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WO2024076723A1 - Traitement de vésicules extracellulaires - Google Patents

Traitement de vésicules extracellulaires Download PDF

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Publication number
WO2024076723A1
WO2024076723A1 PCT/US2023/034623 US2023034623W WO2024076723A1 WO 2024076723 A1 WO2024076723 A1 WO 2024076723A1 US 2023034623 W US2023034623 W US 2023034623W WO 2024076723 A1 WO2024076723 A1 WO 2024076723A1
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WIPO (PCT)
Prior art keywords
evs
bacteria
filter
prevotella
product
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PCT/US2023/034623
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English (en)
Inventor
Fabian B. ROMANO-CHERNAC
Johannes KUNG
Collin MCKENNA
Joseph Shultz
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Evelo Biosciences, Inc.
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Application filed by Evelo Biosciences, Inc. filed Critical Evelo Biosciences, Inc.
Publication of WO2024076723A1 publication Critical patent/WO2024076723A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D43/00—Separating particles from liquids, or liquids from solids, otherwise than by sedimentation or filtration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/10—Separation or concentration of fermentation products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02—Separating microorganisms from their culture media
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms

Definitions

  • Extracellular vesicles are natural lipoprotein nanoparticles produced by many species of bacteria. Their macromolecular content is a complex subset of proteins, glycans, lipids, and LPS. Bacteria can secrete extracellular vesicles into the culture medium.
  • Methods for increasing the production of EVs by bacteria into the medium for a given culture include using perfusion culture systems instead of batch culture systems. Also, increasing the size of the cultures (such as to a commercial scale such as a 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater) (for batch or perfusion cultures) also increases yields.
  • Methods are developed to harvest the increased yields of EVs from bacterial cultures. Such methods include filter systems (such as two-filter systems) and chromatography techniques (such as monoliths) to decrease volumes, increase concentrations, and/or increase purity of EVs after medium (that contains EVs) is removed from cultures.
  • the disclosure provides a method of producing extracellular vesicles (EVs), the method comprising growing EV-producing bacteria in a perfusion culture (e.g., wherein the perfusion culture comprises culture media that comprises EVs) (e.g., under conditions that support bacteria growth and EV production).
  • a perfusion culture e.g., wherein the perfusion culture comprises culture media that comprises EVs
  • the disclosure provides a method of producing extracellular vesicles (EVs), the method comprising growing EV-producing bacteria in a perfusion culture (e.g., wherein the perfusion culture comprises culture media that comprises EVs) (e.g., under conditions that support bacteria growth and EV production).
  • the perfusion culture increases EV yields by at least about 10- fold, e.g., by at least about 15-fold or by at least about 17-fold or by at least about 50-fold, as compared to a batch culture of the same bacteria.
  • the perfusion culture increases EV yields after 24, 48, or 72 hours of culturing, as compared to a batch culture of the same bacteria.
  • EV production of the bacteria is coupled to growth in batch culture.
  • EV production of the bacteria is not coupled to growth in batch culture.
  • the perfusion culture is at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater).
  • a filter system (such as a one-filter system or a two-filter system) removes EVs, metabolites, and waste products of the culture media.
  • the filter system is a two-filter system.
  • the method further comprises filtering the culture media.
  • the method further comprises performing chromatography on the culture media.
  • the method further comprises performing tangential flow filtration on the culture media.
  • the method further comprises drying the culture media.
  • the culture media is dried after the growing step.
  • the culture media is dried after the filtering step.
  • the culture media is dried after the chromatography step.
  • the culture media is dried after the tangential flow filtration step.
  • the method further comprises milling the dried culture media.
  • the disclosure provides a culture media produced by a perfusion culture method provided herein.
  • the disclosure provides a culture media produced by a method of producing extracellular vesicles provided herein.
  • the disclosure provides a method of processing bacterial culture media that comprises extracellular vesicles (EVs), the method comprising passing bacterial culture media that comprises EVs through a two-filter system, wherein the first filter is a product harvest filter (e.g., wherein the output of the product harvest filter comprises EVs) and the second filter is a medium exchange filter.
