometime after that. For example, EVs can be administered at the same time as DSS initiation (day 1), or they can be administered after the first signs of the disease (for example, weight loss or diarrhea),
[00352] [00352] EVs are administered in varying doses and at defined intervals. For example, some mice are injected intravenously with IV at 15, 20 or 15 µg / mouse. Other mice may receive 25, 50 or 100 mg of EV per mouse. Although some mice receive IV via i.v. injection, other mice may receive IV via intraperitoneal injection (i.p.), subcutaneous injection (s.c.), nasal administration, oral gavage or other means of administration. Some mice may receive EV every day (for example, starting on day 1), while others may receive EV at alternate intervals (for example, every other day or once every three days). Additional groups of mice may receive some ratio between bacterial cells and EV. Bacterial cells can be alive, dead or weakened. Bacterial cells can be collected fresh (or frozen) and administered, or they can be irradiated or exterminated by heat before administration.
[00353] [00353] For example, some groups of mice can receive between 1x104 and 5x109 bacterial cells in a separate administration or mixed with the administration of EV. As with EV, the administration of bacterial cells can be varied in relation to the route of administration, dose and schedule. This may include oral gavage, i.v. injection, i.p. or nasal administration.
[00354] [00354] Some groups of mice can be treated with additional anti-inflammatory agent (or additional anti-inflammatory agents) (for example, anti-CD154, blocking TNF family members or other treatment), and / or adequate control ( control vehicle or antibody) at various points in time and in effective doses.
[00355] [00355] In addition, some mice are treated with antibiotics before treatment. For example, vancomycin (0.5 g / l), ampicillin (1.0 g / l), gentamicin (1.0 g / l) and amphotericin B (0.2 g / l) are added to drinking water, and antibiotic treatment is stopped at the time of treatment or a few days before treatment. Some mice receive DSS without receiving antibiotics in advance.
[00356] [00356] At various points in time, mice are subjected to video endoscopy using an endoscope for small animals (Karl Storz Endoskipe, Germany) under anesthesia with isoflurane. Still images and video are recorded to assess the extent of colitis and the response to treatment. Colitis is scored using criteria known in the art. Fecal material is collected for study.
[00357] [00357] At various points in time, mice are sacrificed, and the colon, small intestine, spleen and lymph nodes (eg, mesenteric lymph nodes) are collected. Additionally, blood is collected in serum separation tubes. Tissue damage is examined through histological studies that evaluate, without limitation, crypt architecture, degree of infiltration by inflammatory cells and depletion of goblet cells.
[00358] [00358] The gastrointestinal (GI) tract, lymph nodes and / or other tissues can be removed by histological, cytokine and / or flow cytometric analysis ex vivo using methods known in the art. For example, tissues are collected and can be dissociated using dissociation enzymes according to the manufacturer's instructions. The cells are stained for flow cytometric analysis using techniques known in the art. Staining antibodies can include anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4 and anti-CD103. Other markers that can be analyzed include immune cell pan marker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzima B, CD69, PD-1, CTLA-4) and macrophage / myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4 / 80). In addition to immunophenotyping, serum cytokines are analyzed, including, but not limited to, TNFα, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2 , IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES and MCP-1. Cytokine analysis can be performed on immunological cells obtained from lymph nodes or other tissue, and / or on immunological cells infiltrated into the purified GI CD45 + tract obtained ex vivo. Finally, immunohistochemistry is performed on several sections of tissue to measure T cells, macrophages, dendritic cells and protein expression in the checkpoint molecule.
[00359] [00359] In order to examine the impact and longevity of protection against disease, instead of being sacrificed, some mice can be provoked again with a disease activator. The mice are analyzed for susceptibility to the severity of colitis after the new challenge. Example 19: EV in a delayed-type hypersensitivity (DTH) mouse model
[00360] [00360] Delayed type hypersensitivity (DTH) is an animal model of atopic dermatitis (or allergic contact dermatitis), as reviewed by Petersen et al. (In vivo pharmacological disease models for psoriasis and atopic dermatitis in drug discovery. Basic & Clinical Pharm & Toxicology. 2006. 99 (2): 104 to 115; see also Irving C. Allen (edition) Mouse Models of Innate Immunity: Methods and Protocols, Methods in Molecular Biology, 2013. volume
[00361] [00361] In general, mice are prepared with an antigen administered in the context of an adjuvant (for example, Freund's complete adjuvant) in order to induce a secondary immune (or memory) response measured by swelling and antigen-specific antibody titer .
[00362] [00362] EVs are tested for effectiveness in the mouse model with DTH, alone or in combination with whole bacterial cells, with or without the addition of other anti-inflammatory treatments. For example, C57Bl / 6 mice 6 to 8 weeks old are obtained from Taconic (Germantown, NY) or another supplier. Groups of mice are administered with four subcutaneous injections (s.c.) at four sites on the back (upper and lower) of antigen (eg ovalbumin (OVA)) at an effective dose (50 ul total volume per site). For a DTH response, the animals were injected intradermally (i.d.) into the ears under anesthesia with ketamine / xylazine (approximately 50 mg / kg and 5 mg / kg, respectively). Some mice serve as control animals. Some groups of mice are challenged with 10 ul per ear (vehicle control (0.01% DMSO in saline) in the left ear and antigen (21.2 ug (12 nmol) in the right ear) on day 8. To measure ear inflammation, ear thickness of manually contained animals is measured using a Mitutoyo micrometer. Ear thickness is measured before intradermal challenge as the baseline level for each individual animal. ear is measured twice after intradermal challenge, in approximately 24 hours and 48 hours (ie, days 9 and 10).
[00363] [00363] Treatment with EV starts at some point, close to the time of preparation or close to the time of provoking DTH. For example, EVs can be administered at the same time as subcutaneous injections (day 0), or they can be administered before or after intradermal injection. EVs are administered in varying doses and at defined intervals. For example, some mice are injected intravenously with IV at 15, 20 or 15 µg / mouse. Other mice may receive 25, 50 or 100 mg of EV per mouse. Although some mice receive IV via iv injection, other mice may receive IV via intraperitoneal injection (ip), subcutaneous injection (sc), nasal administration, oral gavage, topical administration, intradermal injection (id) or other means of administration . Some mice may receive EV every day (for example, starting on day 0), while others may receive EV at alternate intervals (for example, every other day or once every three days). Additional groups of mice may receive some ratio between bacterial cells and EV. Bacterial cells can be alive, dead or weakened. Bacterial cells can be collected fresh (or frozen) and administered, or they can be irradiated or exterminated by heat before administration.
[00364] [00364] For example, some groups of mice can receive between 1x104 and 5x109 bacterial cells in a separate administration or mixed with the administration of EV. As with EV, the administration of bacterial cells can be varied in relation to the route of administration, dose and schedule. This may include oral gavage, i.v. injection, i.p. injection, i.d. injection, topical administration or nasal administration.
[00365] [00365] The mice were injected with KLH and CFA i.d at 4 sites along the back (50 µg per KLH mouse prepared in a 1: 1 ratio with CFA in a total volume of 50 µl per site). The mice were dosed for 9 days as follows; 1) oral administration of anaerobic PBS (vehicle); 2) oral administration of 10 mg of Prevotella histicola; 3) oral administration of 100 µg of IV derived from P. histicola; 4) i.p. of PBS; 5) i.p. dexamethasone (positive control); and 6) i.p. 10 µg of EV derived from Prevotella histicola. For EV, total protein was measured using Bio-rad assays (Cat number 5000205) performed according to the manufacturer's instructions. At 24 and 48 hours after challenge with 10 μg of KLH (10 μl by volume), groups receiving Prevotella histicola (live cells) or EV derived from Prevotella histicola, in both oral and ip administration groups, exhibited less inflammation than vehicle groups (Figures 3A and 3B). A dose-dependent DTH response after i.p. of EV derived from Prevotella histicola in 10 µg, 3 µg, 1 µg and 0.1 µg was observed in the reduction of antigen-specific ear swelling (ear thickness) 48 hours after antigen challenge in a mouse model with hypersensitivity of the delayed type based on KLH (Figure 3C).
[00366] [00366] The mice were injected with KLH and CFA i.d at 4 sites along the back (50 µg per KLH mouse prepared in a 1: 1 ratio with CFA in a total volume of 50 µl per site). The mice were dosed for 9 days as follows; 1)
[00367] [00367] The test formulations were prepared for model with delayed type hypersensitivity based on KLH. The DTH model provides a mechanism in vivo to study the cell-mediated immune response and the resulting inflammation after exposure to a specific antigen to which the mice have been sensitized. Several variations of the DTH model have been used and are well known in the art (Irving C. Allen (edition). Mouse Models of Innate Immunity: Methods and Protocols, Methods in Molecular Biology. Volume 1.031, DOI 10.1007 / 978-1-62703- 481- 4_13, Springer Science + Business Media, LLC 2013). For example, the Keyhole Limpet hemocyanin emulsion (KLH) and Freund's complete adjuvant (CFA) is freshly prepared on the day of immunization (day 0). For this purpose, 8 mg of powdered KLH are weighed and carefully resuspended in 16 ml of saline. An emulsion is prepared by mixing the KLH / saline with an equal volume of CFA solution (for example, 10 ml of KLH / saline + 10 ml of CFA solution) using syringes and a luer lock connector. KLH and CFA are vigorously mixed for several minutes to form a white colored emulsion in order to obtain maximum stability. A drop test is performed to verify that a homogeneous emulsion has been obtained, mixing is continued until an intact drop remains visible in the water.
[00368] [00368] On day 0, female C57Bl / 6J mice, approximately 7 weeks old, were prepared with KLH antigen in CFA by subcutaneous immunization (4 sites, 50 μl per site).
[00369] [00369] Dexamethasone, a corticosteroid, is a known anti-inflammatory that improves reactions to DTH in mice, and serves as a positive control to suppress inflammation in this model (Taube and Carlsten, Action of dexamethasone in the suppression of delayed-type hypersensitivity in reconstituted SCID mice.
[00370] [00370] On day 8, the mice were challenged intradermally (i.d.) with 10 μg of KLH in saline solution (in a volume of 10 μl) in the left ear. Inflammatory responses were measured using methods known in the art. Ear pin thickness was measured 24 hours after antigen challenge (Figure 11). As determined by ear thickness, strains of Veillonella were effective in suppressing inflammation compared to mice that received vehicle alone (comparable to treatment with dexamethasone).
[00371] [00371] The efficacy of Veillonella strains can be further studied with the use of varying timing and varying doses. For example, treatment with a bacterial composition of Veillonella can be started at some point, near the time of preparation or near the time of provocation with DTH. For example, Veillonella (1x109 CFU per mouse per day) can be administered at the same time as subcutaneous injections (day 0), or administered before or after intradermal injection. Veillonella strains can be administered in varying doses and at defined intervals, and in various combinations. For example, some mice are injected intravenously with Veillonella in a range between 1x104 and 5x109 bacterial cells per mouse. Some mice receive a mixture of strains. Although some mice receive a Veillonella through iv injection, other mice can receive a Veillonella through intraperitoneal injection (ip), subcutaneous injection (sc), nasal administration, oral gavage, topical administration, intradermal injection (id) or other means administration. Some mice may receive a Veillonella every day (for example, starting on day 0), while others may receive a Veillonella at alternate intervals (for example, every other day or once every three days). Bacterial cells can be alive, dead or weakened. Bacterial cells can be collected fresh (or frozen) and administered, or they can be irradiated or exterminated by heat before administration.
[00372] [00372] Some groups of mice can be treated with anti-inflammatory agent (or anti-inflammatory agents) (for example, anti-CD154, blocking TNF family members or other treatment), and / or adequate control (for example , vehicle or control antibody) at various points in time and in effective doses.
[00373] [00373] In addition, some mice are treated with antibiotics before treatment. For example, vancomycin (0.5 g / l), ampicillin (1.0 g / l), gentamicin (1.0 g / l) and amphotericin B (0.2 g / l) are added to drinking water, and antibiotic treatment is stopped at the time of treatment or a few days before treatment. Some immunized mice are treated without receiving antibiotics.
[00374] [00374] At various points in time, serum samples are extracted. Other groups of mice are sacrificed, and lymph nodes, spleen, mesenteric lymph nodes (MLN), small intestine, colon and other tissues can be removed for histological, histological, cytokine and / or cytometric analysis of ex vivo flow using the methods known in the art. Some mice are exsanguinated from the orbital plexus under anesthesia with O2 / CO2, and ELISA assays are performed.
[00375] [00375] Tissues can be dissociated with the use of dissociation enzymes according to the manufacturer's instructions. The cells are stained for flow cytometric analysis using techniques known in the art. Staining antibodies can include anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4 and anti-CD103. Other markers that can be analyzed include immune cell pan marker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzima B, CD69, PD-1, CTLA-4) and macrophage / myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4 / 80). In addition to immunophenotyping, serum cytokines are analyzed, including, but not limited to, TNFα, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2 , IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES and MCP-1. Cytokine analysis can be performed on immune cells obtained from lymph nodes or other tissue and / or on purified CD45 + infiltrated immune cells obtained ex vivo. Finally, immunohistochemistry is performed on several sections of tissue to measure T cells, macrophages, dendritic cells and protein expression in the checkpoint molecule.
[00376] [00376] The mice were prepared and confronted with KLH as described above and, after measuring the ear swelling in 48 hours, the mice were sacrificed.
[00377] [00377] The ears were removed from the sacrificed animals and placed in a cold EDTA-free protease inhibitor cocktail (Roche). The ears were homogenized with the use of sphere rupture and supernatants analyzed for Il-1β by Luminex kit (EMD Millipore) as per manufacturer's instructions. Mice that were treated with 10 µg of EV of P. histicola (i.p.) showed levels of Il-1β comparable to those observed in the Dexamethasone group (positive control). (Figure 3D). P. histicola-derived EVs are able to suppress pro-inflammatory cytokines.
[00378] [00378] In addition, cervical lymph nodes were dissociated through a cellular marker, washed and stained for FoxP3
[00379] [00379] In order to examine the impact and longevity of DTH protection, instead of being sacrificed, some mice can be challenged again with the contestant antigen (for example, OVA). The mice are analyzed for susceptibility to DTH and severity of response. Example 20: EV in a Type 1 Diabetes (T1D) mouse model
[00380] [00380] Type 1 diabetes (T1D) is an autoimmune disease in which the immune system targets the islets of Langerhans in the pancreas, thereby destroying the body's ability to produce insulin.
[00381] [00381] These are several models of T1D animal models, as analyzed by Belle et al. (Mouse models for type 1 diabetes. Drug Discov Today Dis Models. 2009; 6 (2): 41 to 45; see also Aileen JF King. The use of animal models in diabetes research. Br J Pharmacol. June 2012; 166 (3): 877 to 894. These are models for chemically induced T1D, pathogen-induced T1D, as well as models in which mice spontaneously develop T1D.
[00382] [00382] EV are tested for their effectiveness in a T1D mouse model, alone or in combination with all bacterial cells, with or without the addition of other anti-cancer treatments.
[00383] [00383] Depending on the T1D induction method and / or whether T1D development is spontaneous, treatment with EV is initiated at some point, either around the induction time or after induction, or before onset (or upon initiation) of spontaneous occurrence of T1D. EVs are administered in varying doses and at defined intervals. For example, some mice are injected intravenously with IV at 15, 20 or 15 µg / mouse. Other mice may receive 25, 50 or 100 mg of EV per mouse. Although some mice receive IV via i.v. injection, other mice may receive IV via intraperitoneal injection (i.p.), subcutaneous injection (s.c.), nasal administration, oral gavage or other means of administration. Some mice may receive EV every day, while others may receive EV at alternate intervals (for example, every other day, or once every three days). Additional groups of mice may receive some ratio between bacterial cells and EV. Bacterial cells can be alive, dead or weakened. Bacterial cells can be collected fresh (or frozen) and administered, or they can be irradiated or exterminated by heat before administration.
[00384] [00384] For example, some groups of mice can receive between 1x104 and 5x109 bacterial cells in a separate administration or mixed with the administration of EV. As with EV, the administration of bacterial cells can be varied in relation to the route of administration, dose and schedule. This may include oral gavage, i.v. injection, i.p. or nasal administration.
[00385] [00385] Some groups of mice can be treated with additional treatments and / or an appropriate control (for example, vehicle or antibody control) at various points in time and in effective doses.
[00386] [00386] In addition, some mice are treated with antibiotics before treatment. For example, vancomycin (0.5 g / l), ampicillin (1.0 g / l), gentamicin (1.0 g / l) and amphotericin B (0.2 g / l) are added to drinking water, and antibiotic treatment is stopped at the time of treatment or a few days before treatment. Some immunized mice are treated without receiving antibiotics.
[00387] [00387] Blood glucose is monitored every two weeks before the start of the experiment. At various points in time, subsequently, fasting blood glucose is measured. At various points in time, mice are sacrificed and sites of the pancreas, lymph nodes, or other tissues can be removed for ex vivo analysis of histology, cytokine and / or flow cytometry using methods known in the art. For example, tissues are dissociated using dissociation enzymes according to the manufacturer's instructions. The cells are stained for flow cytometric analysis using techniques known in the art. Staining antibodies can include anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4 and anti-CD103. Other markers that can be analyzed include immune cell pan marker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzima B, CD69, PD-1, CTLA-4) and macrophage / myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4 / 80). In addition to immunophenotyping, serum cytokines are analyzed, including, but not limited to, TNFα, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2 , IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES and MCP-1. Cytokine analysis can be performed on immune cells obtained from lymph nodes or other tissue, and / or on purified tissue filtering immune cells obtained ex vivo. Finally, immunohistochemistry is performed on several sections of tissue to measure T cells, macrophages, dendritic cells and protein expression in the checkpoint molecule. Antibody production can also be assessed by ELISA.
[00388] [00388] In order to examine the impact and longevity of disease protection, instead of being sacrificed, some mice may be again confronted with a disease trigger, or evaluated for susceptibility to relapse. The mice are analyzed for susceptibility to the onset of diabetes and severity after a new confrontation (or relapse of spontaneous occurrence). Example 21: EV in a Primary Sclerosing Cholangitis (PSC) mouse model
[00389] [00389] Primary Sclerosing Cholangitis (PSC) is a chronic liver disease that slowly damages the bile ducts and leads to terminal phase cirrhosis. It is associated with inflammatory bowel disease (IBD).
[00390] [00390] There are several animal models for PSC, as analyzed by Fickert et al. (Characterization of models of animals for primary sclerosing cholangitis (PSC). J Hepatol. June 2014. 60 (6):
[00391] [00391] EV are tested for their effectiveness in a mouse model of PSC, alone or in combination with all bacterial cells, with or without the addition of some other therapeutic agent. DCC-induced cholangitis
[00392] [00392] For example, 6 to 8 week C57bl / 6 mice are obtained from Taconic or another vendor. The mice are fed a diet supplemented with 0.1% DCC for several durations. Some groups receive feed supplemented with DCC for 1 week, others for 4 weeks, others for 8 weeks. Some groups of mice may receive a diet supplemented with CHD for a period of time and then recover later on a normal diet. These mice can be studied for their ability to recover from disease and / or their susceptibility to relapse upon subsequent exposure to CHD. Treatment with EV starts at some point, or around the time of feeding with DCC or subsequent to the initial exposure to DCC. For example, EVs can be administered on day 1, or they can be administered later on. EVs are administered in varying doses and at defined intervals. For example, some mice are injected intravenously with IV at 15, 20 or 15 µg / mouse. Other mice can receive 25, 50, 100 mg of EV per mouse. While some mice receive EV via i.v. injection,
[00393] [00393] In addition, some mice are treated with antibiotics before treatment. For example, vancomycin (0.5 g / l), ampicillin (1.0 g / l), gentamicin (1.0 g / l) and amphotericin B (0.2 g / l) are added to drinking water, and antibiotic treatment is stopped at the time of treatment or a few days before treatment. Some immunized mice are treated without receiving antibiotics. At various points in time, serum samples are analyzed for ALT, AP, bilirubin and serum bile acid (BA) levels.
[00394] [00394] At various points in time, mice are sacrificed, body and liver weight are recorded, and sites of inflammation (eg, liver, large and small intestine, spleen), lymph nodes, or other tissues can be removed for histomorphological characterization ex vivo, cytokine and / or flow cytometry analysis using methods known in the art (see Fickert et al. Characterization of models of animal for primary sclerosing cholangitis (PSC)). J Hepatol. 2014. 60 (6): 1,290 to 1,303). For example, bile ducts are stained for expression of ICAM-1, VCAM-1, MadCAM-1. Some tissues are stained for histological examination, while others are dissociated using dissociation enzymes according to the manufacturer's instructions. The cells are stained for flow cytometric analysis using techniques known in the art. Staining antibodies can include anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4 and anti-CD103. Other markers that can be analyzed include CD45 panimmune cell marker, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzima B, CD69, PD-1, CTLA-4 ), and macrophage / myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4 / 80), as well as adhesion molecule expression (ICAM-1, VCAM-1, MadCAM- 1). In addition to immunophenotyping, serum cytokines are analyzed, including, but not limited to, TNFα, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2 , IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES and MCP-1. The cytokine analysis can be performed on immune cells obtained from lymph nodes or other tissue, and / or on immune cells filtered by purified CD45 + bile duct obtained ex vivo.
[00395] [00395] The liver tissue is prepared for histological analysis, for example, with the use of Sirius red staining followed by quantification of the fibrotic area. At the end of treatment, blood is collected for plasma analysis of liver enzymes, for example, AST or ALT, and to determine Bilirubin levels. The hepatic content of Hydroxyproline can be measured using established protocols. The analysis of liver gene expression of inflammation and fibrosis markers can be performed by qRT-PCR using validated primers. These markers may include, but are not limited to, MCP-1, alpha-SMA, Coll1a1, and TIMP-. Metabolite measurements can be performed on plasma, tissue and fecal samples using established metabolomic methods. Finally, immunohistochemistry is performed on sections of the liver to measure neutrophils, T cells, macrophages, dendritic cells, or other immune cell infiltrates.
[00396] [00396] In order to examine the impact and longevity of protection against disease, instead of being sacrificed, some mice can be re-evaluated with CCD at a later time. Mice are analyzed for susceptibility to cholangitis and severity of cholangitis after further evaluation. BDL-induced cholangitis
[00397] [00397] Alternatively, EVs are tested for their effectiveness in BDL-induced cholangitis. For example, 6 to 8 week old C57Bl / 6J mice are obtained from Taconic or another vendor. After a period of acclimation, the mice undergo a surgical procedure to perform a bile duct connection (BDL). Some control animals receive simulated surgery. The BDL procedure leads to liver damage, inflammation and fibrosis within 7-21 days.
[00398] [00398] Treatment with EV starts at some point, or around the time of surgery or some time after surgery. EVs are administered in varying doses and at defined intervals. For example, some mice are injected intravenously with IV at 15, 20 or 15 µg / mouse. Other mice may receive 25, 50 or 100 mg of EV per mouse. While some mice receive IV via i.v. injection, other mice may receive EV via i.p. injection, subcutaneous injection (s.c.), nasal administration, oral gavage, or other means of administration. Some mice receive EV every day (for example, starting on day 1), while others may receive EV at alternate intervals (for example, every other day, or once every three days). Additional groups of mice may receive some ratio between bacterial cells and EV. Bacterial cells can be alive, dead or weakened. The same bacterial cells can be collected fresh (or frozen) and administered, or they can be irradiated or exterminated by heat before administration. For example, some groups of mice can receive between 1x104 and 5x109 bacterial cells in a separate administration or mixed with the administration of EV. As with EV, bacterial cell administration can be varied through administration, dose and schedule. This may include oral gavage, i.v. injection, i.p. or nasal administration. Some groups of mice can be treated with additional agents and / or an appropriate control (for example, vehicle or antibody) at various points in time and in effective doses.
[00399] [00399] In addition, some mice are treated with antibiotics before treatment. For example, vancomycin (0.5 g / l), ampicillin (1.0 g / l), gentamicin (1.0 g / l) and amphotericin B (0.2 g / l) are added to drinking water, and antibiotic treatment is stopped at the time of treatment or a few days before treatment. Some immunized mice are treated without receiving antibiotics. At various points in time, serum samples are analyzed for ALT, AP, bilirubin and serum bile acid (BA) levels.
[00400] [00400] At various points in time, mice are sacrificed, body and liver weight are recorded, and sites of inflammation (eg, liver, large and small intestine, spleen), lymph nodes, or other tissues can be removed for histomorphological characterization ex vivo, cytokine and / or flow cytometry analysis using methods known in the art (see Fickert et al. Characterization of models of animal for primary sclerosing cholangitis (PSC)). J Hepatol. 2014. 60 (6): 1,290 to 1,303). For example, bile ducts are stained for expression of ICAM-1, VCAM-1, MadCAM-1. Some tissues are stained for histological examination, while others are dissociated using dissociation enzymes according to the manufacturer's instructions. The cells are stained for flow cytometric analysis using techniques known in the art. Staining antibodies can include anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4 and anti-CD103. Other markers that can be analyzed include CD45 panimmune cell marker, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzima B, CD69, PD-1, CTLA-4 ), and macrophage / myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4 / 80), as well as adhesion molecule expression (ICAM-1, VCAM-1, MadCAM- 1). In addition to immunophenotyping, serum cytokines are analyzed, including, but not limited to, TNFα, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2 , IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES and MCP-1. The cytokine analysis can be performed on immune cells obtained from lymph nodes or other tissue, and / or on immune cells filtered by purified CD45 + bile duct obtained ex vivo.
[00401] [00401] The liver tissue is prepared for histological analysis, for example, using Sirius red staining followed by quantification of the fibrotic area. At the end of treatment, blood is collected for plasma analysis of liver enzymes, for example, AST or ALT, and to determine Bilirubin levels. The hepatic content of Hydroxyproline can be measured using established protocols. The analysis of liver gene expression of inflammation and fibrosis markers can be performed by qRT-PCR using validated primers. These markers may include, but are not limited to, MCP-1, alpha-SMA, Coll1a1, and TIMP-. Metabolite measurements can be performed on plasma, tissue and fecal samples using established metabolomic methods. Finally, immunohistochemistry is performed on sections of the liver to measure neutrophils, T cells, macrophages, dendritic cells, or other immune cell infiltrates.
[00402] [00402] In order to examine the impact and longevity of protection against disease, instead of being sacrificed, some mice can be analyzed for recovery. Example 22: EV in a Non-Alcoholic Steatohepatitis (NASH) mouse model
[00403] [00403] Non-alcoholic steatohepatitis (NASH) is a severe form of Non-Alcoholic Fatty Liver Disease (NAFLD), in which the accumulation of hepatic fat (steatosis) and inflammation leads to liver damage and hepatocyte cell death ( ballooning).
[00404] [00404] There are several animal models of NASH, as analyzed by Ibrahim et al. (Animal models of nonalcoholic steatohepatitis: Eat, Delete, and Inflame. Dig Dis Sci. May 2016. 61 (5): 1,325 to 1,336; see also Lau et al. Animal models of non-alcoholic fatty liver disease: current perspectives and recent advances, January 2017. 241 (1): 36 to 44).
[00405] [00405] EV are tested for effectiveness in a mouse model of NASH, alone or in combination with all bacterial cells, with or without the addition of another therapeutic agent. For example, 8-10 week C57Bl / 6J mice, obtained from Taconic (Germantown, NY), or another vendor, are placed on a methionine and choline (MCD) deficient diet for a period of 4-8 weeks during which features of NASH develop, including steatosis, inflammation, ballooning and fibrosis.
[00406] [00406] Prevotella histicola and EVS-derived bacterial cells from P. histicolas are tested for their efficacy in a NASH mouse model, alone or in combination with each other, in varying proportions, with or without the addition of another therapeutic agent. For example, 8 week old C57Bl / 6J mice, obtained from Charles River (France), or another vendor, were acclimated for a period of 5 days, randomized into groups of 10 mice based on body weight, and placed in a deficient methionine and choline diet (MCD), for example, A02082002B from Research Diets (USA), for a period of 4 weeks during which NASH resources developed, including steatosis, inflammation, ballooning and fibrosis. Control chewing mice were fed a normal chewing diet, for example, RM1 (E) 801492 from SDS Diets (UK). Control chewing, MCD diet, and water were provided ad libitum.
[00407] [00407] Treatment with live, frozen P. histicola was started on day 1 of the MCD diet for some mice and continued for 28 consecutive days. Some MCD diet mice were administered with bacterial cells through daily oral gavage of 100 100l of a suspension containing 1.47x109 of bacterial cells.
[00408] [00408] In mice receiving the MCD (NASH induction) diet, orally administered P. histicola were effective in reducing the NAS score compared to vehicle and no treatment group (negative controls) (Figure 4). P. histicola reduced steatosis (Figure 5A), inflammation (Figure 5B and Figure 5C), and ballooning (Figure 5D), as well as total liver cholesterol (Figure 6). P. histicola also reduced the fibrosis score in treated mice (Figure 7A and Figure 7B).
[00409] [00409] Figure 9A shows the effect of P. histicola on free liver fatty acids in mice that were fed a MCD diet, Figure 9B shows the effect of P. histicola on total liver cholesterol in mice that were fed a MCD diet, Figure 9C shows the effect of P. histicola on liver triglycerides in mice that were fed an MCD diet, Figure 9D shows the effect of P. histicola and P. melanogenica on alanine aminotransferase in mice that were fed a MCD diet, Figure 9E shows effect of P. histicola and P. melanogenica in aspartate aminotransferase in mice that were fed an MCD diet.
[00410] [00410] In mice receiving the MCD (NASH induction) diet, administered orally, P. histicola and P. melanogenica were effective in reducing the NAS score compared to vehicle and no treatment group (negative controls) ( Figures 10A and 10B).
[00411] [00411] In other studies, analysis of inflammation of liver gene expression, fibrosis, steatosis, ER stress, or oxidative stress markers can be performed by qRT-PCR using validated primers. These markers may include, but are not limited to, IL-1, TNF-, MCP-1, -SMA, Coll1a1, CHOP, and NRF2.
[00412] [00412] Treatment with EV is started at some point, either at the beginning of the diet, or at some point after the beginning of the diet (for example, a week after). For example, EV can be administered starting on the same day as the initiation of the MCD diet. EVs are administered in varying doses and at defined intervals. For example, some mice are injected intravenously with IV at 15, 20 or 15 µg / mouse. Other mice may receive 25, 50 or 100 mg of EV per mouse. Although some mice receive IV via i.v. injection, other mice may receive IV via intraperitoneal injection (i.p.), subcutaneous injection (s.c.), nasal administration, oral gavage or other means of administration. Some mice may receive EV every day (for example, starting on day 1), while others may receive EV at alternate intervals (for example, every other day or once every three days). Additional groups of mice may receive some ratio between bacterial cells and EV. Bacterial cells can be alive, dead or weakened. Bacterial cells can be collected fresh (or frozen) and administered, or they can be irradiated or exterminated by heat before administration.
[00413] [00413] For example, some groups of mice can receive between 1x104 and 5x109 bacterial cells in a separate administration or mixed with the administration of EV. As with EV, the administration of bacterial cells can be varied in relation to the route of administration, dose and schedule. This may include oral gavage, i.v. injection, i.p. or nasal administration.
[00414] [00414] At various points in time and / or at the end of treatment, mice are sacrificed and liver, intestine, blood, feces or other tissues can be removed for ex vivo histological, biochemical, molecular or cytokine analysis and / or flow cytometry using methods known in the art. For example, liver tissues are weighed and prepared for histological analysis, which can include staining with H&E, Sirius Red and determination of NASH activity scores (NAS). At various points in time, blood is collected for plasma analysis of liver enzymes, for example, AST or ALT, using standard assays. In addition, the liver content of cholesterol, triglycerides, or fatty acid acids can be measured using established protocols. The analysis of liver gene expression of inflammation, fibrosis, steatosis, ER stress, or oxidative stress markers can be performed by qRT-PCR using validated primers. Markers may include, but are not limited to, IL-6, MCP-1, alpha-SMA, Coll1a1, CHOP, and NRF2. Metabolite measurements can be performed on plasma, tissue and faecal samples using established metabolomic methods based on mass spectrometry and biochemistry. Serum cytokines are analyzed, including, but not limited to, TNFα, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL -1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. The cytokine analysis can be performed on immune cells obtained from lymph nodes or other tissue, and / or on immune cells filtered by purified CD45 + bile duct obtained ex vivo. Finally, immunohistochemistry is performed on sections of the liver or intestine to measure neutrophils, T cells, macrophages, dendritic cells, or other immune cell infiltrates.
[00415] [00415] In order to examine the impact and longevity of protection against disease, instead of being sacrificed, some mice can be analyzed for recovery. Example 23: EV in a mouse model of psoriasis
[00416] [00416] Psoriasis is a chronic inflammatory skin disease mediated by T cell. The so-called "plaque-type" psoriasis is the most common form of psoriasis and is typified by dry scales, red plaques and thickening of the skin due to the infiltration of immune cells in the dermis and epidermis. Several animal models contributed to the understanding of this disease, as analyzed by Gudjonsson et al. (Mouse models of psoriasis. J Invest Derm.
[00417] [00417] Psoriasis can be induced in a variety of mouse models, including those using transgenic, knockout, or xenograft models, as well as topical application of imiquimod (IMQ), a TLR7 / 8 ligand.
[00418] [00418] EV are tested for their effectiveness in the mouse model of psoriasis, alone or in combination with all bacterial cells, with or without the addition of other anti-inflammatory treatments. For example, 6 to 8 week old C57Bl / 6 or Balb / c mice are obtained from Taconic (Germantown, NY), or another vendor. The mice are shaved on the back and on the right ear. Groups of mice receive a daily topical dose of 62.5 mg of commercially available IMQ cream (5%) (Aldara; 3M Pharmaceuticals). The dose is applied to the shaved areas for 5 or 6 consecutive days. At regular intervals, mice are classified for erythema, scaling and thickening on a scale of 0 to 4, as described by van der Fits et al. (2009). Mice are monitored for ear thickness using a Mitutoyo micrometer.
[00419] [00419] Treatment with EV starts at some point, or around the time of the first IMQ application, or some subsequent time. For example, EV can be administered at the same time as subcutaneous injections (day 0), or they can be administered before, or by application. EVs are administered in varying doses and at defined intervals. For example, some mice are injected intravenously with IV at 15, 20 or 15 µg / mouse. Other mice may receive 25, 50 or 100 mg of EV per mouse. While some mice receive IV via iv injection, other mice may receive IV via intraperitoneal injection (ip), nasal administration, oral gavage, topical administration, intradermal injection (id), subcutaneous injection (sc) or other means of administration . Some mice may receive EV every day (for example, starting on day 0), while others may receive EV at alternate intervals (for example, every other day or once every three days). Additional groups of mice may receive some ratio between bacterial cells and EV. Bacterial cells can be alive, dead or weakened. Bacterial cells can be collected fresh (or frozen) and administered, or they can be irradiated or exterminated by heat before administration.
[00420] [00420] For example, some groups of mice can receive between 1x104 and 5x109 bacterial cells in a separate administration or mixed with the administration of EV. As with EV, the administration of bacterial cells can be varied in relation to the route of administration, dose and schedule. This may include oral gavage, i.v. injection, i.p. injection, i.d. injection, s.c. injection, topical administration, or nasal administration.
[00421] [00421] Some groups of mice can be treated with anti-inflammatory agent (or anti-inflammatory agents) (for example, anti-CD154, blocking TNF family members or other treatment), and / or adequate control (for example , vehicle or control antibody) at various points in time and in effective doses.
[00422] [00422] In addition, some mice are treated with antibiotics before treatment. For example, vancomycin (0.5 g / l), ampicillin (1.0 g / l), gentamicin (1.0 g / l) and amphotericin B (0.2 g / l) are added to drinking water, and antibiotic treatment is stopped at the time of treatment or a few days before treatment. Some immunized mice are treated without receiving antibiotics.
[00423] [00423] At different points in time, samples of skin from the back and ear are taken by analysis of cryostaining dyeing using methods known in the art. Other groups of mice are sacrificed, and lymph nodes, spleen, mesenteric lymph nodes (MLN), small intestine, colon and other tissues can be removed for histological, histological, cytokine and / or cytometric analysis of ex vivo flow using the methods known in the art. Some tissues can be dissociated using dissociation enzymes according to the manufacturer's instructions. Cryosection samples, tissue samples, or cells obtained ex vivo are stained for analysis by flow cytometry using techniques known in the art. Staining antibodies can include anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4 and anti-CD103. Other markers that can be analyzed include immune cell pan marker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzima B, CD69, PD-1, CTLA-4) and macrophage / myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4 / 80). In addition to immunophenotyping, serum cytokines are analyzed, including, but not limited to, TNFα, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2 , IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES and MCP-1. Cytokine analysis can be performed on immune cells obtained from lymph nodes or other tissue, and / or on immune cells filtered by purified CD45 + skin obtained ex vivo. Finally, immunohistochemistry is performed on several sections of tissue to measure T cells, macrophages, dendritic cells and protein expression in the checkpoint molecule.
[00424] [00424] In order to examine the impact and longevity of protection against psoriasis, instead of being sacrificed, some mice can be studied to assess recovery, or they can be analyzed again with IMQ. The groups of mice analyzed again are analyzed for susceptibility to psoriasis and severity of response. Example 24: Manufacturing conditions
[00425] [00425] Enriched media are used to grow and prepare the bacteria for use in vitro and in vivo. For example, the media may contain sugar, yeast extracts, plant-based peptones, buffers, salts, trace elements, surfactants, anti-foaming agents and vitamins. The composition of complex components, such as yeast extracts and peptones, can be undefined or partially defined (including approximate concentrations of amino acids, sugars, etc.). Microbial metabolism may be dependent on the availability of resources, such as carbon and nitrogen. Various sugars or other sources of carbon can be tested. Alternatively, media can be prepared and selected bacteria cultured as shown by Saarela et al., J. Applied Microbiology. 2005. 99: 1,330 to 1,339, which is incorporated by reference. The influence of the fermentation time, cryoprotectant and neutralization of cellular concentrate on the survival of freeze drying, storage stability and exposure to acid and bile of the selected bacteria produced without milk-based ingredients.
[00426] [00426] On a large scale, the medium is sterilized. Sterilization can be processed at Ultra High Temperature (UHT). UHT processing is carried out at very high temperatures for short periods of time. The UHT range can be from 135 to 180 ° C. For example, the medium can be sterilized for 10 to 30 seconds at 135 ° C.
[00427] [00427] The inoculum can be prepared in flasks or in smaller bioreactors and growth is monitored. For example, the inoculum size can be between approximately 0.5 and 3% of the total volume of the bioreactor. Depending on the application and material requirement, the volume bioreactor can be at least 2 l, 10 l, 80 l, 100 l, 250 l, 1,000 l, 2,500 l, 5,000 l, 10,000 l.
[00428] [00428] Before inoculation, the bioreactor is prepared with medium at the desired pH, temperature and oxygen concentration. The initial pH of the culture medium may differ from the process setpoint. PH stress can be harmful in low cell concentrations; the initial pH could be between pH 7.5 and the process setpoint. For example, the pH can be adjusted between 4.5 and 8.0. During fermentation, the pH can be controlled through the use of sodium hydroxide, potassium hydroxide or ammonium hydroxide. The temperature can be controlled from 25 ° C to 45 ° C, for example, at 37 ° C. Anaerobic conditions are created by reducing the oxygen level in the culture broth from about 8 mg / l to 0 mg / l. For example, nitrogen or gas mixtures (N2, CO2 and H2) can be used in order to establish anaerobic conditions. Alternatively, no gas is used and anaerobic conditions are established by cells that consume the remaining oxygen in the medium. Depending on the strain and size of the inoculum, the fermentation time in a bioreactor can vary. For example, the fermentation time can vary from approximately 5 hours to 48 hours.
[00429] [00429] Reviving microbes from a frozen state may require special considerations. The production medium can stress cells after thawing; a specific defrosting medium may be needed to consistently start a series of defrosted seeds. The kinetics of transferring or passing seed material to fresh media, for the purposes of increasing seed volume or maintaining the state of microbial growth, can be influenced by the current state of microbes (eg exponential growth, stationary growth, without stress, with stress).
[00430] [00430] The inoculation of the production fermenter (or fermenters) can impact growth kinetics and cell activity. The initial state of the bioreactor system needs to be optimized to facilitate successful and consistent production. The fraction between seed culture and the total medium (for example, a percentage) has a drastic impact on growth kinetics. The range can be 1 to 5% of the working volume of the fermenter. The initial pH of the culture medium may differ from the process setpoint. PH stress can be harmful in low cell concentrations; the initial pH can be between pH 7.5 and the process setpoint. The agitation and gas flow in the system during inoculation may differ from the process set points. Physical and chemical stresses due to both conditions can be harmful in low cell concentration.
[00431] [00431] Process conditions and control settings can influence the kinetics of microbial growth and cell activity. Displacements under process conditions can alter the composition of the membrane, the production of metabolites, growth rate, cell stress, etc. The ideal temperature range for growth may vary with the strain. The range can be 20 to 40 C. The ideal pH for growth and downstream activity performance may vary with the strain. The range can be pH 5 to 8. The gases dissolved in the medium can be used by cells for metabolism. It may be necessary to adjust the concentrations of O2, CO2 and N2 throughout the process. The availability of nutrients can displace cell growth. Microbes can have alternative kinetics when excess nutrients are available.
[00432] [00432] The state of microbes at the end of a fermentation and during collection can impact cell survival and activity. Microbes can be preconditioned briefly before collection to better prepare them for the physical and chemical stresses involved in downstream separation and processing. A change in temperature (often reducing to 20 to 5 C) can reduce cell metabolism, delaying growth (and / or death) and physiological change when removed from the fermenter. The effectiveness of centrifugal concentration can be influenced by the pH of the culture. Raising the pH by 1 to 2 points can improve concentration efficiency, but it can also be harmful to cells. Microbes can be stressed briefly before collection by increasing the concentration of salts and / or sugars in the medium. Stressed cells in this way can better survive freezing and freeze-drying during the downstream.
[00433] [00433] Separation methods and technology can impact how efficiently microbes are separated from the culture medium. The solids can be removed using centrifugation techniques. The effectiveness of centrifugal concentration can be influenced by culture pH or with the use of flocculation agents. Raising the pH by 1 to 2 points can improve concentration efficiency, but it can also be harmful to cells. Microbes can be stressed briefly before collection by increasing the concentration of salts and / or sugars in the medium. Stressed cells in this way can better survive freezing and freeze-drying during the downstream. In addition, microbes can also be separated by filtration. Filtration is superior to centrifugation techniques for purification if cells require g minutes for successful centrifugation. Excipients can be added before separation. Excipients can be added for cryoprotection or for protection during lyophilization. Excipients can include, but are not limited to, sucrose, trehalose or lactose, and they can alternatively be mixed with buffer and antioxidants. Before lyophilization, droplets of cell pellets mixed with excipients are submerged in liquid nitrogen.
[00434] [00434] The collection can be performed by continuous centrifugation. The product can be resuspended with various excipients to a desired final concentration. Excipients can be added for cryoprotection or for protection during lyophilization. Excipients can include, but are not limited to, sucrose, trehalose or lactose, and they can alternatively be mixed with buffer and antioxidants. Before lyophilization, droplets of cell pellets mixed with excipients are submerged in liquid nitrogen.
[00435] [00435] Lyophilization of material, including live bacteria, begins with primary drying. During the primary drying phase, the ice is removed. Here, a vacuum is generated and an adequate amount of heat is supplied to the material for the ice to sublimate. During the secondary drying phase, water molecules attached to the product are removed. Here, the temperature is raised until it is higher than in the primary drying phase to break up any physico-chemical interactions that have formed between the water molecules and the product material. The pressure can also be reduced further to increase desorption during this stage. After the freeze drying process is completed, the chamber can be filled with an inert gas, such as nitrogen. The product can be sealed inside the dryer by freezing under dry conditions, preventing exposure to atmospheric water and contaminants. Example 25: A mouse melanoma model
[00436] [00436] Female C57Bl / 6 mice aged 6 to 8 weeks are obtained from Taconic (Germantown, NY). 100,000 B16-F10 tumor cells (ATCC CRL-6475) are resuspended in sterile PBS containing 50% Matrigel and inoculated in a final volume of 100 µl in one rear flank (the first flank) of each mouse. Treatment with Veillonella strains starts at some point after inoculation of tumor cells in varying doses and at defined intervals. For example, some mice receive between 1 and 5x10 ^ 9 CFU (100 μl final volume) per dose. Possible routes of administration include oral gavage (p.o.), intravenous injection, intratumoral (IT) injection, or peritumoral or subtumoral or subcutaneous injection. In order to assess the systemic antitumor effects of treatment with Veillonella, additional mice can be inoculated with tumor cells on the contralateral (untreated, second) flank prior to IT, peritumor or subtumor treatment with Veillonella on the first flank.
[00437] [00437] Some mice may receive Veillonella (p.o.) on day 1 (the day after tumor cell injection). Other mice can receive seven (7) consecutive doses of a bacterial strain (one dose per day on days 14 to 21). Other mice are dosed daily, or alternatively, some mice are dosed every other day. Alternatively, the mice are randomized into several treatment groups at a defined point in time (for example, on the 13th day) or when the tumors reach a certain size (for example, 100 mm3) and treatment is then started accordingly. For example, when tumor volumes reach an average of 100 mm3 (approximately 10 to 12 days after tumor cell inoculation), animals are distributed into groups and treated with a vehicle or a bacterial strain (p.o. or IT). Some additional groups of mice can be treated with an additional cancer therapy or appropriate control antibody. An example of a cancer therapy that can be administered is an immunological checkpoint inhibitor, for example, anti-PD-1, anti-PD-L1, or other treatment that blocks the attachment of an immune checkpoint to their ligand (or ligands). Checkpoint inhibitors anti-PD-1 and anti-PD-L1 can be formulated in PBS and administered intraperitoneally (i.p.) in effective doses. For example, mice received 100 µg of anti-PD-1 (i.p.) every four days, starting on day 1, and continuing for the duration of the study.
[00438] [00438] In addition, some mice are treated with antibiotics before treatment. For example, vancomycin (0.5 g / l),
[00439] [00439] At various points in time, mice are sacrificed, and tumors, lymph nodes or other tissues can be removed by flow cytometric analysis ex vivo using the methods known in the art. For example, tumors are dissociated using a cocktail of Miltenyi tumor dissociating enzymes according to the manufacturer's instructions. Tumor weights are recorded and tumors are cut and then placed in 15 ml tubes containing the enzyme cocktail and placed on ice. The samples are then placed on a gentle shaker at 37 ° C for 45 minutes and briskly cooled with up to 15 ml of complete RPMI. Each cell suspension is strained through a 70 μm filter into a 50 ml falcon tube and centrifuged at 1,000 rpm for 10 minutes. The cells are suspended in FACS buffer and washed to remove remaining debris. If necessary, the samples are strained again through a second 70 μm filter in a new tube. The cells are stained for flow cytometric analysis using techniques known in the art. Staining antibodies can include anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4 and anti-CD103. Other markers that can be analyzed include immune cell pan marker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3, Rorgamat, Granzima B, CD69, PD-1, CTLA-4) and macrophage / myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1). In addition to immunophenotyping, serum cytokines are analyzed,
[00440] [00440] Instead of being sacrificed, some mice can be challenged again by injecting a tumor cell into the contralateral flank (or other area) to determine the impact of the immune system's memory response 'on tumor growth.
[00441] [00441] In mice that received the MCD diet (which induces NASH), Veillonella administered orally was effective in reducing the NAS score compared to vehicle and untreated groups (negative controls) (Figure 16). Veillonella reduced the fibrosis score in treated mice (Figure 17). Veillonella reduced total liver cholesterol (Figure 18) and liver triglycerides (Figure 19). Example 26: A colorectal carcinoma model
[00442] [00442] Female Balb / c mice aged 6 to 8 weeks were obtained from Taconic (Germantown, NY). 100,000 CT-26 c colorectal tumor cells (ATCC CRL-2638) were resuspended in sterile PBS and inoculated in the presence of 50% Matrigel. CT-26 tumor cells were injected subcutaneously into the rear flank of each mouse. When tumor volumes reached an average of 100 mm3 (approximately 10 to 12 days after tumor cell inoculation), the animals were distributed into the following groups: 1) Vehicle; 2) Veillonella strains 3) anti-PD-1 antibody. The antibodies were administered intraperitoneally (ip) at 200 µg / mouse (100 µl final volume) every four days, starting on day 1, for a total of 3 times (Q4Dx3) and Veillonella's EV (5 µg). injected intravenously (iv) every third day, starting on day 1 a total of 4 times (Q3Dx4). Both Veillonella groups showed inhibition of tumor growth greater than that seen in the anti-PD-1 group (Figures 12 and 13).
[00443] [00443] Another example, when the tumor volumes reached an average of 100 mm3 (approximately 10 to 12 days after the inoculation of tumor cells), the animals were distributed in the following groups: 1) Vehicle; 2) Burkholderia pseudomallei; and 3) anti-PD-1 antibody. The antibodies were administered intraperitoneally (ip) at 200 µg / mouse (100 µl of final volume) every four days, starting on day 1, and EV of Burkholderia pseudomallei (5 µg) were injected intravenously (iv) daily , starting on day 1 until the conclusion of the study. The Burkholderia pseudomallei group showed inhibition of tumor growth greater than that observed in the anti-PD-1 group (Figures 20 and 21).
[00444] [00444] Another example, when the tumor volumes reached an average of 100 mm3 (approximately 10 to 12 days after the inoculation of tumor cells), the animals were distributed in the following groups: 1) Vehicle; 2) Evs from Neisseria Meningitidis isolated from the Bexsero® vaccine; and 3) anti-PD-1 antibody. Antibodies were administered intraperitoneally (ip) at 200 µg / mouse (100 µl final volume) every four days, starting on day 1, and Neisseria Meningitidis bacteria (about 1.1x102) were administered intraperitoneally (ip ) daily, starting on day 1 until the conclusion of the study. The Neisseria Meningitidis group showed inhibition of tumor growth greater than that observed in the anti-PD-1 group (Figures 22 and 23). Table 4. Significance test that compares the tumor in the treatment groups vs. control groups on day 11. T test (two-tailed, unpaired, corrected by Welch) calculated on GraphPad. Comparison P-Value Summary IP vs. Vehicle Evs of 0.0004 *** Neisseria Meningitidis.
[00445] [00445] Female Balb / c mice aged 6 to 8 weeks were obtained from Taconic (Germantown, NY). 100,000 CT-26 c colorectal tumor cells (ATCC CRL-2638) were resuspended in sterile PBS and inoculated in the presence of 50% Matrigel. CT-26 tumor cells were injected subcutaneously into the rear flank of each mouse. When tumor volumes reached an average of 100 mm3 (approximately 10 to 12 days after tumor cell inoculation), the animals were distributed into the following groups as highlighted in Table 5. Table 5: Treatment Groups Treatment Group Dose / Route / Schedule 1 Vehicle IV (PBS) N / A / IV / Q3Dx4 2 Vehicle PO (sucrose) N / A / PO / QD 3 Anti-PD-1 200 µg / IP / Q4Dx3 4 EV of Veillonella parvula 10 µg / IV / Q3Dx4 5 Veillonella parvula EV 5 µg / IV / Q3Dx4 6 Veillonella parvula 2 µg / IV / Q3Dx4 EV 7 Veillonella tobetsuensis EV 75 µg / PO. QD 8 Veillonella tobetsuensis EV 5 µg / IV / Q3Dx4
[00446] [00446] As indicated in the table, antibodies were administered intraperitoneally (ip) at 200 µg / mouse (100 µl final volume) every four days, starting on day 1, for a total of 3 times (Q4Dx3) and IV when administered intravenously (iv) received an injection every third day, starting on day 1 a total of 4 times (Q3Dx4). Treatment groups administered per month (p.o.) were administered daily (QD). The efficacy of Veillonella's EV varies based on the source microbe, dose and route of administration (Figures 14 and 15). Incorporation by Reference
[00447] [00447] All publications and patent applications mentioned in this document are incorporated into this document for reference in their entirety as if each individual publication or patent application was specifically and individually indicated and incorporated by reference. In case of conflict, the present request, including the definitions in this document, will prevail. Equivalents
[00448] [00448] Those skilled in the art will recognize, or be able to determine using, no more than routine experimentation, many equivalents of the specific embodiments of the invention described in this document. Such equivalents are intended to be covered by the following claims.