  • the first filter is a product harvest filter (e.g., wherein the output of the product harvest filter comprises EVs)
  • the second filter is a medium exchange filter.
  • the bacterial culture is a perfusion culture.
  • the perfusion culture is at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater).
  • the product harvest filter and the medium exchange filter comprise the same material.
  • the product harvest filter comprises PES (polyethersulfone).
  • the medium exchange filter comprises PES (polyethersulfone).
  • the product harvest filter and the medium exchange filter comprise PES (polyethersulfone).
  • EVs, media, waste and metabolites pass through the product harvest filter.
  • the pore size of the product harvest filter is about 0.5 micron.
  • media, waste and metabolites pass through the medium exchange filter.
  • the pore size of the medium exchange filter is less than about 0.5 micron.
  • the pore size of the medium exchange filter is about 0.05 micron.
  • the pore size of the medium exchange filter is about 0.02 micron.
  • the pore size of the medium exchange filter is about 0.01 micron.
  • the pore size of the medium exchange filter comprises a size cut off of 750kD (kilodalton).
  • the pore size of the medium exchange filter comprises a size cut off of 500kD.
  • the medium exchange filter runs at a higher flux than the product harvest filter.
  • the flux ratio (medium exchange filterproduct harvest filter) is about 5: 1.
  • the flux ratio (medium exchange filterproduct harvest filter) is about 9: 1.
  • the flux ratio (medium exchange filter product harvest filter) is about 10: 1.
  • the flux ratio reduces sieving of the product harvest filter (e.g., as compared to the amount of sieving if the flux ratio was 1 : 1 or if the product harvest filter was used alone).
  • the volume of the output of the product harvest filter is about l/5x the volume than if a single-filter system was used.
  • the volume of the output of the product harvest filter is about l/9x the volume than if a single-filter system was used. [41] In some embodiments, the volume of the output of the product harvest filter is about l/10x the volume than if a single-filter system was used.
  • the output of the product harvest filter comprises a higher concentration of EVs than if a single-filter system was used.
  • the output of the product harvest filter comprises a concentration of EVs that is at least about 5x higher than if a single-filter system was used.
  • the output of the product harvest filter comprises a concentration of EVs that is at least about 9x higher than if a single-filter system was used.
  • the output of the product harvest filter comprises a concentration of EVs that is at least about lOx higher than if a single-filter system was used.
  • the method further comprises performing chromatography on the output of the product harvest filter.
  • the method further comprises performing tangential flow filtration on the output of the product harvest filter.
  • the method further comprises drying the output of the product harvest filter.
  • the output is dried after the filtering step.
  • the output is dried after the chromatography step.
  • the output is dried after the tangential flow filtration step.
  • the method further comprises milling the dried output of the product harvest filter.
  • the disclosure provides an output of a product harvest filter produced by a method of processing bacterial culture media provided herein.
  • the disclosure provides a method of processing a liquid that comprises extracellular vesicles (EVs) to prepare an EV eluate, the method comprising performing chromatography on the liquid.
  • EVs extracellular vesicles
  • the liquid comprises bacterial culture media.
  • the bacterial culture media is from a perfusion culture.
  • the perfusion culture is at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater).
  • the liquid comprises a product-containing volume.
  • the product-containing volume is output from a single-filter system.
  • the product-containing volume is output from a two-filter system.
  • the product-containing volume is output from a product harvest filter.
  • the product harvest filter comprises PES (polyethersulfone).
  • the pore size of the product harvest filter is about 0.5 micron.
  • the chromatography comprises a chromatography column.
  • the chromatography column comprises a monolith.
  • the chromatography column comprises a monolith ion exchange column.
  • the method comprises processing on one column.
  • the liquid is loaded onto the column and then EV-containing eluate is eluted.
  • the method comprises processing on two columns.
  • the liquid is loaded onto a first column, then EV-containing eluate is eluted from the first column, and while EV-containing eluate is eluted from the first column, liquid is loaded onto the second column.
  • EV-containing eluate is eluted from the second column, and while EV-containing eluate is eluted from the second column, liquid is loaded onto the first column.
  • the loading and eluting steps on the first and second columns are alternated to process the liquid continuously.
  • bacterial culture media is filtered prior to performing the chromatography.
  • the chromatography enriches EV yield by greater than about 5-fold.
  • the chromatography enriches EV yield by about 6-fold.
  • the chromatography enriches EV yield by about 12-fold.
  • the yield of EVs from the chromatography is greater than about 50%.
  • the yield of EVs from the chromatography is greater than about 60%.
  • the method further comprises performing tangential flow filtration on the EV eluate. [77] In some embodiments, the method further comprises drying the EV eluate. In some embodiments, the EV eluate is dried after the chromatography step. In some embodiments, the EV eluate is dried after the tangential flow filtration step.
  • the method further comprises milling the dried EV eluate.
  • the disclosure provides an EV eluate produced by a method of processing a liquid that comprises extracellular vesicles (EVs) provided herein.
  • EVs extracellular vesicles
  • the disclosure provides a method comprising:
  • the disclosure provides an output of a product harvest filter produced by a method provided herein.
  • the disclosure provides a method comprising:
  • the disclosure provides an eluate produced by a method provided herein.
  • the disclosure provides a method comprising:
  • EV-producing bacteria in a perfusion culture (e.g., under conditions that support bacteria growth and EV production), wherein the perfusion culture comprises bacterial culture media that comprises EVs (e.g., as described herein);
  • the disclosure provides an eluate produced by a method provided herein.
  • the method comprises EVs from a bacterial strain that is associated with mucus.
  • the method comprises EVs from anaerobic bacteria.
  • the anaerobic bacteria are obligate (e.g., strict) anaerobes.
  • the anaerobic bacteria are facultative anaerobes.
  • the anaerobic bacteria are aerotolerant anaerobes.
  • the EVs are from monoderm bacteria.
  • the EVs are from diderm bacteria.
  • the EVs are from Gram negative bacteria.
  • the EVs are from bacteria of the Prevotellaceae; Veillonellaceae; Tannerellaceae; Rikenellaceae; Selenomonadaceae;
  • the EVs are from Gram positive bacteria.
  • the EVs are from bacteria of the Oscillospiraceae ; Clostridiaceae; or Lachnospiraceae family.
  • the EVs are from bacteria of the genus Prevotella.
  • the EVs are from bacteria of the genus Veillonella.
  • the EVs are from bacteria of the genus Parabacteroides.
  • the EVs are from bacteria of the Oscillospiraceae family. [101] In some embodiments of an aspect provided herein, the EVs are from bacteria of the Tannerellaceae family.
  • the EVs are from bacteria of the Prevotellaceae family.
  • the EVs are from bacteria of the Veillonellaceae family.
  • the EVs are from bacteria of class, order, family, genus, species and/or strain of bacteria provided in Table 1, Table 2, Table 3, and/or Table 4.
  • the disclosure provides a product produced by a method provided herein.
  • Figure l is a schematic showing a process/manufacturing platform for EVs to improve productivity.
  • Figures 2A and 2B are graphs showing comparisons of EV yields (EV product batches (-fold)) from batch culture versus perfusion culture yields over time (hours).
  • Figure 2A shows the batch yields (batch yield; dashed line) and perfusion EV yield (total product; upper solid line with circles) for a first strain of bacteria for which EV production is coupled with growth.
  • Figure 2B shows the batch yields (batch yield; dashed line) and perfusion EV yield (total product; upper solid line with circles) for a first strain of bacteria for which EV production is not coupled with growth.
  • the lower solid line with circles is the EV yield recovered from the perfusion culture (permeated product).
  • FIG. 3 is a schematic showing a set up for a two-filter system.
  • Harvest volume transfers from the fermenter to the two filters: product harvest filter and medium exchange filter.