权利要求:
Claims (161)
[1]
1. Pharmaceutical composition characterized by the fact that it comprises isolated bacterial extracellular (EV) vesicles.
[2]
2. Pharmaceutical composition characterized by the fact that it comprises bacterial extracellular vesicles (EV) and bacteria.
[3]
3. Pharmaceutical composition according to claim 2, characterized by the fact that at least about or not more than 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%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42 %, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75% , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total particles of EV or bacteria in the pharmaceutical composition are EV.
[4]
4. Pharmaceutical composition according to claim 2, characterized by the fact that at least about or more than 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%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42 %, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75% , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total particles of EV or bacteria in the pharmaceutical composition are bacteria.
[5]
5. Pharmaceutical composition according to claim 2, characterized by the fact that at least about or more than 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%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42 %, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75% , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total EV protein and bacteria in the pharmaceutical composition is EV protein.
[6]
6. In some modalities, at least about or not more than 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 %, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62% , 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79 %, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total EV and bacteria protein in the pharmaceutical composition is a bacterial protein.
[7]
7. Pharmaceutical composition according to claim 2, characterized by the fact that at least about or not more than 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%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42 %, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,
72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total EV lipids and bacteria in the pharmaceutical composition are EV lipids.
[8]
8. Pharmaceutical composition according to claim 2, characterized by the fact that at least about or not more than 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%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42 %, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75% , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total lipids of EV and bacteria in the pharmaceutical composition are lipids from bacteria.
[9]
9. Pharmaceutical composition characterized by the fact that it comprises bacteria isolated from EVs.
[10]
Pharmaceutical composition according to any one of claims 2 to 9, characterized in that the composition comprises live, dead or debilitated bacteria.
[11]
11. Pharmaceutical composition according to any one of claims 1 to 10, characterized by the fact that the EV and / or bacteria are from defective Abiotrophia, Abiotrophia para_adiacens, Abiotrophia sp. oral clone P4PA_155 P1, Acetanaerobacterium elongatum, Acetivibrio cellulolyticus, Acetivibrio ethanolgignens, Acetobacter aceti, Acetobacter fabarum, Acetobacter lovaniensis, Acetobacter malorum, Acetobacteris, Acetobacter pasteur, Acetobacter pasteur, Acetobacter pasteur, Acetobacter pasteur, Acetobacter pasteur , Achromobacter piechaudii, Achromobacter xylosoxidans, Acidaminococcus fermentans, Acidaminococcus intestini, Acidaminococcus sp.
D21, Acidilobus saccharovorans, Acidithiobacillus ferrivorans, Acidovorax sp. 98_63833, Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter genomosp.
C1, Acinetobacter haemolyticus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter lwoffii, Acinetobacter parvus, Acinetobacter radioresistens, Acinetobacter schindleri, Acinetobacter sp. 56A1, Acinetobacter sp.
CIP 101934, Acinetobacter sp.
CIP 102143, Acinetobacter sp.
CIP 53.82, Acinetobacter sp.
M16_22, Acinetobacter sp.
RUH2624, Acinetobacter sp.
SH024, Actinobacillus actinomycetemcomitans, Actinobacillus minor, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus ureae, Actinobaculum massiliae, Actinobaculum schaalii, Actinobaculum sp.
M L # 101342, Actinobaculum sp.
P2P_19 P1, Actinomyces cardiffensis, Actinomyces europaeus, Actinomyces funkei, Actinomyces genomosp.
C1, Actinomyces genomosp.
C2, Actinomyces genomosp.
P1 MB6_C03 oral clone, Actinomyces georgiae, Actinomyces israelii, Actinomyces massiliensis, Actinomyces meyeri, Actinomyces naeslundii, Actinomyces nasicola, Actinomyces neuii, Actinomyces odontolyticus, Actinomyces oricola, Actinomyces orihominisomy, Actinomyces orihominisomy, Actinomyces orihominisomy. 7400942, Actinomyces sp. c109, Actinomyces sp.
CCUG 37290, Actinomyces sp.
ChDC B197, Actinomyces sp.
GEJ15, Actinomyces sp.
HKU31, Actinomyces sp.
ICM34, Actinomyces sp.
ICM41, Actinomyces sp.
ICM47, Actinomyces sp.
ICM54, Actinomyces sp.
M2231_94_1, Actinomyces sp. oral clone GU009, Actinomyces sp. oral clone GU067, Actinomyces sp. oral clone IO076, Actinomyces sp. oral clone 1077, Actinomyces sp. oral clone IP073, Actinomyces sp. oral clone IP081, Actinomyces sp. oral clone JA063, Actinomyces sp. oral tax 170, Actinomyces sp.
oral taxon 171, Actinomyces sp. oral tax 178, Actinomyces sp. oral taxon 180, Actinomyces sp. oral taxon 848, Actinomyces sp. oral tax C55, Actinomyces sp.
TeJ5, Actinomyces urogenitalis, Actinomyces viscosus, Adlercreutzia equolifaciens, Aerococcus sanguinicola, Aerococcus urinae, Aerococcus urinaeequi, Aerococcus viridans, Aeromicrobium marinum, Aeromicrobium sp.
JC14, Aeromonas allosaccharophila, Aeromonas enteropelogenes, Aeromonas hydrophila, Aeromonas jandaei, Aeromonas salmonicida, Aeromonas trota, Aeromonas veronii, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotellacaotella, Prevla dentoteli, Prevla Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella saccharolytica, Prevotella scopos, Prevotella shahiior, Prevotella shahiior, zen 4, Aggregatibacter actinomycetemcomitans, Aggregatibacter aphrophilus, Aggregatibacter segnis, Agrobacterium radiobacter, Agrobacterium tumefaciens, Agrococcus jenensis, Akkermansia muciniphila, Alcaligenes faecalis, Alcaligenes sp.
CO14, Alcaligenes sp.
S3, Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, Alicyclobacillus contaminans, Alicyclobacillus cycloheptanicus, Alicyclobacillus herbarius, Alicyclobacillus pomorum, Alicyclobacillus sp.
CCUG 53762, Alistipes finegoldii, Alistipes indistinctus, Alistipes onderdonkii, Alistipes putredinis, Alistipes shahii, Alistipes sp.
HGB5, Alistipes sp.
JC50, Alistipes sp.
RMA 9912, Alkaliphilus metalliredigenes, Alkaliphilus oremlandii, Alloscardovia omnicolens, Alloscardovia sp.
OB7196, Anaerobaculum hydrogeniformans, Anaerobiospirillum succiniciproducens, Anaerobiospirillum thomasii, Anaerococcus hydrogenalis, Anaerococcus lactolyticus, Anaerococcus octavius,
Anaerococcus prevotii, Anaerococcus sp. 8404299, Anaerococcus sp. 8405254, Anaerococcus sp. 9401487, Anaerococcus sp. 9403502, Anaerococcus sp. gpac104, Anaerococcus sp. gpac126, Anaerococcus sp. gpac155, Anaerococcus sp. gpac199, Anaerococcus sp. gpac215, Anaerococcus tetradius, Anaerococcus vaginalis, Anaerofustis stercorihominis, Anaeroglobus geminatus, Anaerosporobacter mobilis, Anaerostipes caccae, Anaerostipes sp. 3_2_56FAA, Anaerotruncus colihominis, Anaplasma, Anaplasma phagocytophilum, aneurinilyticus Aneurinibacillus, Aneurinibacillus danicus, migulanus Aneurinibacillus, Aneurinibacillus terranovensis, Aneurinibacillus thermoaerophilus, Anoxybacillus contaminans, flavithermus Anoxybacillus, Arcanobacterium haemolyticum, pyogenes Arcanobacterium, Arcobacter butzleri, cryaerophilus Arcobacter, agilis Arthrobacter, Arthrobacter arilaitensis, Arthrobacter bergerei, Arthrobacter globiformis, Arthrobacter nicotianae, Atopobium minutum, Atopobium parvulum, Atopobium rimae, Atopobium sp.
BS2, Atopobium sp.
F0209, Atopobium sp.
ICM42b10, Atopobium sp.
ICM57, Atopobium vaginae, Aurantimonas coralicida, altamirensis Aureimonas, ignavus Auritibacter, Averyella dalhousiensis, aeolius Bacillus, Bacillus aerophilus Bacillus aestuarii, alcalophilus Bacillus, Bacillus amyloliquefaciens, Bacillus anthracis, Bacillus atrophaeus, Bacillus badius, Bacillus Cereus, circulans Bacillus, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus flexus, Bacillus fordii, Bacillus gelatini, Bacillus halmapalus, Bacillus halodurans, Bacillus herbersteinensis, Bacillus horti, Bacillus idriensis, Bacillus lentus, Bacillus licheniformis, Bacillus nichillonini, Bacillus megaterealis, Bacillus megatereali , Bacillus pumilus, Bacillus safensis, Bacillus simplex, Bacillus sonorensis, Bacillus sp. 10403023 MM10403188, Bacillus sp. 2_A_57_CT2, Bacillus sp. 2008724126, Bacillus sp. 2008724139,
Bacillus sp. 7_16AIA, Bacillus sp. 9_3AIA, Bacillus sp.
AP8, Bacillus sp.
B27 (2008), Bacillus sp.
BT1B_CT2, Bacillus sp.
GB1.1, Bacillus sp.
GB9, Bacillus sp.
HU19.1, Bacillus sp.
HU29, Bacillus sp.
HU33.1, Bacillus sp.
JC6, Bacillus sp. oral taxon F26, Bacillus sp. oral taxon F28, Bacillus sp. oral tax F79, Bacillus sp.
SRC_DSF1, Bacillus sp.
SRC_DSF10, Bacillus sp.
SRC_DSF2, Bacillus sp.
SRC_DSF6, Bacillus sp. tc09, Bacillus sp. zh168, Bacillus sphaericus, Bacillus sporothermodurans, Bacillus subtilis, Bacillus thermoamylovorans, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacteroidales bacterium ph8, Bacteroidales genomosp.
P1, Bacteroidales genomosp.
P2 oral clone MB1_G13, Bacteroidales genomosp.
P3 MB1_G34 oral clone, Bacteroidales genomosp.
P4 MB2_G17 oral clone, Bacteroidales genomosp.
P5 oral clone MB2_P04, Bacteroidales genomosp.
P6 MB3_C19 oral clone, Bacteroidales genomosp.
P7 oral clone MB3_P19, Bacteroidales genomosp.
P8 MB4_G15 oral clone, Bacteroides acidifaciens, Bacteroides barnesiae, Bacteroides caccae, Bacteroides cellulosilyticus, Bacteroides clarus, Bacteroides coagulans, Bacteroides coprocola, Bacteroides coprophilus, Bacteroides dorei, Bacteroides bacteroides, Bacteroides bacteroides, Bacteroides bacteroides Bacteroides helcogenes, Bacteroides heparinolyticus, Bacteroides intestinalis, Bacteroides massiliensis, Bacteroides nordii, Bacteroides oleiciplenus, Bacteroides ovatus, Bacteroides pectinophilus, Bacteroides plebeius, Bacteroides pyogenes, Bacteroides sp. 1_1_14, Bacteroides sp. 1_1_30, Bacteroides sp. 1_1_6, Bacteroides sp. 2_1_22, Bacteroides sp. 2_1_56FAA, Bacteroides sp. 2_2_4, Bacteroides sp. 20_3, Bacteroides sp. 3_1_19, Bacteroides sp. 3_1_23, Bacteroides sp. 3_1_33FAA, Bacteroides sp. 3_1_40A, Bacteroides sp. 3_2_5, Bacteroides sp. 315_5, Bacteroides sp. 31SF15, Bacteroides sp. 31SF18, Bacteroides sp. 35AE31, Bacteroides sp. 35AE37, Bacteroides sp. 35BE34, Bacteroides sp. 35BE35, Bacteroides sp. 4_1_36, Bacteroides sp. 4_3_47FAA, Bacteroides sp. 9_1_42FAA, Bacteroides sp.
AR20, Bacteroides sp.
AR29, Bacteroides sp.
B2, Bacteroides sp.
D1, Bacteroides sp.
D2, Bacteroides sp.
D20, Bacteroides sp.
D22, Bacteroides sp.
F_4, Bacteroides sp.
NB_8, Bacteroides sp.
WH2, Bacteroides sp.
XB12B, Bacteroides sp.
XB44A, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides ureolyticus, Bacteroides vulgatus, Bacteroides xylanisolvens, Bacteroidetes bacterium taxa oral D27, Bacteroidetes bacterium taxa oral F31, Bacteroidetes bacterium, bacterial bacterium, bacterial taxa, bacterium Bartonella henselae, Bartonella quintana, Bartonella tamiae, Bartonella washoensis, Bdellovibrio sp.
MPA, Bifidobacteriaceae genomosp.
C1, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium gallicum, Bifidobacterium infantis, Bifidobacterium kashiwanohense, Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum, Bifidobacterium scardovii, Bifidobacterium sp.
HM2, Bifidobacterium sp.
HMLN12, Bifidobacterium sp.
M45, Bifidobacterium sp.
MSX5B, Bifidobacterium sp.
TM_7, Bifidobacterium thermophilum, Bifidobacterium urinalis, Bilophila wadsworthia, Bisgaard Taxon, Bisgaard Taxon, Bisgaard Taxon, Bisgaard Taxon, Blastomonas natatoria, Blautia coccoides, Blautia glucerasea, Blautia glucerasei, Blautiautautia, Blautiautia, Blautia sp.
M25, Blautia stercoris, Blautia wexlerae, Bordetella bronchiseptica, Bordetella holmesii, Bordetella parapertussis,
Bordetella pertussis, Borrelia afzelii, Borrelia burgdorferi, Borrelia crocidurae, Borrelia duttonii, Borrelia garinii, Borrelia hermsii, Borrelia hispanica, Borrelia persica, Borrelia recurrentis, Borrelia sp.
NE49, Borrelia spielmanii, Borrelia turicatae, Borrelia valaisiana, Brachybacterium alimentium, Brachybacterium conglomeratum, Brachybacterium tyrofermentans, Brachyspira aalborgi, Brachyspira pilosicoli, Brachyspira sp.
HIS3, Brachyspira sp.
HIS4, Brachyspira sp.
HIS5, Brevibacillus agri, Brevibacillus brevis, Brevibacillus centrosporus, Brevibacillus choshinensis, Brevibacillus invocatus, Brevibacillus laterosporus, Brevibacillus parabrevis, Brevibacillus reuszeri, Brevibacillus sp. phR, Brevibacillus thermoruber, Brevibacterium aurantiacum, Brevibacterium casei, Brevibacterium epidermidis, Brevibacterium frigoritolerans, Brevibacterium linens, Brevibacterium mcbrellneri, Brevibacterium paucivorans, Brevibacterium sanguinis, Brevibacterium sanguinis.
H15, Brevibacterium sp.
JC43, Brevundimonas subvibrioides, Brucella abortus, Brucella canis, Brucella ceti, Brucella melitensis, Brucella microti, Brucella ovis, Brucella sp. 83_13, Brucella sp.
BO1, Brucella suis, Bryantella formatexigens, Buchnera aphidicola, Bulleidia extructa, Burkholderia ambifaria, Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia mallei, Burkholderia multivorans, Burkholderia oklahomensis, Burkholderia pseudomalk, Burkholderia pseudomallei, Burkholderia pseudomalk, Burkholderia pseudomallei, Burkholderia pseudomallei, Burkholderia pseudomalk, Burkholderia pseudomalk, Burkholderia pseudomalk, Burkholderia pseudomalk, Burkholderia pseudomalk, Burkholderia pseudomalk. 383, Burkholderia xenovorans, Burkholderiales bacterium 1_1_47, Butyricicoccus pullicaecorum, Butyricimonas virosa, Butyrivibrio crossotus, Butyrivibrio fibrisolvens, Caldimonas manganoxidans, Caminicella sporogenes, Campylobacteri coli, Campylobacteris, Campylobacteris, curvylylacteris, Campylobacteris, , Campylobacter rectus, Campylobacter showae, Campylobacter sp.
FOBRC14, Campylobacter sp.
FOBRC15,
Campylobacter sp. oral clone BB120, Campylobacter sputorum, Campylobacter upsaliensis, Candidatus Arthromitus sp.
SFB_mouse_Yit, Candidatus Sulcia muelleri, Capnocytophaga canimorsus, Capnocytophaga genomosp.
C1, Capnocytophaga gingivalis, Capnocytophaga granulosa, Capnocytophaga ochracea, Capnocytophaga sp.
GEJ8, Capnocytophaga sp. oral clone AH015, Capnocytophaga sp. oral clone ASCH05, Capnocytophaga sp. oral clone ID062, Capnocytophaga sp. oral strain A47ROY, Capnocytophaga sp. oral strain S3, Capnocytophaga sp. oral taxon 338, Capnocytophaga sp.
S1l, Capnocytophaga sputigena, Cardiobacterium hominis, Cardiobacterium valvarum, Carnobacterium divergens, Carnobacterium maltaromaticum, Catabacter hongkongensis, Catenibacterium mitsuokai, Catonella genomosp.
P1 MB5_P12 oral clone, Catonella morbi, Catonella sp. oral clone FL037, Cedecea davisae, Cellulosimicrobium funkei, Cetobacterium somerae, Chlamydia muridarum, Chlamydia psittaci, Chlamydia trachomatis, Chlamydiales bacterium NS11, Chlamydiales bacterium NS13, Chlamydiales bacterium NS16, Chlamydophylophylophila
P1, Christensenella minuta, Chromobacterium violaceum, Chryseobacterium anthropi, Chryseobacterium gleum, Chryseobacterium hominis, Citrobacter amalonaticus, Citrobacter braakii, Citrobacter farmeri, Citrobacter freundii, Citrobacter gillenii, Citrobacter, bacterium, Citrobacter, bacterium, Citrobacter, bacterium 30_2, Citrobacter sp.
KMSI_3, Citrobacter werkmanii, Citrobacter youngae, Cloacibacillus evryensis, Clostridiaceae bacterium END_2, Clostridiaceae bacterium JC13, Clostridiales bacterium 1_7_47FAA, Clostridiales bacterium 9400853, Clostridialesium bacterial, bacterial bacterial 9403326 Clostridiales bacterium ph2, Clostridiales bacterium SY8519, Clostridiales genomosp.
BVAB3, Clostridiales sp.
SM4_1, Clostridiales sp.
SS3_4, Clostridiales sp.
SSC_2, Clostridium acetobutylicum, Clostridium aerotolerans, aldenense Clostridium, Clostridium aldrichii, algidicarnis Clostridium, Clostridium algidixylanolyticum, Clostridium aminovalericum, Clostridium amygdalinum, argentinense Clostridium, Clostridium asparagiforme, Clostridium baratii, Clostridium bartlettii, Clostridium beijerinckii, Clostridium bifermentans, bolteae Clostridium, Clostridium botulinum, Clostridium butyricum, Clostridium Cadaveris, Clostridium carboxidivorans, Clostridium carnis, Clostridium celatum, Clostridium celerecrescens, Clostridium cellulosi, Clostridium chauvoei, Clostridium citroniae, Clostridium clariflavum, Clostridium clostridiiformes, Clostridium clostridioforme, Clostridium coccoides, Clostridium cochlearium, Clostridium cocleatum, Clostridium colicanis, Clostridium colinum , Clostridium difficile, Clostridium disporicum, Clostridium estertheticum, Clostridium fallax, Clostridium favososporum, Clostridium felsineum, Clostridium frigidicarnis, Clostr idium gasigenes, Clostridium ghonii, Clostridium glycolicum, Clostridium glycyrrhizinilyticum, Clostridium haemolyticum, Clostridium hathewayi, Clostridium hiranonis, Clostridium histolyticum, Clostridium hylemonae, Clostridium indolis, Clostridium innocuum, Clostridium irregulare, isatidis Clostridium, Clostridium kluyveri, Clostridium lactatifermentans, lavalense Clostridium, Clostridium leptum , Clostridium limosum, Clostridium magnum, Clostridium malenominatum, Clostridium mayombei, Clostridium methylpentosum, Clostridium nexile, Clostridium novyi, Clostridium orbiscindens, Clostridium oroticum, Clostridium paraputridium, clostridium, clostridium, clostridium, clostridium, clostridium, clostridium perfringens, Clostridium, rectum,
Clostridium saccharogumia, Clostridium saccharolyticum, Clostridium sardiniense, Clostridium sartagoforme, Clostridium scindens, Clostridium septicum, Clostridium sordellii, Clostridium sp. 7_2_43FAA, Clostridium sp.
D5, Clostridium sp.
HGF2, Clostridium sp.
HPB_46, Clostridium sp.
JC122, Clostridium sp.
L2_50, Clostridium sp.
LMG 16094, Clostridium sp.
M62_1, Clostridium sp.
MLG055, Clostridium sp.
MT4 E, Clostridium sp.
NMBHI_1, Clostridium sp.
NML 04A032, Clostridium sp.
SS2_1, Clostridium sp.
SY8519, Clostridium sp.
TM_40, Clostridium sp.
YIT 12069, Clostridium sp.
YIT 12070, Clostridium sphenoides, Clostridium spiroforme, Clostridium sporogenes, Clostridium sporosphaeroides, Clostridium stercorarium, Clostridium sticklandii, Clostridium straminisolvens, subterminale Clostridium, sulfidigenes Clostridium, Clostridium symbiosum, Clostridium tertium, Clostridium tetani, Clostridium thermocellum, Clostridium tyrobutyricum, Clostridium viride, Clostridium xylanolyticum , Collinsella aerofaciens, Collinsella intestinalis, Collinsella stercoris, Collinsella tanakaei, Comamonadaceae bacterium NML000135, Comamonadaceae bacterium NML790751, Comamonadaceae bacterium NML910035, Comamonadaceae bacterium NML910036, Comamonadaceae sp.
NSP5, Conchiformibius kuhniae, Coprobacillus cateniformis, Coprobacillus sp. 29_1, Coprobacillus sp.
D7, Coprococcus catus, Coprococcus comes, Coprococcus eutactus, Coprococcus sp.
ART55_1, Coriobacteriaceae bacterium BV3Ac1, Coriobacteriaceae bacterium JC110, Coriobacteriaceae bacterium PHI accolens Corynebacterium, Corynebacterium ammoniagenes, Corynebacterium Amycolatum, Corynebacterium appendicis, argentoratense Corynebacterium, Corynebacterium atypicum, Corynebacterium aurimucosum, bovis, Corynebacterium, canis Corynebacterium, Corynebacterium casei, Corynebacterium confusum, Corynebacterium coyleae,
Corynebacterium diphtheriae, Corynebacterium durum, efficiens Corynebacterium, Corynebacterium falsenii, Corynebacterium flavescens, Corynebacterium genitalium, Corynebacterium glaucum, Corynebacterium glucuronolyticum, Corynebacterium glutamicum, Corynebacterium hansenii, Corynebacterium imitans, Corynebacterium jeikeium, Corynebacterium kroppenstedtii, Corynebacterium lipophiloflavum, Corynebacterium macginleyi, mastitidis Corynebacterium, Corynebacterium matruchotii , Corynebacterium minutissimum, Corynebacterium mucifaciens, Corynebacterium propinquum, Corynebacterium pseudodiphtheriticum, Corynebacterium pseudogenitalium, Corynebacterium pseudotuberculosis, Corynebacterium pyruviciprodacterens, Corynebacterium resistor, Corynebacterium, Corynebacterium 1 ex ewe, Corynebacterium sp.
L_2012475, Corynebacterium sp.
NML 93_0481, Corynebacterium sp.
NML 97_0186, Corynebacterium sp.
NML 99_0018, Corynebacterium striatum, Corynebacterium sundsvallense, tuberculostearicum Corynebacterium, Corynebacterium tuscaniae, Corynebacterium ulcerans, Corynebacterium urealyticum, Corynebacterium ureicelerivorans, Corynebacterium variabile, xerosis Corynebacterium, Coxiella burnetii, malonaticus Cronobacter, Cronobacter sakazakii, turicensis Cronobacter, curtum Cryptobacterium, Cupriavidus metallidurans, Cytophaga xylanolytica , Deferribacteres sp. oral clone JV001, Deferribacteres sp. oral clone JV006, Deferribacteres sp. oral clone JV023, Deinococcus radiodurans, Deinococcus sp.
R_43890, Delftia acidovorans, Dermabacter hominis, Dermacoccus sp.
Ellin185, Active Desmospora, Desmospora sp. 8437, Desulfitobacterium frappieri, Desulfitobacterium hafniense, Desulfobulbus sp. oral clone CH031, Desulfotomaculum nigrificans, Desulfovibrio desulfuricans, Desulfovibrio fairfieldensis,
Desulfovibrio piger, Desulfovibrio sp. 3_1_syn3, Desulfovibrio vulgaris, Dialister invisus, Dialister micraerophilus, Dialister microaerophilus, Dialister pneumosintes, Dialister propionicifaciens, Dialister sp. oral taxon 502, Dialister succinatiphilus, Dietzia natronolimnaea, Dietzia sp.
BBDP51, Dietzia sp.
CA149, Dietzia timorensis, Dorea formicigenerans, Dorea longicatena, Dysgonomonas gadei, Dysgonomonas mossii, Edwardsiella tarda, Slow Eggerthella, Eggerthella sinensis, Eggerthella sp. 1_3_56FAA, Eggerthella sp.
HGA1, Eggerthella sp.
YY7918, Ehrlichia chaffeensis, Eikenella corrodens, Enhydrobacter aerosaccus, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter cancerogenus, Enterobacter cloacae, Enterobacter cowanii, Enterobacter hormaechei, Enterobacter sp. 247BMC, Enterobacter sp. 638, Enterobacter sp.
JC163, Enterobacter sp.
SCSS, Enterobacter sp.
TSE38, Enterobacteriaceae bacterium 9_2_54FAA, Enterobacteriaceae bacterium CF01Ent_1, Enterobacteriaceae bacterium Smarlab 3,302,238, Enterococcus avium, Enterococcus caccae, Enterococcus casseliflavus, durans Enterococcus, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, gilvus Enterococcus, Enterococcus hawaiiensis, Enterococcus hirae, Enterococcus italicus, Enterococcus mundtii , Enterococcus raffinosus, Enterococcus sp.
BV2CASA2, Enterococcus sp.
CCRI_16620, Enterococcus sp.
F95, Enterococcus sp.
RfL6, Enterococcus thailandicus, Eremococcus coleocola, Erysipelothrix inopinata, Erysipelothrix rhusiopathiae, Erysipelothrix tonsillarum, Erysipelotrichaceae bacterium 3_1_53, Erysipelotrichaceae bacterium 5 Escher, Escheria, Escheria, Escherichi, 5, 1_1_43, Escherichia sp. 4_1_40B, Escherichia sp.
B4, Escherichia vulneris, Ethanoligenens harbinense, Eubacteriaceae bacterium P4P_50 P4, Eubacterium barkeri, Eubacterium biforme, Eubacterium brachy, Eubacterium budayi, Eubacterium callanderi, Eubacterium cellulosolvens, Eubacterium contubum, Eubacterium contubum, Eubacterium cohort, Eubacterium coil, Eubacterium coil fissicatena, Eubacterium hadrum, Eubacterium hallii, Eubacterium infirmum, Eubacterium limosum, Eubacterium moniliforme, Eubacterium multiforme, Eubacterium nitritogenes, Eubacterium nodatum, Eubacterium ramulus, Eubacterium rectale, Eubacterium ruminantium, Eubacterum ruminantium, Eubacterium ruminantium, Eubacterium ruminantium, Eubacterium ruminantium, Eubacterium ruminantium. 3_1_31, Eubacterium sp.
AS15l, Eubacterium sp.
OBRC9, Eubacterium sp. oral clone GI038, Eubacterium sp. oral clone IR009, Eubacterium sp. oral clone JH012, Eubacterium sp. oral clone JI012, Eubacterium sp. oral clone JN088, Eubacterium sp. oral clone JS001, Eubacterium sp. OH3A oral clone, Eubacterium sp.
WAL 14571, Eubacterium tenue, Eubacterium tortuosum, Eubacterium ventriosum, Eubacterium xylanophilum, Eubacterium yurii, Ewingella americana, Exiguobacterium acetylicum, Facklamia hominis, Faecalibacterium prausnitzii, Filifactor alocis, Filifactor magosusis, Filifactor villosusus.
C1, Flavobacterium sp.
NF2_1, Flavonifractor plautii, Flexispira rappini, Flexistipes sinusarabici, Francisella novicida, Francisella philomiragia, Francisella tularensis, Fulvimonas sp.
NML 060897, Fusobacterium canifelinum, Fusobacterium genomosp.
C1, Fusobacterium genomosp.
C2, Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium naviforme, Fusobacterium necrogenes, Fusobacterium necrophorum, Fusobacterium nucleatum, Fusobacterium periodonticum, Fusobacterium russii, Fusobacterium sp. 1_1_41FAA, Fusobacterium sp. 11_3_2, Fusobacterium sp. 12_1B, Fusobacterium sp. 2_1_31, Fusobacterium sp. 3_1_27, Fusobacterium sp. 3_1_33, Fusobacterium sp. 3_1_36A2, Fusobacterium sp. 3_1_5R,
Fusobacterium sp.
AC18, Fusobacterium sp.
ACB2, Fusobacterium sp.
AS2, Fusobacterium sp.
CM1, Fusobacterium sp.
CM21, Fusobacterium sp.
CM22, Fusobacterium sp.
D12, Fusobacterium sp. oral clone ASCF06, Fusobacterium sp. oral clone ASCF11, Fusobacterium ulcerans, Fusobacterium varium, Gardnerella vaginalis, Gemella haemolysans, Gemella morbillorum, Gemella morbillorum, Gemella sanguinis, Gemella sp. oral clone ASCE02, Gemella sp. oral clone ASCF04, Gemella sp. oral clone ASCF12, Gemella sp.
WAL 1945J, Gemmiger formicilis, Geobacillus kaustophilus, Geobacillus sp.
E263, Geobacillus sp.
WCH70, stearothermophilus Geobacillus, thermocatenulatus Geobacillus, thermodenitrificans Geobacillus, thermoglucosidasius Geobacillus, thermoleovorans Geobacillus, bemidjiensis Geobacter, violaceus gloeobacter, azotocaptans Gluconacetobacter, diazotrophicus Gluconacetobacter, Gluconacetobacter entanii, europaeus Gluconacetobacter, Gluconacetobacter hansenii, Gluconacetobacter johannae, oboediens Gluconacetobacter, xylinus Gluconacetobacter, Gordonia bronchialis, Gordonia polyisoprenivorans, Gordonia sp.
KTR9, Gordonia sputi, Gordonia terrae, Gordonibacter pamelaeae, Gordonibacter pamelaeae, Gracilibacter thermotolerans, Gramella forsetii, Granulicatella adiacens, Granulicatella elegans, Granulicatella paradiacens, Granulicatella sp.
M658_99_3, Granulicatella sp. oral clone ASC02, Granulicatella sp. oral clone ASCA05, Granulicatella sp. oral clone ASCB09, Granulicatella sp. oral clone ASCG05, Grimontia hollisae, Haematobacter sp.
BC14248, Haemophilus aegyptius, Haemophilus ducreyi, Haemophilus genomosp.
P2 MB3_C24 oral clone, Haemophilus genomosp.
P3 MB3_C38 oral clone, Haemophilus haemolyticus, Haemophilus influenzae, Haemophilus parahaemolyticus, Haemophilus parainfluenzae, Haemophilus paraphrophaemolyticus, Haemophilus parasuis, Haemophilus somnus,
Haemophilus sp. 70334, Haemophilus sp.
HK445, Haemophilus sp. oral clone ASCA07, Haemophilus sp. oral clone ASCG06, Haemophilus sp. oral clone BJ021, Haemophilus sp. oral clone BJ095, Haemophilus sp. oral clone JM053, Haemophilus sp. oral taxon 851, Haemophilus sputorum, Hafnia alvei, Halomonas elongata, Halomonas johnsoniae, Halorubrum lipolyticum, Helicobacter bilis, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter pullorum, Helicobacter pylori, Helicobacter sp.
None, Helicobacter winghamensis, Heliobacterium modesticaldum, Herbaspirillum seropedicae, Herbaspirillum sp.
JC206, Histophilus somni, Holdemania filiformis, Hydrogenoanaerobacterium saccharovorans, Hyperthermus butylicus, Hyphomicrobium sulfonivorans, Hyphomonas neptunium, Ignatzschineria indica, Ignatzschineria sp.
NML 95_0260, Ignicoccus islandicus, Inquilinus limosus, Janibacter limosus, Janibacter melonis, Janthinobacterium sp.
SY12, Johnsonella ignored, Jonquetella anthropi, Kerstersia gyiorum, Kingella denitrificans, Kingella genomosp.
P1 MB2_C20 oral clone, Kingella kingae, Kingella oralis, Kingella sp. oral clone ID059, Klebsiella oxytoca, Klebsiella pneumoniae, Klebsiella sp.
AS10, Klebsiella sp.
Co9935, Klebsiella sp. enrichment culture clone SRC_DSD25, Klebsiella sp.
OBRC7, Klebsiella sp.
SP_BA, Klebsiella sp.
SRC_DSD1, Klebsiella sp.
SRC_DSD11, Klebsiella sp.
SRC_DSD12, Klebsiella sp.
SRC_DSD15, Klebsiella sp.
SRC_DSD2, Klebsiella sp.
SRC_DSD6, Klebsiella variicola, ascorbata Kluyvera, cryocrescens Kluyvera, marina Kocuria palustris Kocuria, rhizophila Kocuria, rosea Kocuria, varians Kocuria, bovis Lachnobacterium, multipara Lachnospira, Lachnospira pectinoschiza, Lachnospiraceae bacterium 1_1_57FAA, Lachnospiraceae bacterium 1_4_56FAA, Lachnospiraceae bacterium 2_1_46FAA, Lachnospiraceae bacterium 2_1_58FAA , Lachnospiraceae bacterium 3_1_57FAA_CT1, Lachnospiraceae bacterium 4_1_37FAA,
Lachnospiraceae bacterium 5_1_57FAA, Lachnospiraceae bacterium 5_1_63FAA, Lachnospiraceae bacterium 6_1_63FAA, Lachnospiraceae bacterium 8_1_57FAA, Lachnospiraceae bacterium 9_1_43BFAA, Lachnospiraceae bacterium A4, Lachnospiraceae bacterium DJF VP30, Lachnospiraceae bacterium ICM62, Lachnospiraceae bacterium MSX33, Lachnospiraceae bacterium oral taxon 107, Lachnospiraceae bacterium oral taxon F15, Lachnospiraceae genomosp.
C1 acidipiscis Lactobacillus acidophilus Lactobacillus alimentarius Lactobacillus amylolyticus Lactobacillus, Lactobacillus amylovorus, Lactobacillus Antri, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, catenaformis Lactobacillus coleohominis Lactobacillus coryniformis Lactobacillus crispatus Lactobacillus curvatus Lactobacillus, Lactobacillus delbrueckii, Lactobacillus dextrinicus, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus gastricus, Lactobacillus genomosp.
C1, Lactobacillus genomosp.
C2, Lactobacillus helveticus, Lactobacillus hilgardii, hominis Lactobacillus iners Lactobacillus, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kalixensis, kefiranofaciens Lactobacillus, Lactobacillus kefiri, Lactobacillus kimchii, Lactobacillus leichmannii, Lactobacillus mucosae, murinus Lactobacillus nodensis Lactobacillus, Lactobacillus oeni, oris Lactobacillus Lactobacillus parabrevis, Lactobacillus parabuchneri, Lactobacillus paracasei, Lactobacillus parakefiri, Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus plantarum, pontis Lactobacillus, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus rogosae, ruminis Lactobacillus, Lactobacillus sakei, salivarius Lactobacillus, Lactobacillus saniviri, Lactobacillus senioris, Lactobacillus sp . 66c, Lactobacillus sp.
BT6, Lactobacillus sp.
KLDS 1.0701, Lactobacillus sp.
KLDS 1.0702, Lactobacillus sp.
KLDS 1.0703, Lactobacillus sp.
KLDS 1.0704, Lactobacillus sp.
KLDS 1.0705, Lactobacillus sp.
KLDS
1.0707, Lactobacillus sp.
KLDS 1.0709, Lactobacillus sp.
KLDS 1.0711, Lactobacillus sp.
KLDS 1.0712, Lactobacillus sp.
KLDS 1.0713, Lactobacillus sp.
KLDS 1.0716, Lactobacillus sp.
KLDS 1.0718, Lactobacillus sp.
KLDS 1.0719, Lactobacillus sp. oral clone HT002, Lactobacillus sp. oral clone HT070, Lactobacillus sp. oral taxon 052, Lactobacillus tucceti, Lactobacillus ultunensis, Lactobacillus vaginalis, Lactobacillus vini, Lactobacillus vitulinus, Lactobacillus zeae, Lactococcus garvieae, Lactococcus lactis, Lactococcus lactis, Lactocacis raffinolactisis lactis, lactactis oral clone AP009, Legionella hackeliae, Legionella longbeachae, Legionella pneumophila, Legionella sp.
D3923, Legionella sp.
D4088, Legionella sp.
H63, Legionella sp.
NML 93L054, Legionella steelei, Leminorella grimontii, Leminorella richardii, Leptospira borgpetersenii, Leptospira broomii, Leptospira interrogans, Leptospira licerasiae, Leptotrichia buccalis, Leptotrichia genomosp.
C1, Leptotrichia goodfellowii, Leptotrichia hofstadii, Leptotrichia shahii, Leptotrichia sp. neutropenicPatient, Leptotrichia sp. oral clone GT018, Leptotrichia sp. oral clone GT020, Leptotrichia sp. oral clone HE012, Leptotrichia sp. oral clone IK040, Leptotrichia sp. oral clone P2PB_51 P1, Leptotrichia sp. Oral taxon 223, Leuconostoc carnosum, Leuconostoc citreum, Leuconostoc gasicomitatum, Leuconostoc inhae, Leuconostoc kimchii lactis, Leuconostoc, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Listeria grayi, Listeria innocua, Listeria ivanovii, Listeria monocytogenes, Listeria welshimeri, sanguinis Luteococcus, Lutispora thermophila, Lysinibacillus fusiformis, Lysinibacillus sphaericus, Macrococcus caseolyticus, Mannheimia haemolytica, Marvinbryantia formatexigens, Massilia sp.
CCUG 43427A, Megamonas funiformis, Megamonas hypermegale, Megasphaera elsdenii, Megasphaera genomosp.
C1, Megasphaera genomosp. type_1, Megasphaera micronuciformis, Megasphaera sp.
BLPYG_07, Megasphaera sp.
UPII 199_6, Metallosphaera Sedula, Methanobacterium Formicicum, Methanobrevibacter acididurans, arboriphilus Methanobrevibacter, curvatus Methanobrevibacter, cuticularis Methanobrevibacter, filiformis Methanobrevibacter, Methanobrevibacter gottschalkii, Methanobrevibacter millerae, olleyae Methanobrevibacter, oralis Methanobrevibacter, Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter thaueri, Methanobrevibacter woesei, Methanobrevibacter wolinii , Methanosphaera stadtmanae, Methylobacterium extorquens, Methylobacterium podarium, Methylobacterium radiotolerans, Methylobacterium sp. South, Methylobacterium sp.
MM4, Methylocella silvestris, Methylophilus sp.
ECd5, Microbacterium chocolatum, Microbacterium flavescens, Microbacterium gubbeenense, Microbacterium lacticum, Microbacterium oleivorans, Microbacterium oxydans, Microbacterium paraoxydans, Microbacterium phyllosphaerae, Microbacterium schleiferi, Microbacterium sp. 768, Microbacterium sp. oral strain C24KA, Microbacterium testaceum, Micrococcus antarcticus, Micrococcus luteus, Micrococcus lylae, Micrococcus sp. 185, Microcystis aeruginosa, Mitsuokella jalaludinii, Mitsuokella multacida, Mitsuokella sp. oral taxon 521, Mitsuokella sp. oral tax G68, Mobiluncus curtisii, Mobiluncus mulieris, Moellerella wisconsensis, Mogibacterium diversum, Mogibacterium neglectum, Mogibacterium pumilum, Mogibacterium timidum, Mollicutes bacterium pACH93, Moorella thermoacetica, Moraxella catarrhalis, Moraxella lincolni, Moraxella lincolni, Moraxella lincolni. 16285, Moraxella sp.
GM2, Morganella morganii, Morganella sp.
JB_T16, Morococcus cerebrosus, Moryella indoligenes, Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium alsiensis, Mycobacterium avium, Mycobacterium chelonae, Mycobacterium colombiense,
Mycobacterium elephantis, Mycobacterium gordonae, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium lacus, Mycobacterium leprae, Mycobacterium lepromatosis, Mycobacterium mageritense, Mycobacterium mantenii, Mycobacterium marinum, Mycobacterium microti, Mycobacterium neoaurum, Mycobacterium parascrofulaceum, Mycobacterium paraterrae, Mycobacterium phlei, Mycobacterium seoulense, Mycobacterium smegmatis , Mycobacterium sp. 1761, Mycobacterium sp. 1776, Mycobacterium sp. 1781, Mycobacterium sp. 1791, Mycobacterium sp. 1797, Mycobacterium sp.
AQ1GA4, Mycobacterium sp.
B10_07,09,0206, Mycobacterium sp.
GN_10546, Mycobacterium sp.
GN_10827, Mycobacterium sp.
GN_11124, Mycobacterium sp.
GN_9188, Mycobacterium sp.
GR_2007_210, Mycobacterium sp.
HE5, Mycobacterium sp.
NLA001000736, Mycobacterium sp.
W, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycobacterium vulneris, Mycoplasma agalactiae, Mycoplasma amphoriforme, Mycoplasma arthritidis, Mycoplasma bovoculi, Mycoplasma faucium, Mycoplasma, Mycoplasma, mycoplasma, mycoplasma, mycoplasma, genoplasma Mycoplasma putrefaciens, Mycoplasma salivarium, Mycoplasmataceae genomosp.
P1 MB1_G23 oral clone, Myroides odoratimimus, Myroides sp.
MY15, Neisseria bacilliformis, Neisseria cinerea, Neisseria elongata, Neisseria flavescens, Neisseria genomosp.
P2 MB5_P15 oral clone, Neisseria gonorrhoeae, Neisseria lactamica, Neisseria macacae, Neisseria meningitidis, Neisseria mucosa, Neisseria pharyngis, Neisseria polysaccharea, Neisseria sicca, Neisseria sp.
KEM232, Neisseria sp. oral clone AP132, Neisseria sp. oral clone JC012, Neisseria sp. oral strain B33KA, Neisseria sp. oral taxon 014, Neisseria sp.
SMC_A9199, Neisseria sp.
TM10_1, Neisseria subflava, Neorickettsia risticii,
Neorickettsia sennetsu, Nocardia brasiliensis, Nocardia cyriacigeorgica, Nocardia farcinica, Nocardia puris, Nocardia sp. 01_Je_025, Nocardiopsis dassonvillei, Novosphingobium aromaticivorans, Oceanobacillus caeni, Oceanobacillus sp.
Ndiop, Ochrobactrum anthropi, Ochrobactrum intermedium, Ochrobactrum pseudintermedium, Odoribacter laneus, Odoribacter splanchnicus, Okadaella gastrococcus, Oligella ureolytica, Oligella urethralis, Olsenella genomosp.
C1, Olsenella profusa, Olsenella sp.
F0004, Olsenella sp. oral taxon 809, Olsenella uli, Opitutus terrae, Oribacterium sinus, Oribacterium sp.
ACB1, Oribacterium sp.
ACB7, Oribacterium sp.
CM12, Oribacterium sp.
ICM51, Oribacterium sp.
OBRC12, Oribacterium sp. oral taxon 078, Oribacterium sp. oral tax 102, Oribacterium sp. oral taxon 108, Orientia tsutsugamushi, Ornithinibacillus bavariensis, Ornithinibacillus sp. 7_10AIA, Oscillibacter sp.
G2, Oscillibacter valericigenes, Oscillospira guilliermondii, formigenes Oxalobacter, barcinonensis Paenibacillus, Paenibacillus barengoltzii, Paenibacillus chibensis, Paenibacillus cookii, Paenibacillus durus, glucanolyticus Paenibacillus, Paenibacillus lactis, Paenibacillus lautus, Paenibacillus pabuli, Paenibacillus polymyxa, Paenibacillus popilliae, Paenibacillus sp.
CIP 101062, Paenibacillus sp.
HGF5, Paenibacillus sp.
HGF7, Paenibacillus sp.
JC66, Paenibacillus sp. oral taxon F45, Paenibacillus sp.
R_27413, Paenibacillus sp.
R_27422, Paenibacillus timonensis, Pantoea agglomerans, Pantoea ananatis, Pantoea brenneri, Pantoea citrea, Pantoea conspicua, Pantoea septica, Papillibacter cinnamivorans, Parabacteroides distasonis, Parabacteroides goldsteinii, Parabacteroides gordonii, Parabacteroides gordonii, Parabacteroides gordonii
D13, Parabacteroides sp.
NS31_3, Parachlamydia sp.
UWE25, Paracoccus denitrificans, Paracoccus marcusii, Paraprevotella clara, Paraprevotella xylaniphila,
Parascardovia denticolens, Parasutterella excrementihominis, Parasutterella secunda, Parvimonas micra, Parvimonas sp. oral taxon 110, Pasteurella bettyae, Pasteurella dagmatis, Pasteurella multocida, Pediococcus acidilactici, Pediococcus pentosaceus, Peptococcus niger, Peptococcus sp. oral clone JM048, Peptococcus sp. oral tax 167, Peptoniphilus asaccharolyticus, Peptoniphilus duerdenii, Peptoniphilus harei, Peptoniphilus indolicus, Peptoniphilus ivorii, Peptoniphilus lacrimalis, Peptoniphilus sp. gpac007, Peptoniphilus sp. gpac018A, Peptoniphilus sp. gpac077, Peptoniphilus sp. gpac148, Peptoniphilus sp.
JC140, Peptoniphilus sp. oral tax 386, Peptoniphilus sp. oral tax 836, Peptostreptococcaceae bacterium ph1, Peptostreptococcus anaerobius, Peptostreptococcus micros, Peptostreptococcus sp. 9succ1, Peptostreptococcus sp. oral clone AP24, Peptostreptococcus sp. oral clone FJ023, Peptostreptococcus sp.
P4P_31 P3, Peptostreptococcus stomatis, Phascolarctobacterium faecium, Phascolarctobacterium sp.
YIT 12068, Phascolarctobacterium succinatutens, Phenylobacterium zucineum, Photorhabdus asymbiotica, Pigmentiphaga daeguensis, Planomicrobium koreense, Plesiomonas shigelloides, Porphyromonadaceae bacterium NML 060648, Porphyromonas porphyromonas, porphyromonas poracromonas, porphyromonas asaccharolytica oral clone BB134, Porphyromonas sp. oral clone F016, Porphyromonas sp. oral clone P2PB_52 P1, Porphyromonas sp. oral clone P4GB_100 P2, Porphyromonas sp.
UQD 301, Porphyromonas uenonis, Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella corporis, Prevotella dentalis, Prevotella denticola, Prevotella denticola, Prevotella genicom.
C1, Prevotella genomosp.
C2, Prevotella genomosp.
P7 oral clone
MB2_P31, Prevotella genomosp.
P8 oral clone MB3_P13, Prevotella genomosp.
P9 oral clone MB7_G16, Prevotella heparinolytica, Prevotella histicola, Prevotella intermedia, Prevotella loescheii, Prevotella marculi, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella multisaccharivorax, Prevotella multisaccharivorax, Prevotella nanceiensis, orotelisisisigris, Prevotella pallens, Prevotella ruminicola, Prevotella salivae, Prevotella sp.
BI_42, Prevotella sp.
CM38, Prevotella sp.
ICM1, Prevotella sp.
ICM55, Prevotella sp.
JCM 6330, Prevotella sp. oral clone AA020, Prevotella sp. oral clone ASCG10, Prevotella sp. oral clone ASCG12, Prevotella sp. oral clone AU069, Prevotella sp. oral clone CY006, Prevotella sp. oral clone DA058, Prevotella sp. oral clone FL019, Prevotella sp. oral clone FU048, Prevotella sp. oral clone FW035, Prevotella sp. oral clone GI030, Prevotella sp. oral clone GI032, Prevotella sp. oral clone GI059, Prevotella sp. oral clone GU027, Prevotella sp. oral clone HF050, Prevotella sp. oral clone ID019, Prevotella sp. oral clone IDR_CEC_0055, Prevotella sp. oral clone IK053, Prevotella sp. oral clone IK062, Prevotella sp. oral clone P4PB_83 P2, Prevotella sp. oral taxon 292, Prevotella sp. oral taxon 299, Prevotella sp. oral taxon 300, Prevotella sp. oral taxon 302, Prevotella sp. oral taxon 310, Prevotella sp. oral taxon 317, Prevotella sp. oral taxon 472, Prevotella sp. oral taxon 781, Prevotella sp. oral taxon 782, Prevotella sp. oral tax F68, Prevotella sp. oral tax G60, Prevotella sp. oral tax G70, Prevotella sp. oral tax G71, Prevotella sp.
SEQ053, Prevotella sp.
SEQ065, Prevotella sp.
SEQ072, Prevotella sp.
SEQ116, Prevotella sp.
SG12, Prevotella sp. sp24, Prevotella sp. sp34, Prevotella stercorea, Prevotella tannerae, Prevotella timonensis, Prevotella veroralis, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis, Prevotella dentasini, Prevotella enoeca,
Prevotella falsenii, Prevotella fusca, Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella saccharolyi, Prevotella saccharolytica, Prevotella saccharolytica, Prevla Propionibacteriaceae bacterium NML 02_0265, Propionibacterium acidipropionici, Propionibacterium acnes, Propionibacterium avidum, Propionibacterium freudenreichii, Propionibacterium granulosum, Propionibacterium jensenii, Propionibacterium propionicum, Propionibacterium sp. 434_HC2, Propionibacterium sp.
H456, Propionibacterium sp.
LG, Propionibacterium sp. oral taxon 192, Propionibacterium sp.
S555a, Propionibacterium thoenii, Proteus mirabilis, Proteus penneri, Proteus sp.
HS7514, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia rustigianii, Providencia stuartii, Pseudoclavibacter sp.
Timone, Pseudoflavonifractor capillosus, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas gessardii, Pseudomonas mendocina, Pseudomonas monteilii, Pseudomonas poae, Pseudomonas pseudoalalkigenes, Pseudomonas putida, Pseudomonas putida, Pseudomonas putida, Pseudomonas putida, Pseudomonas putida. 2_1_26, Pseudomonas sp.
G1229, Pseudomonas sp.
NP522l, Pseudomonas stutzeri, Pseudomonas tolaasii, Pseudomonas viridiflava, Pseudoramibacter alactolyticus, Psychrobacter arcticus, Psychrobacter cibarius, Psychrobacter cryohalolentis, Psychrobacter faecalis, Psychrobacter nivimaris, Psychrobacter sp. 13983, Pyramidobacter piscolens, Ralstonia pickettii, Ralstonia sp. 5_7_47FAA, Raoultella ornithinolytica, Raoultella planticola, Raoultella terrigena, Rhodobacter sp. oral taxon C30, Rhodobacter sphaeroides, Rhodococcus corynebacterioides, Rhodococcus equi, Rhodococcus erythropolis,
Rhodococcus fascians, Rhodopseudomonas palustris, Rickettsia akari, Rickettsia conorii, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia slovaca, Rickettsia typhi, Robinsoniella peoriensis, Roseburia cecicola, Roseburia faecalis, Roseburia, Roseburia, Roseburia fae 11SE37, Roseburia sp. 11SE38, Roseiflexus castenholzii, Roseomonas cervicalis, Roseomonas mucosa, Roseomonas sp.
NML94_0193, Roseomonas sp.
NML97_0121, Roseomonas sp.
NML98_0009, Roseomonas sp.
NML98_0157, Rothia aeria, Rothia dentocariosa, Rothia mucilaginosa, Rothia nasimurium, Rothia sp. oral taxon 188, Ruminobacter amylophilus, Ruminococcaceae bacterium D16, Ruminococcus albus, Ruminococcus bromii, Ruminococcus callidus, Ruminococcus champanellensis, Ruminococcus flavefaciens, Ruminococcus gnavus, Ruminococcus rnococcus Rnsocococcus rnsocococcus rnsococcus rocococcus hansenii. 18P13, Ruminococcus sp. 5_1_39BFAA, Ruminococcus sp. 9SE51, Ruminococcus sp.
ID8, Ruminococcus sp.
K_1, Ruminococcus torques, Saccharomonospora viridis, Salmonella bongori, Salmonella enterica, Salmonella enterica, Salmonella enterica, Salmonella enterica, Salmonella enterica, Salmonella enterica, Salmonella enterica, Salmonella enterica, Salmonella enterica, Salmonella enterica, Salmonella enterica, Salmonella enterica, Salmonella enterica, Salmonella enterica, Salmonella enterica, Salmonella enterica, Salmonella enterica Salmonella typhimurium, Sarcina ventriculi, Scardovia inopinata, Scardovia wiggsiae, Segniliparus rotundus, Segniliparus rugosus, Selenomonas artemidis, Selenomonas dianae, Selenomonas flueggei, Selenomonas genomosp.
C1, Selenomonas genomosp.
C2, Selenomonas genomosp.
P5, Selenomonas genomosp.
P6 MB3_C41 oral clone, Selenomonas genomosp.
P7 oral clone MB5_C08, Selenomonas genomosp.
P8 oral clone MB5_P06, Selenomonas infelix, Selenomonas noxia, Selenomonas ruminantium, Selenomonas sp.
FOBRC9, Selenomonas sp. oral clone FT050, Selenomonas sp. oral clone GI064, Selenomonas sp. oral clone GT010, Selenomonas sp. oral clone HU051, Selenomonas sp. oral clone IK004, Selenomonas sp. oral clone IQ048, Selenomonas sp. oral clone JI021, Selenomonas sp. oral clone JS031, Selenomonas sp. OH4A oral clone, Selenomonas sp. oral clone P2PA_80 P4, Selenomonas sp. oral tax 137, Selenomonas sp. oral taxon 149, Selenomonas sputigena, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Serratia odorifera, Serratia proteamaculans, Shewanella putrefaciens, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Shuttleworthia satelles.