  • Captured product from the product harvest filter transfers to a product reservoir and can be further processed, such as through a capture step(s). Metabolites and waste products that passed through the medium exchange filter transfer to a waste reservoir.
  • Figure 4 is a graph showing a theoretical result of using a two-filter (dual membrane perfusion) system. Shifting flux from the “Product Harvest” filter to the “Medium Exchange” filter increases the Flux Ratio, and results in product concentration (-fold; upward sloping line) increasing and permeate volume (downward sloping line; volume (vol)/day) decreasing with increasing ratio. DETAILED DESCRIPTION
  • the disclosure provides methods developed to harvest EVs (such as increased yields of EVs) from bacterial cultures. Yields can be increased, for example, by using perfusion culture systems instead of batch culture systems and/or by increasing the size of the cultures (such as to a commercial scale such as a 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater) (for batch or perfusion cultures).
  • Such harvest methods include filter systems (such as two-filter systems) and chromatography techniques (such as monoliths) to decrease volumes, increase concentrations, and/or increase purity of EVs after medium (that contains EVs) is removed from cultures.
  • perfusion culture can increase EV yields by at least 10-fold, e.g., by at least 15-fold or 17-fold or 50-fold, after 72 hours of culturing.
  • a filter system removes metabolites, and waste products of the culture, yet does not remove the bacterial cells.
  • the filter system also removes product (EVs).
  • the volumes of media from a perfusion culture are greater than for a batch process (for example, up to ten times greater per day).
  • filter area and flow rates need to be managed to ensure sufficient removal of product, metabolites, and waste products, and to avoid the need for expensive enlarged filter areas.
  • flow rates and volumes need to be managed to minimize filter sieving.
  • a two-filter system can address one or more of these considerations.
  • Additional further processing of the output of the perfusion culture can be performed.
  • additional further processing can include filtration (such as with a two-filter system), chromatography, tangential flow filtration, drying (such as by lyophilization or spray drying), and/or milling of a dried product.
  • a two-filter system can be used to process the culture media.
  • the two-filter system can be used as part of a continuous process (e.g., as opposed to a batch or intermittent process).
  • the two filters can be run at the same time, e.g., but at different flow rates.
  • One filter functions to collect product (e.g., EVs) (the product harvest filter).
  • the pore size of the product harvest filter is selected to allow product (e.g., EVs) to pass through, such as a 0.5 micron pore size.
  • Product e.g., EVs
  • the second filter functions to collect media, metabolites, and waste products (the medium exchange filter), yet product (e.g., EVs) does not pass through.
  • the pore size of the medium exchange filter is selected to allow media, metabolites, and waste products to pass through but to not allow product to pass through, such as a 0.05 micron (or smaller, such as 0.02 micron or 0.01 micron) pore size.
  • the medium exchange filter can be selected based on size cut-off: such as a 750kD or 500kD size limits for what can pass through.
  • size cut-off such as a 750kD or 500kD size limits for what can pass through.
  • Both the product harvest filter and the medium exchange filter can be made of the same material, such as PES (polyethersulfone).
  • the medium exchange filter runs at a higher flux than the product harvest filter.
  • the flux ratio (medium exchange filterproduct harvest filter) can be 5: 1, or 9: 1, or 10: 1.
  • this allows the product harvest filter to collect higher concentration product (EVs) in a smaller volume, such as 1/5, 1/9 or 1/10 the volume than if a single-filter system was used.
  • EVs concentration product
  • a smaller volume such as 12,000 liters is further processed.
  • This reduced volume provides advantages for further processing of the product-containing volume (e.g., the output of the product harvest filter when a two-filter system is used).
  • a two-filter (dual-membrane) perfusion system in place of a single-filter perfusion system reduces downstream volume and increases product concentration.
  • Additional further processing of the output of the two-filter system can be performed.
  • Such additional further processing can include chromatography, tangential flow filtration, drying (such as by lyophilization or spray drying), and/or milling of a dried product.