MSX8B, Shuttleworthia sp. oral tax G69, Simonsiella muelleri, Slackia equolifaciens, Slackia exigua, Slackia faecicanis, Slackia heliotrinireducens, Slackia isoflavoniconvertens, Slackia piriformis, Slackia sp.
NATTS, Solobacterium moorei, Sphingobacterium faecium, Sphingobacterium mizutaii, Sphingobacterium multivorum, Sphingobacterium spiritivorum, Sphingomonas echinoides, Sphingomonas sp. oral clone FI012, Sphingomonas sp. oral clone FZ016, Sphingomonas sp. oral tax A09, Sphingomonas sp. oral taxon F71, Sphingopyxis alaskensis, Spiroplasma insolitum, Sporobacter termitidis, Sporolactobacillus inulinus, Sporolactobacillus nakayamae, Sporosarcina newyorkensis, Sporosarcina sp. 2681, Staphylococcaceae bacterium NML 92_0017, Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus carnosus, Staphylococcus cohnii, Staphylococcus condimenti, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus fleurettii, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus pasteuri Staphylococcus pseudintermedius, Staphylococcus saccharolyticus, Staphylococcus saprophyticus, Staphylococcus sciuri, Staphylococcus sp. bottae7 clone, Staphylococcus sp.
H292, Staphylococcus sp.
H780, Staphylococcus succinus, Staphylococcus vitulinus, Staphylococcus warneri, Staphylococcus xylosus, Stenotrophomonas maltophilia, Stenotrophomonas sp.
FG_6, Streptobacillus moniliformis, Streptococcus agalactiae, Streptococcus alactolyticus, Streptococcus anginosus, Streptococcus australis, Streptococcus bovis, Streptococcus canis, Streptococcus constellatus, Streptococcus cristatus, Streptococcus downei, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equinus, Streptococcus gallolyticus, Streptococcus genomosp.
C1, Streptococcus genomosp.
C2, Streptococcus genomosp.
C3, Streptococcus genomosp.
C4, Streptococcus genomosp.
C5, Streptococcus genomosp.
C6, Streptococcus genomosp.
C7, Streptococcus genomosp.
C8, Streptococcus gordonii, Streptococcus infantarius, Streptococcus infantis, Streptococcus intermedius, Streptococcus lutetiensis, Streptococcus massiliensis, Streptococcus milleri, Streptococcus mitis, Streptococcus mutans, Streptococcus oligofermentans, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus pasteurianus, Streptococcus peroris, Streptococcus pneumoniae, Streptococcus porcinus, Streptococcus pseudopneumoniae, Streptococcus pseudoporcinus, Streptococcus pyogenes, Streptococcus ratti, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sinensis, Streptococcus sp. 16362, Streptococcus sp. 2_1_36FAA, Streptococcus sp. 2285_97, Streptococcus sp. 69130, Streptococcus sp.
AC15, Streptococcus sp.
ACS2, Streptococcus sp.
AS20, Streptococcus sp.
BS35a, Streptococcus sp.
C150, Streptococcus sp.
CM6, Streptococcus sp.
CM7, Streptococcus sp.
ICM10, Streptococcus sp.
ICM12, Streptococcus sp.
ICM2, Streptococcus sp.
ICM4, Streptococcus sp.
ICM45, Streptococcus sp.
M143, Streptococcus sp.
M334, Streptococcus sp.
OBRC6, Streptococcus sp.
oral clone ASB02, Streptococcus sp. oral clone ASCA03, Streptococcus sp. oral clone ASCA04, Streptococcus sp. oral clone ASCA09, Streptococcus sp. oral clone ASCB04, Streptococcus sp. oral clone ASCB06, Streptococcus sp. oral clone ASCC04, Streptococcus sp. oral clone ASCC05, Streptococcus sp. oral clone ASCC12, Streptococcus sp. oral clone ASCD01, Streptococcus sp. oral clone ASCD09, Streptococcus sp. oral clone ASCD10, Streptococcus sp. oral clone ASCE03, Streptococcus sp. oral clone ASCE04, Streptococcus sp. oral clone ASCE05, Streptococcus sp. oral clone ASCE06, Streptococcus sp. oral clone ASCE09, Streptococcus sp. oral clone ASCE10, Streptococcus sp. oral clone ASCE12, Streptococcus sp. oral clone ASCF05, Streptococcus sp. oral clone ASCF07, Streptococcus sp. oral clone ASCF09, Streptococcus sp. oral clone ASCG04, Streptococcus sp. oral clone BW009, Streptococcus sp. oral clone CH016, Streptococcus sp. oral clone GK051, Streptococcus sp. oral clone GM006, Streptococcus sp. oral clone P2PA_41 P2, Streptococcus sp. oral clone P4PA_30 P4, Streptococcus sp. oral taxon 071, Streptococcus sp. oral tax G59, Streptococcus sp. oral tax G62, Streptococcus sp. oral tax G63, Streptococcus sp.
SHV515, Streptococcus suis, Streptococcus thermophilus, Streptococcus uberis, Streptococcus urinalis, Streptococcus vestibularis, Streptococcus viridans, Streptomyces albus, Streptomyces griseus, Streptomyces sp. 1 AIP_2009, Streptomyces sp.
SD 511, Streptomyces sp.
SD 524, Streptomyces sp.
SD 528, Streptomyces sp.
SD 534, Streptomyces thermoviolaceus, Subdoligranulum variabile, Succinatimonas hippei, Sutterella morbirenis, Sutterella parvirubra, Sutterella sanguinus, Sutterella sp.
YIT 12072, Sutterella stercoricanis, Sutterella wadsworthensis, Synergistes genomosp.
C1, Synergistes sp.
RMA 14551, Synergistetes bacterium ADV897, Synergistetes bacterium LBVCM1157,
Synergistetes bacterium taxon oral 362, Synergistetes bacterium taxon oral D48, Syntrophococcus sucromutans, Syntrophomonadaceae genomosp.
P1, Tannerella forsythia, Tannerella sp. 6_1_58FAA_CT1, Tatlockia micdadei, Tatumella ptyseos, Tessaracoccus sp. oral taxon F04, Tetragenococcus halophilus, Tetragenococcus koreensis, Thermoanaerobacter pseudethanolicus, Thermobifida fusca, Thermofilum pendens, Thermus aquaticus, Tissierella praeacuta, Trabulsiella guamensis, Treponema denticola, Treponema genicom, Treponema
P1, Treponema genomosp.
P4 oral clone MB2_G19, Treponema genomosp.
P5 oral clone MB3_P23, Treponema genomosp.
P6 MB4_G11 oral clone, Treponema lecithinolyticum, Treponema pallidum, Treponema parvum, Treponema phagedenis, Treponema putidum, Treponema refringens, Treponema socranskii, Treponema sp. 6: H: D15A_4, Treponema sp. clone DDKL_4, Treponema sp. oral clone JU025, Treponema sp. oral clone JU031, Treponema sp. oral clone P2PB_53 P3, Treponema sp. oral taxon 228, Treponema sp. oral taxon 230, Treponema sp. oral taxon 231, Treponema sp. oral taxon 232, Treponema sp. oral taxon 235, Treponema sp. oral taxon 239, Treponema sp. oral taxon 247, Treponema sp. oral taxon 250, Treponema sp. oral tax 251, Treponema sp. oral taxon 254, Treponema sp. oral taxon 265, Treponema sp. oral taxon 270, Treponema sp. oral taxon 271, Treponema sp. oral taxon 508, Treponema sp. oral taxon 518, Treponema sp. oral tax G85, Treponema sp. rot cases of sheep, Treponema Vincentii, Tropheryma whipplei, pyogenes Trueperella, paurometabola Tsukamurella, Tsukamurella tyrosinosolvens, Turicibacter sanguinis, Ureaplasma parvum, Ureaplasma urealyticum, Ureibacillus composti, Ureibacillus suwonensis, Ureibacillus Terrenus, Ureibacillus thermophilus, Ureibacillus thermosphaericus, fluvialis Vagococcus, Veillonella atypica , Veillonella dispar, Veillonella genomosp.
P1 MB5_P17 oral clone, Veillonella montpellierensis, Veillonella parvula, Veillonella sp. 3_1_44, Veillonella sp. 6_1_27, Veillonella sp. ACP1, Veillonella sp. AS16, Veillonella sp. BS32l, Veillonella sp. ICM51a, Veillonella sp. MSA12, Veillonella sp. NVG 100cf, Veillonella sp. OK11, Veillonella sp. oral clone ASCA08, Veillonella sp. oral clone ASCB03, Veillonella sp. oral clone ASCG01, Veillonella sp. oral clone ASCG02, Veillonella sp. OH1A oral clone, Veillonella sp. oral tax 158, Veillonellaceae bacterium oral tax 131, Veillonellaceae bacterium oral tax 155, Vibrio cholerae, Vibrio fluvialis, Vibrio furnissii, Vibrio mimicus, Vibrio parahaemolyticus, Vibrio sp. RC341, Vibrio vulnificus, Victivallaceae bacterium NML 080035, Victivallis vadensis, Virgibacillus proomii, Weissella beninensis, Weissella cibaria, Weissella confusa, Weissella hellenica, Weissella kandleri, Weissella kamesensis, Weissella paramesenteroides. KLDS 7.0701, Wolinella succinogenes, Xanthomonadaceae bacterium NML 03_0222, Xanthomonas campestris, Xanthomonas sp. kmd_489, Xenophilus aerolatus, Yersinia aldovae, Yersinia aleksiciae, Yersinia bercovieri, Yersinia enterocolitica, Yersinia frederiksenii, Yersinia intermedia, Yersinia kristensenii, Yersinia mollaretii, Yersinia pestis, Yersinia pysis, Yersinia pysudotubis, Yersinia pseudotubis, Yersinia pseis Paraclostridium benzoelyticum, Dielma fastidiosa, Longicatena caecimuris, and Veillonella tobetsuensis.
[12]
12. Pharmaceutical composition according to claim 11, characterized by the fact that the EV and bacteria are of the same species or strain.
[13]
13. Pharmaceutical composition according to claim 11, characterized by the fact that the EV and bacteria are of different species or strains.
[14]
Pharmaceutical composition according to any one of claims 1 to 13, characterized in that the pharmaceutical composition is formulated for oral administration.
[15]
Pharmaceutical composition according to any one of claims 1 to 14, characterized in that the composition further comprises an additional therapeutic.
[16]
16. Pharmaceutical composition, according to claim 15, characterized by the fact that the additional treatment is a cancer treatment.
[17]
17. Pharmaceutical composition according to claim 16, characterized by the fact that the cancer therapy comprises a chemotherapeutic agent.
[18]
18. Pharmaceutical composition, according to claim 17, characterized by the fact that the chemotherapeutic agent is selected from the group consisting of thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carbocone, meturedopa, uredopa, altretamine, triethylenomelamine, triethylenophosphoramide, triethylenothiophosphoramide, trimethylolomelamine, bulatacin, bulatacinone, camptothecin, topotecan, briostatin, calistatin, CC-1065, cryptoficin 1, cryptophine, clothephine, chloroquine, chloroquine, sarcobutine, panathistatin, panthystatin mecloretamine, meclorethamine oxide hydrochloride, melphalan, novembiquin, phenesterin, prednimustine, trophosphamide, uracil mustard, carmustine, chloro zotocin, photemustine, lomustine, nimustine, ranimnustine, calicheamicin, dinamycin, clodronate; neocarzinostatin chromophore, aclacinomisins, actinomycin, autrarnicin, azasserine, bleomycins, cactinomycin, carabicin, Caminomycin, carzinophylline, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxorubin, idrubicin, lorubin, and
marcelomycin, mitomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, chelamycin, rhodorubicin, streptozine, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, methotrexate, methotrexate, 5-fluorourate, 5-fluourour , pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, tiamiprine, thioguanine, ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, testosterone, floxuridine, detoxoxin, ammonium, propolis, calusterone, caluster , trilostane, frolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, ansacrine, bestrabucil, bisanthrene, edatraxate, defofamine, demecolcin, diaziquone, elformitin, eliptinium, hydroxy, nitrate, epithelone, hydroxy, acetate, epithelone, hydroxy maytansine, ansamitocins, mitoguazone, mitoxantrone, mopidanmol, nitraerin, pen tostatin, fenamet, pyrarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, PSK polysaccharide complex, razoxane, rhizoxin, sizofuran, spirogermanium, tenuazonic acid, triaziquone; 2,2 ', 2' '- trichlorotriethylamine, trichothecene, T-2 toxin, verracurin A, roridin A, anguidine, urethane, vindesine, dacarbazine, manomustine, mitobronitol, mitolactol, pipobroman, gacitosin, arabinoside, cyclophosphamide, cyclophosphamide, ti doxetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, cisplatin, oxaliplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, iratine, amine, xenotype, edatrexine, RFS 2000, difluoromethylomitine, retinoic acid and capecitabine.
[19]
19. Pharmaceutical composition according to any one of claims 16 to 18, characterized by the fact that the cancer therapy comprises an immunotherapeutic agent against cancer.
[20]
20. Pharmaceutical composition according to claim 19, characterized by the fact that the cancer immunotherapeutic agent comprises an immune checkpoint inhibitor.
[21]
21. Pharmaceutical composition according to claim 20, characterized in that the immune checkpoint inhibitor is an antibody or antigen binding fragment thereof that specifically binds to an immune checkpoint protein.
[22]
22. Pharmaceutical composition according to claim 21, characterized by the fact that the immune checkpoint protein is selected from the group consisting of CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7- H3, B7-H4, BTLA, KIR, LAG3, TIM-3 or VISTA.
[23]
23. Pharmaceutical composition according to claim 20, characterized by the fact that the immune checkpoint inhibitor is selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010.
[24]
24. Pharmaceutical composition according to any one of claims 19 to 23, characterized by the fact that the cancer immunotherapeutic agent comprises a cancer specific antibody or antigen-binding fragment thereof.
[25]
25. Pharmaceutical composition according to claim 24, characterized by the fact that the cancer-specific antibody or antigen-binding fragment thereof specifically binds to a cancer-associated antigen.
[26]
26. Pharmaceutical composition according to claim 25, characterized by the fact that the cancer-associated antigen is selected from the group consisting of adipophyllin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein ("AFP "), ARTC1, B- RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (" CEA "), CASP- 5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen ("ETA"), ETV6-AML1, EZH2, FGF5, FLT3-ITD, FN1, G250 / MN fusion protein / CAIX, GAGE-1,2,8, GAGE- 3,4,5,6,7, GAS7, glypican-3, GnTV, gp100 / Pmel17, GPNMB, HAUS3, hepsin, HER-2 / neu, HERV-K -MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralfa2, intestinal carboxyl esterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN- 1, KMHN1 also known o such as CCDC110, LAGE-1, LDLR-fucosyltransferase AS fusion protein, lengsine, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, melan-A / MART-1, Meloe, midquine, MMP-2, MMP -7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, myosin, myosin class I, N-crude, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO -1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RARalpha fusion protein, polymorphic epithelial mucin ("PEM"), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secernina 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1 , survivin, SYT-SSX1 fusion protein or
SYT-SSX2, TAG-1, TAG-2, telomerase, TGF-betaRII, TPBG, TRAG-3, triosphosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase ("TYR"), VEGF, WT1 and XAGE-1b / GAGED2a.
[27]
27. Pharmaceutical composition, according to claim 26, characterized by the fact that the cancer-associated antigen is a neo-antigen.
[28]
28. Pharmaceutical composition according to any one of claims 19 to 27, characterized by the fact that the cancer immunotherapeutic agent comprises a cancer vaccine.
[29]
29. Pharmaceutical composition according to claim 28, characterized in that the cancer vaccine comprises a polypeptide comprising an epitope of a cancer-associated antigen.
[30]
30. Pharmaceutical composition according to claim 29, characterized by the fact that the cancer-associated antigen is selected from the group consisting of adipophyllin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein ("AFP "), ARTC1, B- RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (" CEA "), CASP- 5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen ("ETA"), ETV6-AML1, EZH2, FGF5, FLT3-ITD, FN1, G250 / MN fusion protein / CAIX, GAGE-1,2,8, GAGE- 3,4,5,6,7, GAS7, glypican-3, GnTV, gp100 / Pmel17, GPNMB, HAUS3, hepsin, HER-2 / neu, HERV-K -MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralfa2, intestinal carboxyl esterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN- 1, KMHN1 also known o such as CCDC110, LAGE-1, LDLR-fucosyltransferase AS fusion protein, lengsine, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, melan-A / MART-1, Meloe, midquine, MMP-2, MMP -7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, myosin, myosin class I, N-crude, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO -1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RARalpha fusion protein, polymorphic epithelial mucin ("PEM"), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secernina 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1 , survivin, SYT-SSX1 or SYT-SSX2 fusion protein, TAG-1, TAG-2, telomerase, TGF-betaRII, TPBG, TRAG-3, triosphosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2 , tyrosinase, tyrosinase ("TYR"), VEGF, WT1 and XAGE-1b / GAGED2a.
[31]
31. Pharmaceutical composition according to claim 29, characterized by the fact that the cancer-associated antigen is a neo-antigen.
[32]
32. Pharmaceutical composition according to any one of claims 29 to 31, characterized in that the polypeptide is a fusion protein.
[33]
33. Pharmaceutical composition according to claim 28, characterized by the fact that the cancer vaccine comprises a nucleic acid that encodes an epitope of a cancer-associated antigen.
[34]
34. Pharmaceutical composition according to claim 33, characterized by the fact that the cancer-associated antigen is selected from the group consisting of adipophyllin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein ("AFP "), ARTC1, B-
RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen ("CEA"), CASP-5, CASP-8, CD274 , CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen ("ETA"), ETV6-AML1, EZH2, FGF5, FLT3-ITD, FN1, G250 / MN / CAIX, GAGE-1, fusion protein, GAGE-1, 2.8, GAGE- 3,4,5,6,7, GAS7, glypican-3, GnTV, gp100 / Pmel17, GPNMB, HAUS3, hepsin, HER-2 / neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralfa2, intestinal carboxyl esterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110 , LAGE-1, LDLR-fucosyltransferase AS fusion protein, lengsine, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9 , MAGE-C1, MAGE-C2, malic enzyme, mamaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, melan- A / MART-1, Meloe, midquine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, myosin, myosin class I, N-crude, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1 / LAGE-2, OA1, OGT, OS-9, polypeptide P, p53, PAP, PAX5, PBF, pml-RARalpha fusion protein, epithelial mucin polymorphic ("PEM"), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secernina 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or SYT-SSX2 fusion protein, TAG-1, TAG-2, telomerase, TGF-betaRII, TPBG, TRAG-3, triosphosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase ("TYR"), VEGF, WT1 and XAGE-1b / GAGED2a.
[35]
35. Pharmaceutical composition according to claim 33, characterized by the fact that the cancer-associated antigen is a neo-antigen.
[36]
36. Pharmaceutical composition according to any one of claims 33 to 35, characterized by the fact that the nucleic acid is DNA.
[37]
37. Pharmaceutical composition according to any one of claims 33 to 35, characterized in that the nucleic acid is RNA.
[38]
38. Pharmaceutical composition according to claim 37, characterized by the fact that RNA is mRNA.
[39]
39. Pharmaceutical composition according to any one of claims 36 to 38, characterized by the fact that the nucleic acid is in a vector.
[40]
40. Pharmaceutical composition according to claim 39, characterized by the fact that the vector is a bacterial vector.
[41]
41. Pharmaceutical composition according to claim 40, characterized by the fact that the bacterial vector is selected from the group consisting of Mycobacterium bovis (BCG), Salmonella Typhimurium ssp., Salmonella Typhi ssp., Clostridium sp. Spores, Escherichia coli Nissle 1917, Escherichia coli K-12 / LLO, Listeria monocytogenes, and Shigella flexneri.
[42]
42. Pharmaceutical composition according to claim 39, characterized by the fact that the vector is a viral vector.
[43]
43. Pharmaceutical composition according to claim 42, characterized by the fact that the viral vector is selected from the group consisting of vaccinia, adenovirus, RNA virus, and avipox with replication defect, fowlpox with replication defect, canarypox with replication defect, MVA with replication defect and adenovirus with replication defect.
[44]
44. Pharmaceutical composition according to any one of claims 19 to 43, characterized in that the immunotherapeutic agent comprises an antigen presenting cell (APC) prepared with a cancer specific antigen.
[45]
45. Pharmaceutical composition according to claim 44, characterized by the fact that APC is a dendritic cell, a macrophage or a B cell.
[46]
46. Pharmaceutical composition according to claim 44 or 45, characterized by the fact that the cancer-associated antigen is selected from the group consisting of adipophyllin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein ( "AFP"), ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABL b3a2 fusion protein, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen ("CEA"), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin-A1, k-can fusion protein, DKK1, EFTUD2, elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen ("ETA"), ETV6-AML1, EZH2, FGF5, FLT3-ITD, FN1, G250 fusion protein / MN / CAIX, GAGE-1,2,8, GAGE- 3,4,5,6,7, GAS7, glypican-3, GnTV, gp100 / Pmel17, GPNMB, HAUS3, hepsin, HER-2 / neu, HERV -K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralfa2, intestinal carboxyl esterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM- HN-1, KMHN1 also know as CCDC110, LAGE-1, LDLR-fucosyltransferase AS fusion protein, lengsine, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, melan-A / MART-1, Meloe, midquine, MMP-2, MMP -7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, myosin, myosin class I, N-crude, NA88-A, neo-PAP, NFYC,
NY-BR-1, NY-ESO-1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RARalfa fusion protein, polymorphic epithelial mucin ("PEM") , PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secernina 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or SYT-SSX2, TAG-1, TAG-2, telomerase, TGF-betaRII, TPBG, TRAG-3, triosphosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase ("TYR"), VEGF, WT1 and XAGE-1b / GAGED2a.
[47]
47. Pharmaceutical composition according to claim 44 or 45, characterized by the fact that the cancer-associated antigen is a neo-antigen.
[48]
48. Pharmaceutical composition according to any one of claims 19 to 47, characterized in that the immunotherapeutic agent comprises a cancer-specific chimeric antigen (CAR) receptor.
[49]
49. Pharmaceutical composition according to claim 48, characterized by the fact that the CAR is administered on the surface of a T cell.
[50]
50. Pharmaceutical composition according to claim 48 or 49, characterized by the fact that the CAR binds specifically to a cancer-associated antigen.
[51]
51. Pharmaceutical composition according to claim 50, characterized by the fact that the cancer-associated antigen is selected from the group consisting of adipophyllin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein ("AFP "), ARTC1, B- RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (" CEA "), CASP- 5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2,
cyclin D1, cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen ("ETA"), ETV6 fusion protein -AML1, EZH2, FGF5, FLT3-ITD, FN1, G250 / MN / CAIX, GAGE-1,2,8, GAGE- 3,4,5,6,7, GAS7, glypican-3, GnTV, gp100 / Pmel17 , GPNMB, HAUS3, hepsin, HER-2 / neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralfa2, intestinal carboxyl esterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1, LDLR-fucosyltransferase AS fusion protein, lengsine, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mamaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2 , ME1, melan-A / MART-1, Meloe, midquina, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, myosin, myosin class I, N-crude , NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, protein f use pml-RARalfa, polymorphic epithelial mucin ("PEM"), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secernina 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or SYT-SSX2, TAG-1, TAG-2, telomerase, TGF-betaRII fusion protein, TPBG, TRAG-3, triosphosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase ("TYR"), VEGF, WT1 and XAGE-1b / GAGED2a.
[52]
52. Pharmaceutical composition according to claim 49, characterized by the fact that the cancer-associated antigen is a neo-antigen.
[53]
53. Pharmaceutical composition according to any one of claims 19 to 52, characterized in that the immunotherapeutic agent comprises a cancer-specific T cell.
[54]
54. Pharmaceutical composition according to claim 53, characterized in that the T cell is a CD4 + T cell.
[55]
55. Pharmaceutical composition according to claim 54, characterized in that the CD4 + T cell is a TH1 T cell, a TH2 T cell or a TH17 T cell.
[56]
56. Pharmaceutical composition according to any one of claims 53 to 55, characterized in that the T cell expresses a specific T cell receptor for a cancer-associated antigen.
[57]
57. Pharmaceutical composition according to claim 56, characterized by the fact that the cancer-associated antigen is selected from the group consisting of adipophyllin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein ("AFP "), ARTC1, B- RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (" CEA "), CASP- 5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen ("ETA"), ETV6-AML1, EZH2, FGF5, FLT3-ITD, FN1, G250 / MN fusion protein / CAIX, GAGE-1,2,8, GAGE- 3,4,5,6,7, GAS7, glypican-3, GnTV, gp100 / Pmel17, GPNMB, HAUS3, hepsin, HER-2 / neu, HERV-K -MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralfa2, intestinal carboxyl esterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN- 1, KMHN1 also known o such as CCDC110, LAGE-1, LDLR-fucosyltransferase AS fusion protein, lengsine, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9,
MAGE-C1, MAGE-C2, malic enzyme, mamaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, melan-A / MART-1, Meloe, midquina, MMP-2, MMP-7, MUC1 , MUC5AC, mucin, MUM-1, MUM-2, MUM-3, myosin, myosin class I, N-crude, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1 / LAGE -2, OA1, OGT, OS-9, polypeptide P, p53, PAP, PAX5, PBF, fusion protein pml-RARalfa, polymorphic epithelial mucin ("PEM"), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secernina 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, protein fusion system SYT-SSX1 or SYT-SSX2, TAG-1, TAG-2, telomerase, TGF-betaRII, TPBG, TRAG-3, triosphosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase ("TYR"), VEGF, WT1 and XAGE-1b / GAGED2a.
[58]
58. Pharmaceutical composition according to any one of claims 19 to 57, characterized by the fact that the immunotherapeutic agent comprises an immune activating protein.
[59]
59. Pharmaceutical composition according to claim 58, characterized by the fact that the immune activating protein is a cytokine or chemokine.
[60]
60. Pharmaceutical composition according to claim 59, characterized by the fact that the immune activating protein is selected from the group consisting of B lymphocyte chemoattractant ("BLC"), chemokine with CC 11 motif ("Eotaxin-1 "), eosinophilic chemotactic protein 2 (" Eotaxin-2 "), granulocyte colony stimulating factor (" G-CSF "), granulocyte macrophage colony stimulating factor (" GM-CSF), 1-309, molecule of intercellular adhesion 1 ("ICAM-1"), interferon alfa ("IFN-alpha"), interferon beta ("IFN-beta"), interferon gamma ("IFN-gamma"), interlucin-1 alpha ("IL-1 alpha "), interlucin-1 beta (" IL-1 beta "), interleukin 1 receptor antagonist (" IL-1 ra "), interleukin-2 (" IL-2 "), interleukin-4 (" IL- 4 "), interleukin-5 (" IL-5 "), interleukin-6 (" IL-6 "), interleukin-6 soluble receptor (" IL-6 sR "), interleukin-7 (" IL-7 " ), interleukin-8 ("IL-8"), interleukin-10 ("IL-10"), interleukin-11 ("IL-11"), interleukin-12 beta subunit ("IL-12 p40 "or" IL-12 p70 "), interleukin-13 (" IL-13 "), interleukin-15 (" IL-15 "), interleukin-16 (" IL-16 "), interleukin-17A-F ( "IL-17A-F"), interleukin-18 ("IL-18"), interleukin-21 ("IL-21"), interleukin-22 ("IL-22"), interleukin-23 ("IL-23 "), interleukin-33 (" IL-33 "), chemokine ligand (with CC motif) 2 (" MCP-1 "), macrophage colony stimulating factor (" M-CSF "), gamma interferon-induced monocin ("MIG"), chemokine 2 ligand (with CC motif) ("MIP-1 alpha"), chemokine 4 ligand (with CC motif) ("MIP-1 beta"), macrophage inflammatory protein-1-delta ("MIP-1 delta"), platelet-derived growth factor B subunit ("PDGF-BB"), chemokine 5 ligand (with CC motif), expressed and secreted normal T cell regulated at activation ("RANTES") , TIMP metallopeptidase 1 ("TIMP-1") inhibitor, TIMP metallopeptidase 2 ("TIMP-2") inhibitor, tumor necrosis factor-lymphotoxin-alpha ("TNF alpha"), tumor necrosis factor-beta lymphotoxin ("TNF beta"), r soluble TNF type 1 receptor ("sTNFRI"), sTNFRIIAR, brain-derived neurotrophic factor ("BDNF"), basic fibroblast growth factor ("bFGF"), bone morphogenetic protein 4 ("BMP-4"), protein bone morphogenetics 5 ("BMP-5"), bone morphogenetic protein 7 ("BMP-7"), nerve growth factor ("b-NGF"), epidermal growth factor ("EGF"), growth factor receptor epidermal ("EGFR"), vascular endothelial growth factor derived from the endocrine gland ("EG-VEGF"), fibroblast growth factor 4 ("FGF-4"), keratinocyte growth factor ("FGF-7") , growth differentiation factor 15 ("GDF-15"), glial cell-derived neurotrophic factor ("GDNF"), growth hormone-like EGF-like growth factor ("HB-EGF"), hepatocyte growth factor ("HGF"), insulin-like growth factor-1 binding protein ("IGFBP-1"), insulin-like growth factor-binding protein ina 2 ("IGFBP-2"), insulin-like growth factor-binding protein 3 ("IGFBP-3"), insulin-like growth factor-binding protein 4 ("IGFBP-4"), protein binding to insulin-like growth factor 6 ("IGFBP-6"), insulin-like growth factor 1 ("IGF-1"), insulin, macrophage colony stimulating factor ("M-CSF R"), receptor nerve growth factor ("NGF R"), neurotrophin-3 ("NT-3"), neurotrophin-4 ("NT-4"), osteoclastogenesis inhibitory factor ("osteoprotegerin"), derived growth factor receptors platelet ("PDGF-AA"), phosphatidylinositol-glycan biosynthesis ("PIGF"), Skp, culina, F-box containing complex ("SCF"), stem cell factor receptor ("SCF R"), transforming growth factor alpha ("TGFalfa"), transforming growth factor beta-1 ("TGF beta 1"), transforming growth factor beta-3 ("TGF beta 3"), vascular endothelial growth factor ("VEGF" ), receptor of the vascular endothelial growth 2 ("VEGFR2"), receptor for vascular endothelial growth factor 3 ("VEGFR3"), VEGF-D 6Ckine, protein tyrosine kinase UFO receptor ("Axl"), beta cellulin ("BTC"), epithelial chemokine mucosal associated ("CCL28"), chemokine 27 ligand (with CC motif) ("CTACK"), chemokine 16 ligand (with CXC motif) ("CXCL16"), chemokine with CXC 5 motif ("ENA- 78" ), chemokine 26 ligand (with CC motif) ("Eotaxin-3"), granulocyte chemotactic protein 2 ("GCP-2"), GRO, chemokine 14 ligand (with CC motif) ("HCC-l") , chemokine 16 linker (with CC motif) ("HCC-4"), interleukin-9 ("IL-9"), interleukin-17 F ("IL-17F"), interleukin-18 binding protein (" IL-18 BPa "), interleukin-
28 A ("IL-28A"), interleukin 29 ("IL-29"), interleukin 31 ("IL-31"), chemokine with motif CXC 10 ("IP-10"), chemokine receptor CXCR3 ("I -TAC "), leukemia inhibiting factor (" LIF "), light chemokine ligands (motif C) (" lymphotactin "), monocyte chemo-attracting protein 2 (" MCP-2 "), monocyte chemo-attracting protein 3 ( "MCP-3"), monocyte 4 chemoattracting protein ("MCP-4"), macrophage-derived chemokine ("MDC"), macrophage migration inhibiting factor ("MIF"), chemokine ligand (with CC motif) 20 ("MIP-3 alpha"), chemokine with CC motif 19 ("MIP-3 beta"), chemokine ligand (with CC motif) 23 ("MPIF-1"), macrophage-stimulating alpha protein chain (" MSPalfa "), type 4 nucleosome assembly protein 1 (" NAP-2 "), secreted phosphoprotein 1 (" osteopontin "), pulmonary and activation-regulated cytokine (" PARC "), platelet factor 4 (" PF4 "), stromal cell-derived factor-1 alpha ("SDF-1 alpha"), chemokine ligand (with CC motif) 17 ( "TARC"), chemokine expressed in the thymus ("TECK"), thymic stromal lymphopoietin ("TSLP 4- IBB"), CD 166 antigen ("ALCAM"), differentiation cluster 80 ("B7-1"), member of tumor necrosis factor receptor superfamily 17 ("BCMA"), differentiation cluster 14 ("CD14"), differentiation cluster 30 ("CD30"), differentiation cluster 40 ("CD40 ligand"), adhesion molecule cell-related carcinoembryonic antigen 1 (bile glycoprotein) ("CEACAM-1"), death receptor 6 ("DR6"), deoxythymidine kinase ("Dtk"), type 1 membrane glycoprotein ("endogline"), protein tyrosine kinase erbB-3 receptor ("ErbB3"), endothelium-leukocyte 1 adhesion molecule ("E-selectin"), apoptosis antigen 1 ("Fas"), tyrosine kinase similar to Fms 3 ("Flt-3L"), member of the tumor necrosis factor 1 receptor ("GITR") superfamily, member of the tumor necrosis factor 14 superfamily ("HVEM"), intercellular adhesion molecule 3 ("ICAM-3"), IL-1 R4 , IL-1
RI, IL-10 Rbeta, IL-17R, IL-2Rgama, IL-21R, lysosome 2 membrane protein ("LIMPII"), lipocalin associated with neutrophilic gelatinase ("lipocalin-2"), CD62L ("L-selectin "), lymphatic endothelium (" LYVE-1 "), sequence related to the MHC class IA polypeptide (" MICA "), sequence related to the MHC class IB (" MICB ") polypeptide, NRGl-betal, growth factor receptor derived from beta-type platelets ("PDGF Rbeta"), platelet endothelial cell adhesion molecule ("PECAM-1"), RAGE, hepatitis A 1 cell receptor ("TIM-1"), member of the factor receptor superfamily tumor necrosis IOC ("TRAIL R3"), trapin protein transglutaminase binding domain ("trapin-2"), urokinase receptor ("uPAR"), vascular cell adhesion protein 1 ("VCAM-1"), XEDARActivin A, Agouti-related protein ("AgRP"), ribonuclease 5 ("angiogenin"), angiopoietin 1, angiostatin, catheprine S, CD40, protein from the IB cryptic family ("crypto-1"), DAN, related protein aa Dickkopf 1 ("DKK-1"), E-cadherin, epithelial cell adhesion molecule ("EpCAM"), Fas ligand (FasL or CD95L), Fcg RIIB / C, FoUistatin, galectin-7, intercellular adhesion molecule 2 ("ICAM-2"), IL-13 Rl, IL-13R2, IL-17B, IL-2 Ra, IL-2 Rb, IL-23, LAP, neuronal cell adhesion molecule ("NrCAM"), inhibitor plasminogen activator-1 ("PAI-1"), platelet-derived growth factor receptors ("PDGF-AB"), resistin, stromal cell-derived factor 1 ("SDF-1 beta"), sgpl30, protein related to secreted frizzled 2 ("ShhN"), sialic acid-binding immunoglobulin-type lectins ("Siglec-5"), ST2, transforming growth factor-beta 2 ("TGF beta 2"), Tie-2, thrombopoietin ("TPO"), member of the tumor necrosis factor 10D receptor superfamily ("TRAIL R4"), myeloid 1-expressed trigger receptor ("TREM-1"), vascular endothelial growth factor C ("VEGF- C "), VEGFR1Adiponectin, adipsin (" AND "), alpha-fetoprotein (" AFP "), angiopoie type 4 ("ANGPTL4"), beta-2-
microglobulin ("B2M"), basal cell adhesion molecule ("BCAM"), carbohydrate antigen 125 ("CA125"), cancer antigen 15-3 ("CA15-3"), carcinoembryonic antigen ("CEA") , cAMP receptor protein ("CRP"), human epidermal growth factor 2 receptor ("ErbB2"), follistatin, follicle stimulating hormone ("FSH"), chemokine 1 ligand (with CXC motif) ("GRO alpha "), human chorionic gonadotropin (" beta HCG "), insulin-like growth factor receptor 1 (" IGF-1 sR "), IL-1 sRII, IL-3, IL-18 Rb, IL-21, leptin , metalloproteinase-1 ("MMP-1"), metalloproteinase-2 ("MMP-2"), metalloproteinase-3 ("MMP-3"), metalloproteinase-8 ("MMP-8"), matrix metalloproteinase-9 ("MMP-9"), metalloproteinase-10 ("MMP-10") matrix, metalloproteinase-13 ("MMP-13") matrix, neural cell adhesion molecule ("NCAM-1"), entactin ("Nidogen-1"), neuron-specific enolase ("NSE"), oncostatin M ("OSM"), procalcitonin, prolactin, specific antigen prostate cancer ("PSA"), sialic acid binding Ig type 9 ("Siglec-9"), ADAM 17 endopeptidase ("TACE"), thyroglobulin, metalloproteinase 4 inhibitor ("TIMP-4"), TSH2B4, protein containing disintegrin domain and metalloproteinase 9 ("ADAM-9"), angiopoietin 2, member of the tumor necrosis factor 13 ligand superfamily / member of the nuclear phosphoprotein 32 superfamily rich in acid leucine B ("APRIL"), bone morphogenetic protein 2 ("BMP-2"), bone morphogenetic protein 9 ("BMP-9"), complement component 5a ("C5a"), cathepsin L, CD200, CD97, queerine, member of the Factor receptor superfamily tumor necrosis 6B ("DcR3"), fatty acid binding protein 2 ("FABP2"), fibroblast activation protein, alpha ("FAP"), fibroblast growth factor 19 ("FGF-19"), galectin -3, hepatocyte growth factor receptor ("HGF R"), IFN-gamma / beta R2, insulin-like growth factor 2 ("IGF-2"), seed growth receptor similar to insulin 2 ("IGF-2 R"), interleukin-1 6 receptor ("IL-1R6"), interleukin 24 ("IL-24"), interleukin 33 ("IL-33", kallikrein 14, asparaginil endopeptidase ("legumaine"), oxidized low-density lipoprotein receptor 1 ("LOX-1"), mannose-binding lectin ("MBL"), neprilisin ("NEP"), Notch 1 homologue associated with translocation (drosophyll ) ("Notch-1"), overexpressed nephroblastoma ("NOV"), osteoactivin, programmed cell death protein 1 ("PD-1"), N-acetylmuramoyl-L-alanine amidase ("PGRP-5"), serpine A4, secreted frizzled-related protein a 3 ("sFRP-3"), thrombomodulin, Toll type 2 receptor ("TLR2"), member of the tumor necrosis factor 10A receptor ("TRAIL Rl"), transferrin ( "TRF"), WIF-lACE-2, albumin, AMICA, angiopoietin 4, B cell activation factor ("BAFF"), carbohydrate antigen 19-9 ("CA19-9"), CD 163, clusterin, CRT AM, chemokine 14 ("CXCL14") ligand ("CXCL14"), cystatin C, decorin ("DCN"), protein related to Dickkopf 3 ("Dkk-3"), delta type 1 protein ("DLL1"), fetuin A, heparin-binding growth factor 1 ("aFGF"), alpha folate receptor ("FOLR1"), furin , GPCR-associated classification protein ("GASP-1"), GPCR-associated classification protein ("GASP-2"), granulocyte colony stimulating factor receptor ("GCSF R"), hepsin serine protease ( "HAI-2"), interleukin-17B receptor ("IL-17B R"), interleukin 27 ("IL-27"), lymphocyte activation gene 3 ("LAG-3"), apolipoprotein AV ("LDL R "), pepsinogen I, retinol-binding protein 4 (" RBP4 "), SOST, heparan sulfate proteoglycate (" Syndecan-1 "), member of the tumor necrosis factor 13B (" TACI ") receptor superfamily , tissue factor pathway inhibitor ("TFPI"), TSP-1, tumor necrosis factor receptor superfamily, member 10b ("TRAIL R2"), TRANCE, troponin I, urokinase plasminogen activator ("uPA") , cadherin 5, cadherin type 2 or VE (vascular endothelial) also co known as CD144 ("VE-cadherin"), protein of the signaling pathway inducible by WNTl 1 ("WISP-1") and activator of the nuclear factor receptor κ B ("RANK").
[61]
61. Pharmaceutical composition according to any one of claims 19 to 60, characterized in that the immunotherapeutic agent comprises an adjuvant.
[62]
62. Pharmaceutical composition according to claim 61, characterized by the fact that the adjuvant is selected from the group consisting of an immunomodulatory protein, adjuvant 65, α-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, β-glucan peptide, CpG DNA, GPI-0100, lipid A, lipopolysaccharide, Lipovant, montanide, N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A and trehalose dimicolate.
[63]
63. Pharmaceutical composition according to any one of claims 16 to 62, characterized in that the therapeutic agent against cancer comprises an angiogenesis inhibitor.
[64]
64. Pharmaceutical composition according to claim 63, characterized by the fact that the angiogenesis inhibitor is selected from the group consisting of bevacizumab (Avastin®), ziv-aflibercept (Zaltrap®), sorafenib (Nexavar®), sunitinib (Sutent®), pazopanib (Votrient®), regorafenib (Stivarga®) and cabozantinib (Cometriq ™).
[65]
65. Pharmaceutical composition according to any one of claims 16 to 64, characterized in that the therapeutic agent against cancer comprises an antibiotic.
[66]
66. Pharmaceutical composition according to claim 65, characterized by the fact that the antibiotic is selected from the group consisting of aminoglycosides,
ansamycins, carbacefemas, carbapenemas, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptide antibiotics, quinolones, fluoroquinolone, sulfonamides, tetracycline compounds and antimicrobial compounds.
[67]
67. Pharmaceutical composition according to any one of claims 16 to 66, characterized in that the therapeutic agent against cancer comprises therapeutic bacteria.
[68]
68. Pharmaceutical composition according to claim 67, characterized by the fact that the composition further comprises a prebiotic.
[69]
69. Pharmaceutical composition according to claim 68, characterized in that the prebiotic is a fruit-oligosaccharide, a galacto-oligosaccharide, a trans-galacto-oligosaccharide, a xylo-oligosaccharide, a chito-oligosaccharide, an oligosaccharide of soy, a gentile-oligosaccharide, an isomalto-oligosaccharide, a mano-oligosaccharide, a malto-oligosaccharide, a mannano-oligosaccharide, lactulose, lactosaccharose, palatinose, glycosyl sucrose, guar gum, arabic gum, tagalose, amyline, amyline, amyline, amyline, amylin xylan or a cyclodextrin.
[70]
70. Pharmaceutical composition according to claim 15, characterized in that the additional therapeutic agent comprises an antibiotic.
[71]
71. Pharmaceutical composition according to claim 70, characterized by the fact that the antibiotic is selected from the group consisting of aminoglycosides, ansamycins, carbacefemas, carbapenemas, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones , penicillins, polypeptide antibiotics, quinolones, fluoroquinolone, sulfonamides, tetracyclines,
antimycobacterial compounds and combinations thereof.
[72]
72. Pharmaceutical composition according to claim 70 or 71, characterized in that the additional therapeutic agent comprises therapeutic bacteria.
[73]
73. Pharmaceutical composition according to claim 15, characterized in that the additional therapeutic agent comprises 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.
[74]
74. Pharmaceutical composition according to claim 74, characterized by the fact that the additional therapeutic agent is selected from the group consisting of cyclosporine, retinoids, corticosteroids, derived from propionic acid, derived from acetic acid, derived from enolic acid, phenamic acid derivatives, Cox-2 inhibitors, lumiracoxib, ibuprofen, magnesium choline salicylate, fenprofen, salsalate, difunisal, tolmetin, ketoprofen, flurbiprofen, oxaprozine, indomethacin, sulindac, etodolac, ketorolac, nabumetone, valoxide, nabumetone, valoxide, nabumetone, valoxide, MK0966; rofecoxib, acetominophen, celecoxib, diclofenac, tramadol, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefanamic acid, meclofenamic acid, flufenamic acid, tolfenamic, valdecoxib, parecoxib, metodol, aspirin, metodol, ), antimalarial drugs (for example, hydroxychloroquine and chloroquine), sulfasalazine, leflunomide, azathioprine, cyclosporine, gold salts, minocycline, cyclophosphamide, D-penicillamine, minocycline, auranofin, tacrolimus, for example, myocrisine, , TNF alpha antagonists or TNF alpha receptor antagonists), for example, ADALIMUMABE (Humira®), ETANERCEPT (Enbrel®), INFLIXIMABE
(Remicade®; TA-650), CERTOLIZUMABE PEGOL (Cimzia®; CDP870), GOLIMUMABE (Simpom®; CNTO 148), ANAKINRA (Kineret®), RITUXIMABE (Rituxan®; MabThera®), ABATACEPT (Orencia®), TOCILIZE RoActemra / Actemra®), integrin antagonists (TYSABRI® (natalizumab)), IL-1 antagonists (ACZ885 (Ilaris)), Anakinra (Kineret®)), CD4 antagonists, IL-23 antagonists, IL- antagonists 20, IL-6 antagonists, BLyS antagonists (eg Atacicept, Benlysta® / LymphoStat-B® (belimumab)), p38 inhibitors, CD20 antagonists (ocrelizumab, ofatumumab (Arzerra®)), gamma interferon antagonists (fontolizumab), prednisolone, prednisone, dexamethasone, cortisol, cortisone, hydrocortisone, methylprednisolone, betamethasone, triamcinolone, beclometasoma, fludrocortisone, deoxycorticosterone, aldosterone, doxycycline, vancomycin, 46-pioglitazone viusida, TwHF, methoxsalene, vitamin D - ergocalciferol, milnacipran, paclitaxel, rosig tazona, tacrol imus (Prograf®), RADOOl, rapamune, rapamycin, fostamatinib, fentanyl, XOMA 052, disodium fostamatinib, rosigtazone, curcumin (Longvida ™), rosuvastatin, maraviroc, ramipnl, milnacipran, cobiprostone, somatropin, gene therapy vector , GW856553, esomeprazole, everolimus, trastuzumab, JAKl and JAK2 inhibitors, JAK pan inhibitors, for example, tetracyclic pyridone 6 (P6), 325, PF-956980, denosumab, IL-6 antagonists, CD20 antagonists, CD20 antagonists CTLA4, IL-8 antagonists, IL-21 antagonists, IL-22 antagonists, integrin antagonists (Tysarbri® (natalizumab)), VGEF antagonists, CXCL antagonists, MMP antagonists, defensin antagonists, IL antagonists -1 (including IL-1 beta antagonists) and IL-23 antagonists (for example, receptor decoys, antagonist antibodies).
[75]
75. Pharmaceutical composition according to claim 74, characterized by the fact that the immunosuppressive agent is acorticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporine A, mercaptopurine, azathiopurine, prednisone, methotrexate, anti-histotrexate, anti-histotrexate, anti-histamine. , glucocorticoids, epinephrine, theophylline, sodium chromoline, antileukotrienes, anticholinergic drugs for rhinitis, TLR antagonists, inflammasome inhibitors, anticholinergic decongestants, mast cell stabilizers, anti-IgE monoclonal antibodies, vaccines (for example, vaccines used for vaccines used in vaccines for amount of an allergen is gradually increased), cytokine inhibitors, such as anti-IL-6 antibodies, TNF inhibitors, such as infliximab, adalimumab, certolizumab pegol, golimumab or etanercept, and combinations thereof.
[76]
76. Method for treating a disease in a subject, characterized in that it comprises administering to the subject a pharmaceutical composition, as defined in any one of claims 1 to 15.
[77]
77. Method according to claim 76, characterized by the fact that the disease is an autoimmune disease, an inflammatory disease, a metabolic disease or a cancer.
[78]
78. Method for treating cancer in a subject, characterized by the fact that it comprises administering to the subject a pharmaceutical composition, as defined in any one of claims 1 to
69.
[79]
79. Method for increasing a microbiome in a subject who has cancer, characterized by the fact that it comprises administering to the subject a pharmaceutical composition, as defined in any one of claims 1 to 69, to the subject, so that the EVs and / or bacteria are added to a niche in the subject.
[80]
80. Method for depleting a tumor of bacteria associated with cancer in a subject, characterized by the fact that it comprises administering to the subject a pharmaceutical composition, as defined in any one of claims 1 to 69, to the subject, so that the EVs and / or bacteria are added to a niche in the subject.
[81]
81. Method for altering a tumor microbiome in a subject, characterized in that it comprises administering, to the subject, a pharmaceutical composition, as defined in any one of claims 1 to 69, to the subject, so that the EVs and / or bacteria are added to a niche in the subject.
[82]
82. Method for altering a mesenteric lymph node microbiome in a subject, characterized in that it comprises administering to the subject a pharmaceutical composition, as defined in any one of claims 1 to 69, to the subject, so that the EVs and / or bacteria are added to a niche in the subject.
[83]
83. Method for altering an antigen presentation by dendritic cells in a subject, characterized by the fact that it comprises administering, to the subject, a pharmaceutical composition, as defined in any one of claims 1 to 69, to the subject, so that the EVs and / or bacteria are added to a niche in the subject.
[84]
84. Method for activating epithelial cells in a subject, characterized by the fact that it comprises administering to the subject a pharmaceutical composition, as defined in any one of claims 1 to 69, to the subject, so that the EVs and / or bacteria are added to a niche in the subject.
[85]
85. Method according to any of claims 79 to 84, characterized by the fact that the niche is in the subject's gastrointestinal tract.
[86]
86. Method according to any of claims 79 to 84, characterized by the fact that the niche is in the subject's urogenital tract.
[87]
87. Method according to any of claims 79 to 84, characterized by the fact that the niche is in the subject's respiratory tract.
[88]
88. Method according to any of claims 79 to 87, characterized by the fact that the EVs and / or bacteria come from a cancer-associated bacterium.
[89]
89. Method, according to claim 88, characterized by the fact that the cancer-associated bacteria is of a species selected from the group consisting of the bacterial species listed in Table 2.
[90]
90. Method according to any of claims 88 and 89, characterized by the fact that the EVs and / or bacteria are from a mandatory anaerobic bacterium.
[91]
91. Method according to claim 90, characterized by the fact that the obligatorily anaerobic bacterium is a genus selected from the group consisting of Bacteroides, Prevotella, Porphyromonas, Fusobacterium, Bilophila, Sutterella, Peptostreptococcus, Clostridium, Actinomyces Propionibacterium, Eubacterium, Lactobacillus, Streptococcus, Veillonella, Agathobaculum, Atopobium, Blautia, Burkholderia, Dielma, Longicatena, Paraclostridium, Turicibacter and Tyzzerella.
[92]
92. Method according to any one of claims 79 to 87, characterized by the fact that the EVs and / or bacteria come from a bacterium of a genus selected from the group consisting of Escherichia, Klebsiella, Lactobacillus,
Shigella and Staphylococcus.
[93]
93. Method according to any one of claims 79 to 92, characterized by the fact that the cancer is selected from the group consisting of hematological malignancy, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T cell leukemia, leukemia leukemia, leukemia leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, skin leukemia, embryonic leukemia, eosinophilic leukemia, Gross leukemia, Gross leukemia Rieder, Schilling leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hair cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, acute monocytic leukemia, leukemia leukemia, leukemia leukemia, leukemia leukemia, leukemia leukemia lymphoblastic, lymphocytic leukemia, lymphogenic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, mycelial leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, leukemia leukemia, plasmacytic leukemia, navel cell acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adenomatous carcinoma, adrenal cortex carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basal cell carcinoma, basaloid carcinoma, bronchial carcinoma, basal cell carcinoma, basal cell carcinoma bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, body carcinoma, cribriform carcinoma, carcinoma en cuirasse, skin carcinoma, cylindrical carcinoma, carcinoma of cylindrical cells, carcinoma duct, hard carcinoma, embryonic carcinoma, encephaloid carcinoma, squamous cell carcinoma, adenoid epithelial carcinoma, exophytic carcinoma, ex ulcere carcinoma, fibrous carcinoma, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, ring cell carcinoma, sinplex ring carcinoma , small cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, spongy carcinoma, squamous carcinoma, squamous cell carcinoma, string carcinoma, telangiectatic carcinoma, telangiectoid carcinoma, transitional cell carcinoma, carcinoma of tuberous cell carcinoma tuberous, verrucous carcinoma, villous carcinoma, gigantocellular carcinoma, glandular carcinoma, granular cell carcinoma, capillary matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hyperernionary carcinoma, infantile carcinoma, situational carcinoma intrae carcinoma pidermal, intraepithelial carcinoma, Krompecher carcinoma, Kulchitzky cell carcinoma, large cell carcinoma, lenticular carcinoma, lenticular carcinoma, lipomatous carcinoma, lymphepithelial carcinoma, medullary carcinoma, medullary carcinoma, melanotic carcinoma, mucinous carcinoma, mucinous carcinoma mucocellular carcinoma, mucoepidermoid carcinoma, mucous carcinoma, mucus carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat grain cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, pre-invasive carcinoma, squamous cell carcinoma, pre-invasive carcinoma kidney cell carcinoma, reserve cell carcinoma, sarcomatoid carcinoma, schneiderian carcinoma, squamous carcinoma,
scrotum carcinoma, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, large cell sarcoma, abemethar sarcoma, liposarcoma, liposarcoma, liposarcoma moles, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonic sarcoma, Wilms tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic B-cell sarcoma, lymphoma, sarcoma Jensen, Kaposi's sarcoma, Kupffer's cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymal sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, myocardial, sarcoma, hygienic, sarcoma 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, cancer of the genitourinary tract, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, Harding-Passey melanoma, juvenile melanoma, malignant lentigo melanoma, malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma, subungual melanoma, plasmacytoma, colorectal cancer, rectal cancer and superficial spreading melanoma.
[94]
94. Method according to any of claims 76 to 93, characterized in that the pharmaceutical composition is administered orally.
[95]
95. Method according to any one of claims 76 to 93, characterized in that the pharmaceutical composition is administered intravenously.
[96]
96. Method according to any of claims 76 to 93, characterized in that the pharmaceutical composition is administered intratumorally.
[97]
97. Method according to any one of claims 76 to 93, characterized in that the pharmaceutical composition is administered sub-tumorally.
[98]
98. Method according to any one of claims 76 to 93, characterized in that the pharmaceutical composition is administered by subcutaneous, intradermal or intraperitoneal injection.
[99]
99. Method according to any of claims 76 to 93, characterized in that the pharmaceutical composition is administered intratumorally with a controlled release matrix.
[100]
100. Method according to any one of claims 78 to 99, characterized in that the administration of the pharmaceutical composition treats cancer.
[101]
101. Method according to any of claims 78 to 100, characterized in that the administration of the pharmaceutical composition induces an anti-tumor immune response.
[102]
102. Method according to any one of claims 78 to 101, characterized in that the cancer therapy comprises radiation therapy.
[103]
103. Method according to any of claims 76 to 102, characterized in that it further comprises administering an antibiotic to the subject.
[104]
104. Method according to claim 103, characterized by the fact that the antibiotic is selected from the group consisting of aminoglycosides, ansamycins, carbacefemas, carbapenemas, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptide antibiotics, quinolones, fluoroquinolone, sulfonamides, tetracyclines, antimycobacterial compounds and combinations thereof.
[105]
105. Method according to any one of claims 78 to 104, characterized in that the cancer therapy comprises administering therapeutic bacteria to the subject.
[106]
106. Method for treating an immune disorder in a subject, characterized in that it comprises administering to the subject a pharmaceutical composition, as defined in any one of claims 1 to 15 and 71 to 75.
[107]
107. Method, according to claim 106, characterized by the fact that the immune disorder is selected from the group consisting of acute disseminated universal alopecia, Behcet's disease, Chagas' disease, chronic fatigue syndrome, dysautonomy, encephalomyelitis, spondylitis ankylosing, aplastic anemia, suppurative hidradenitis, autoimmune hepatitis, autoimmune oophoritis, celiac disease, Crohn's disease, type 1 diabetes mellitus, giant cell arteritis, Goodpasture's syndrome, Grave's disease, Guillain-Barre syndrome, Hashimoto's disease, purple Henoch-Schonlein disease, Kawasaki disease, lupus erythematosus, microscopic colitis, microscopic polyarteritis, mixed connective tissue disease, Muckle-Wells syndrome, multiple sclerosis, myasthenia gravis, opsoclonus-myoclonus syndrome, optic neuritis, Ord thyroiditis, pemphigus , polyarteritis nodosa, polymyalgia, rheumatoid arthritis, Reiter's syndrome, Sjogren's syndrome, temporal arteritis, Wege's granulomatosis ner, hot autoimmune hemolytic anemia, interstitial cystitis, lyme disease, morphia, psoriasis, sarcoidosis, scleroderma, ulcerative colitis, vitiligo, contact hypersensitivity, contact dermatitis (including that caused by poison ivy), hives, skin allergies, respiratory allergies (hay fever, allergic rhinitis, allergy to residential mites) and gluten-sensitive enteropathy (celiac disease), appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, hepatitis, suppurative hidradenitis, iritis, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, percarditis, peritonitis, pharyngitis, pleuritis, pneumonitis, prostatitis, pyelonephritis and stomatitis, transplant rejection (involving organs such as kidney, liver, heart, lung, pancreas (for example, islet cells) , bone marrow, cornea, small intestine, cutaneous allografts, cutaneous homografts and heart valve xenografts, serum disease and graft vs. disease. host), acute pancreatitis, chronic pancreatitis, acute respiratory distress syndrome, Sexary syndrome, congenital adrenal hyperplasia, non-suppurative thyroiditis, cancer-associated hypercalcemia, pemphigus, bullous dermatitis herpetiformis, severe erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, allergic rhinitis seasonal or perennial, bronchial asthma, contact dermatitis, atopic dermatitis, hypersensitivity reactions to drugs, allergic conjunctivitis, keratitis, herpes zoster ophthalmic, iritis and oiridocyclitis, chorioretinitis, optic neuritis, symptomatic sarcoidosis, chemotherapy of pulmonary pulmonary fulminant or pulmonary fulminant idiopathic thrombocytopenia in adults, secondary thrombocytopenia in adults, acquired hemolytic anemia (autoimmune), leukemia and lymphomas in adults, acute childhood leukemia, regional enteritis, autoimmune vasculitis, multiple sclerosis, chronic obstructive pulmonary disease, rejection of solid organ transplantation, sepsis . Preferred treatments include transplant rejection treatment, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, type 1 diabetes, asthma, inflammatory bowel disease, systemic lupus erythematosus, psoriasis, chronic obstructive pulmonary disease and inflammation accompanied by infectious diseases (for example, sepsis) .
[108]
108. Method according to any of claims 106 and 107, characterized in that the pharmaceutical composition is administered orally.
[109]
109. Method according to any one of claims 106 and 107, characterized in that the pharmaceutical composition is administered intravenously.
[110]
110. Method according to any of claims 106 and 107, characterized in that the pharmaceutical composition is administered by subcutaneous, intradermal or intraperitoneal injection.
[111]
111. Method according to any one of claims 70 to 110, characterized by the fact that it also comprises administering a prebiotic to the subject.
[112]
112. Method according to claim 111, characterized by the fact that the prebiotic is a fructo-oligosaccharide, a galacto-oligosaccharide, a trans-galacto-oligosaccharide, a xylooligosaccharide, a chito-oligosaccharide, a soy oligosaccharide , a gentile-oligosaccharide, an isomalto-oligosaccharide, a mano-oligosaccharide, a malto-oligosaccharide, a mannano-oligosaccharide, lactulose, lactosaccharose, palatinose, glycosyl sucrose, guar gum, arabic gum, tagalose, amylose, amylose, amylose, amylose, amylose, amylose, amylose, amylose or a cyclodextrin.
[113]
113. Method according to any one of claims 70 to 112, characterized by the fact that the subject is a human being.
[114]
114. Method according to any one of claims 70 to 112, characterized in that the subject is a non-human mammal.
[115]
115. Method according to claim 114, characterized by the fact that the mammal is selected from the group consisting 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.
[116]
116. Method for generating a genetically modified bacterium, characterized by the fact that it comprises introducing, in the bacterium, a modification that results in increased production of EVs.
[117]
117. Method according to claim 116, characterized by the fact that the bacterium is a species selected from the group consisting of the bacterial species listed in Table 1, Blautia massiliensis, Paraclostridium benzoelyticum, Dielma fastidiosa, Longicatena caecimuris and Veillonella tobetsuensis .
[118]
118. Method according to any of claims 116 and 117, characterized in that the bacterium is a mandatory anaerobic bacterium.
[119]
119. Method, according to claim 118, characterized by the fact that the obligatorily anaerobic bacterium is a genus selected from the group consisting of Bacteroides, Prevotella, Porphyromonas, Fusobacterium, Bilophila, Sutterella, Peptostreptococcus, Clostridium, Actinomyces,
Propionibacterium, Eubacterium, Lactobacillus, Streptococcus, Veillonella Agathobaculum, Atopobium, Blautia, Burkholderia, Dielma, Longicatena, Paraclostridium, Turicibacter and Tyzzerella.
[120]
120. Method, according to claim 115, characterized by the fact that the bacterium is of a genus selected from the group consisting of Escherichia, Klebsiella, Lactobacillus, Shigella and Staphylococcus.
[121]
121. Method according to any of claims 116 to 119, characterized in that the bacterium is modified by directed evolution.
[122]
122. Method according to claim 121, characterized by the fact that directed evolution comprises the exposure of the bacterium to an environmental condition under which improved EV production improves bacterial survival.
[123]
123. Method according to claim 122, characterized by the fact that the environmental condition requires the stability of the EV at a pH less than or equal to 4.
[124]
124. Method according to any one of claims 116 to 123, characterized in that it comprises a screening of mutagenized bacteria using an assay that detects the increase in the production of EV.
[125]
125. Method according to claim 124, characterized by the fact that it further comprises mutagenizing the bacteria.
[126]
126. Method according to claim 124 or 125, characterized in that the bacteria are mutagenized upon exposure to a chemical mutagen or UV radiation.
[127]
127. Method for generating a genetically modified bacterium, characterized by the fact that it comprises introducing, in the bacterium, a modification that results in the production of EVs with a therapeutic property improved by the bacterium.
[128]
128. Method according to claim 127, characterized in that the improved therapeutic property comprises improved oral administration.
[129]
129. Method according to claim 127, characterized in that the improved therapeutic property comprises stability at a pH less than or equal to 4.
[130]
130. The method of claim 127, characterized in that the improved therapeutic property comprises stability at a concentration of bile acid between 0.2% and 2%.
[131]
131. Method according to any one of claims 127 to 130, characterized in that the improved therapeutic property comprises increased immunological activation.
[132]
132. Method according to any of claims 127 to 131, characterized by the fact that the bacterium is of a species selected from the group consisting of the bacterial species listed in Table 1, Blautia massiliensis, Paraclostridium benzoelyticum, Dielma fastidiosa, Longicatena caecimuris and Veillonella tobetsuensis.
[133]
133. Method according to any of claims 127 to 131, characterized in that the bacterium is a cancer-associated bacterium.
[134]
134. Method according to claim 133, characterized by the fact that the cancer-associated bacteria is of a species selected from the group consisting of the bacterial species listed in Table 2.
[135]
135. Method according to any one of claims 127 to 133, characterized in that the bacterium is a mandatory anaerobic bacterium.
[136]
136. Method according to claim 134, characterized by the fact that the obligatorily anaerobic bacterium is of a genus selected from the group consisting of Bacteroides, Prevotella, Porphyromonas, Fusobacterium, Bilophila, Sutterella, Peptostreptococcus, Clostridium, Actinomyces, Propionibacterium , Eubacterium, Lactobacillus, Streptococcus, Veillonella Agathobaculum, Atopobium, Blautia, Burkholderia, Dielma, Longicatena, Paraclostridium, Turicibacter and Tyzzerella.
[137]
137. Method according to any of claims 127 to 134, characterized by the fact that the bacterium is of a genus selected from the group consisting of Escherichia, Klebsiella, Lactobacillus, Shigella and Staphylococcus.
[138]
138. Method according to any one of claims 127 to 137, characterized in that the bacterium is modified by directed evolution.
[139]
139. Method according to claim 138, characterized in that the directed evolution comprises the exposure of the bacterium to an environmental condition under which the stability of EVs at a pH less than or equal to 4 improves bacterial survival.
[140]
140. Method according to any one of claims 127 to 139, characterized in that it comprises a screening of mutagenized bacteria using an assay that detects the increased activation of an immune response.
[141]
141. Method according to claim 140, characterized by the fact that it also comprises mutagenizing the bacteria.
[142]
142. Method according to claim 140 or 141, characterized in that the bacteria are mutagenized upon exposure to a chemical mutagen or UV radiation.
[143]
143. Method according to any one of claims 140 to 142, characterized in that the assay is an in vivo assay, an ex vivo assay or an in vitro assay.
[144]
144. Method according to any of claims 138 to 143, characterized in that the assay is an in vivo immune response assay.
[145]
145. Method according to claim 144, characterized by the fact that the tumor extermination assay in vivo is carried out on mice.
[146]
146. Method according to any one of claims 140 to 142, characterized in that the assay is an in vitro immune response assay.
[147]
147. Modified bacterium, characterized by the fact that it is generated in accordance with the method, as defined in any one of claims 127 to 146.
[148]
148. Method for the cultivation of a bacterium for improved production of EV, characterized by the fact that it comprises cultivating the bacteria under stress-inducing growth conditions.
[149]
149. Method according to claim 148, characterized by the fact that stress-inducing growth conditions comprise growth in the presence of subinhibitory concentrations of an antibiotic.
[150]
150. Method according to claim 149, characterized by the fact that the antibiotic is selected from the group consisting of aminoglycosides, ansamycins, carbacefemas, carbapenemas, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptide antibiotics, quinolones, fluoroquinolone, sulfonamides, tetracyclines, antimycobacterial compounds and combinations thereof.
[151]
151. Method according to claim 148, characterized by the fact that stress-inducing growth conditions comprise growth in the presence of subinhibitory concentrations of a host antimicrobial peptide.
[152]
152. Method according to claim 151, characterized in that the host's antimicrobial peptide is a lysozyme, defensin or Reg protein.
[153]
153. Method according to claim 148, characterized in that the stress-inducing growth conditions comprise growth in the presence of subinhibitory concentrations of an antimicrobial peptide produced by bacteria.
[154]
154. Method according to claim 153, characterized in that the antimicrobial peptide produced by bacteria is a bacteriocin or a microcin.
[155]
155. Method according to claim 148, characterized by the fact that stress-inducing growth conditions comprise growth under temperature stress.
[156]
156. Method according to claim 148, characterized by the fact that stress-inducing growth conditions comprise growth under conditions of carbon limitation.
[157]
157. Method according to claim 148, characterized by the fact that stress-inducing growth conditions comprise growth in the presence of subinhibitory salt concentrations.
[158]
158. Method according to claim 148, characterized by the fact that stress-inducing growth conditions comprise growth in the presence of UV light.
[159]
159. The method of claim 148,
characterized by the fact that stress-inducing growth conditions comprise growth in the presence of hydrogen peroxide.
[160]
160. Method according to any one of claims 148 to 159, characterized by the fact that the bacterium is a species selected from the group consisting of the bacterial species listed in Table 1, Blautia massiliensis, Paraclostridium benzoelyticum, Dielma fastidiosa, Longicatena caecimuris and Veillonella tobetsuensis.
[161]
161. Bioreactor, characterized by the fact that it comprises a modified bacterium, as defined in the claim
147.

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

GB0130123D0|2001-12-17|2002-02-06|Microbiological Res Agency|Outer membrane vesicle vaccine and its preparation|

---

KR101430283B1|2009-09-01|2014-08-14|주식회사이언메딕스|Extracellular vesicles derived from gut microbiota, and disease models, vaccines, drug screening methods, and diagnostic methods using the same|

---

WO2014152484A1|2013-03-14|2014-09-25|Regents Of The University Of Minnesota|Freeze dried fecal microbiota for use in fecal microbial transplantation|WO2017217753A1|2016-06-14|2017-12-21|한국생명공학연구원|Strain of genus agathobaculum having effects of preventing or treating degenerative brain diseases, and use thereof|

---

DK3436054T3|2016-09-13|2019-11-11|Allergan Inc|STABILIZED NON-PROTEIN CLOSTRID TOXIN COMPOSITIONS|

---

WO2019178487A2|2018-03-15|2019-09-19|Evelo Biosciences, Inc.|Compositions and methods for treating disease using klebsiella quasipneumoniae subsp. similipneumoniae|

---

WO2019178494A1|2018-03-15|2019-09-19|Evelo Biosciences, Inc.|Compositions and methods for treating cancer and inflammation using tyzzerella nexilis|

---

WO2019178490A1|2018-03-15|2019-09-19|Evelo Biosciences, Inc.|Compositions and methods for treating cancer and inflammation using klebsiella oxytoca|

---

WO2019232122A1|2018-05-30|2019-12-05|University Of Florida Research Foundation, Inc.|Mechanistic bacterial efector to prevent apoptosis of human beta cells|

---

CN108949699B|2018-08-03|2020-09-04|江南大学|Hybridoma cell strain secreting meloxicam monoclonal antibody and application thereof|

---

AU2019351019A1|2018-09-27|2021-04-22|Research Development Foundation|Methods and probiotic compositions for the treatment of bone disorders|

---

CN109207425A|2018-09-29|2019-01-15|中国医科大学附属口腔医院|Porphyromonas gingivalis inducing macrophage excretion body rna expression research method|

---

CN109762762A|2019-01-11|2019-05-17|江苏大学|A kind of superior strain of lipopeptid and its preparation method and application|

---

TW202045192A|2019-02-22|2020-12-16|美商艾弗洛生物科技股份有限公司|Bacterial membrane preparations|

---

CN110195028B|2019-03-08|2022-03-01|大连理工大学|Preparation method and application of compost low-temperature compound fermentation inoculant|

---

WO2020214961A1|2019-04-17|2020-10-22|Codiak Biosciences, Inc.|Methods of treating tuberculosis|

---

CN110317744B|2019-05-08|2021-11-05|安徽农业大学|Marseillea fungus for producing blue-violet pigment and method for producing blue-violet pigment by using Marseillea fungus|

---

KR20220020894A|2019-06-11|2022-02-21|에벨로 바이오사이언시즈, 인크.|Treated microbial extracellular vesicles|

---

CA3144350A1|2019-06-20|2020-12-24|Locus Ip Company, Llc|Co-cultivation of a myxobacterium and acinetobacter for enhanced production of emulsan|

---

WO2021026130A1|2019-08-05|2021-02-11|Evelo Biosciences, Inc.|Compositions and methods of treating psoriasis and atopic dermatitis using prevotella histicola|

---

CN110577910B|2019-09-17|2022-02-01|南京农业大学|Brevibacillus laterosporus, antibacterial lipopeptide and application of antibacterial lipopeptide in agriculture and food|

---

US20210077549A1|2019-09-18|2021-03-18|Research Development Foundation|Methods and probiotic compositions for the treatment of metabolic diseases and disorders|

---

CN110982729A|2019-10-23|2020-04-10|江苏省中国科学院植物研究所|Composite microbial inoculum for degrading poplar bark cellulose which is byproduct of wood processing|

---

WO2021138571A1|2019-12-31|2021-07-08|Assembly Biosciences, Inc.|Compositions comprising bacterial species and methods related thereto|

---

WO2021137494A1|2020-01-02|2021-07-08|삼육대학교산학협력단|Composition for preventing or treating diabetes comprising butyricimonas sp. strain as active ingredient|

---

WO2021142279A1|2020-01-10|2021-07-15|Evelo Biosciences, Inc.|Compositions and methods of treatment using veillonella parvula|

---

WO2021146523A1|2020-01-17|2021-07-22|Evelo Biosciences, Inc.|Solid dosage forms with improved disintegration profiles|

---

WO2021158797A1|2020-02-04|2021-08-12|University Of Louisville Research Foundation, Inc.|Use of probiotic bacterium-derived extracellular micro- and/or nanoparticles for the treatment of disease|

---

US11253534B2|2020-03-23|2022-02-22|Sabine Hazan|Method of preventing COVID-19 infection|

---

WO2021203083A2|2020-04-02|2021-10-07|Dupont Nutrition Biosciences Aps|Compositions for metabolic health|

---

WO2021205444A1|2020-04-06|2021-10-14|Yeda Research And Development Co. Ltd.|Methods of diagnosing cancer and predicting responsiveness to therapy|

---

KR102273232B1|2020-05-08|2021-07-06|코스맥스 주식회사|Micrococcus lylae strain and skin condition improving uses of thereof|

---

KR102273234B1|2020-05-08|2021-07-06|코스맥스 주식회사|Kocuria varians strain and skin condition improving uses of thereof|

---

WO2021242056A1|2020-05-28|2021-12-02|주식회사 바이오뱅크힐링|Bifidobacterium sp. strain and extracellular vesicle derived therefrom, and anti-inflammatory and anti-bacterial uses thereof|

---

WO2021252838A1|2020-06-11|2021-12-16|Evelo Biosciences, Inc.|Compositions and methods for treating diseases and disorders using fournierella massiliensis|

---

WO2021252877A1|2020-06-11|2021-12-16|Evelo Biosciences, Inc.|Compositions and methods for treating diseases and disorders using oscillospiraceae microbial extracellular vesicles|

---

WO2021252861A1|2020-06-11|2021-12-16|Evelo Biosciences, Inc.|Compositions and methods for treating diseases and disorders using megasphaera sp|

---

WO2021261891A1|2020-06-22|2021-12-30|로제타엑소좀|Method and composition for enhancing cancer treatment efficacy of bacteria extracellular vesicles|

---

CN111743922A|2020-07-14|2020-10-09|华熙生物科技股份有限公司|Composition for improving colonization and activity of probiotics in nasal cavity and application of composition in nasal cavity care|

---

WO2022020326A1|2020-07-21|2022-01-27|Evelo Biosciences, Inc.|Veillonella parvula strain as an oral therapy for neuroinflammatory diseases|

---

CN111876359A|2020-08-04|2020-11-03|榆林沙漠王生物科技有限公司|Compound microbial inoculum for recycling vegetable residues and preparation method and application thereof|

---

WO2022035269A1|2020-08-14|2022-02-17|주식회사 고바이오랩|Faecalibacterium prausnitzii strain and uses thereof|

---

CN112094820B|2020-09-17|2021-12-07|扬州大学|Enterobacter hollisae phage YZU.P.A-5 and application thereof|

---

CN112094328A|2020-09-21|2020-12-18|华南农业大学|Extraction and purification method of Campylobacter jejuni outer membrane vesicles|

---

CN112094778A|2020-09-24|2020-12-18|千禾味业食品股份有限公司|Weissella LAS1, preparation method and application thereof|

---

KR102269962B1|2020-12-11|2021-06-28|주식회사 바이오뱅크힐링|Eubacterium limosum strain, and vesicles from thereof and anti-inflammation and anti-bacteria uses of thereof|

法律状态:
2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762556015P| true| 2017-09-08|2017-09-08|

---

US62/556,015|2017-09-08|

---

US201862669151P| true| 2018-05-09|2018-05-09|

---

US62/669,151|2018-05-09|

---

PCT/US2018/050211|WO2019051380A1|2017-09-08|2018-09-10|Bacterial extracellular vesicles|

---