  • chromatography Downstream of growing a bacterial culture for EV production (such as by perfusion culture (particularly when perfusion culturing is performed at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater))), chromatography can be used to process culture media or a product-containing volume.
  • Chromatography can be used to process a product-containing volume, such as after culture media containing product (e.g., EVs) is filtered, such as through a single-filter system or a two-filter system.
  • a monolith can be used (e.g., such as a monolith supplied by Sartorius).
  • a monolith is cast as a single block and is inserted into a chromatographic housing. The monolith is characterized by a highly interconnected network of channels.
  • Considerations include the feasibility and specificity of chromatography to capture the product (e.g., EVs); the large size of the chromatography matrices (such as monoliths); binding capacity; process volumes (including load, buffers and waste); and pool volume management.
  • One or two columns can be used to process culture media (e.g., from a perfusion culture) or a product-containing volume, such as after culture media containing product (e.g., EVs) is filtered, such as through a single-filter system or a two-filter system.
  • culture media or product-containing volume is loaded onto the column and then the product is eluted.
  • culture media or product-containing volume is loaded onto a first column. While product is being eluted from the first column, culture media or product-containing volume is loaded onto a second column.
  • culture media or product-containing volume is loaded onto the first column while product is being eluted from the second column.
  • the loading and eluting steps on the first and second columns can continue to be alternated to process culture media or product-containing volume continuously. This allows for continuous capture, such as to achieve pure and highly concentrated product (this may be considered a process intermediate if additional further processing is performed).
  • pH can affect column loading capacity, such as by three-fold. Enrichment factors can be greater than about 5-fold, e.g., 6- or 12-fold. Yields from the chromatography can be, for example, greater than about 50%, e.g., greater than about 60%.
  • Additional further processing of the output of the chromatography can be performed.
  • Such additional further processing can include tangential flow filtration, drying (such as by lyophilization or spray drying), and/or milling of a dried product.
  • EVs Extracellular vesicles
  • EVs may be naturally-produced vesicles derived from bacteria. EVs are comprised of bacterial lipids and/or bacterial proteins and/or bacterial nucleic acids and/or bacterial carbohydrate moieties, and are isolated from culture supernatant. The natural production of these vesicles can be artificially enhanced (for example, increased) or decreased through manipulation of the environment in which the bacterial cells are being cultured (for example, by media or temperature alterations).
  • EV compositions may be modified to reduce, increase, add, or remove bacterial components or foreign substances to alter efficacy, immune stimulation, stability, immune stimulatory capacity, stability, organ targeting (for example, lymph node), absorption (for example, gastrointestinal), and/or yield (for example, thereby altering the efficacy).
  • purified EV composition or “EV composition” refers to a preparation of 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 the EVs in any process used to produce the preparation. It can also refer to a composition that has been significantly enriched for specific components.
  • Extracellular vesicles may also be obtained from mammalian cells and from can be obtained from microbes such as archaea, fungi, microscopic algae, protozoans, and parasites.
  • “Identity” as between nucleic acid sequences of two nucleic acid molecules can be determined as a percentage of identity using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., et al., J Molec Biol 215:403 (1990); Guide to Huge Computers, Martin J.
  • isolated or “enriched” encompasses a microbe, an EV (such as a bacterial EV) or other entity or substance that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by the hand of man.
  • Isolated bacteria or EVs may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated.
  • isolated bacteria or 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, for example, substantially free of other components.
  • Metal refers to any and all molecular compounds, compositions, molecules, ions, co-factors, catalysts or nutrients used as substrates in any cellular or bacterial metabolic reaction or resulting as product compounds, compositions, molecules, ions, co-factors, catalysts or nutrients from any cellular or bacterial metabolic reaction.
  • a substance is “pure” if it is substantially free of other components.
  • the terms “purify,” “purifying,” and “purified” refer to an EV (such as an EV from bacteria) preparation or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (for example, whether in nature or in an experimental setting), or during any time after its initial production.
  • An EV preparation or compositions may be considered purified if it is isolated at or after production, such as from one or more other bacterial components, and a purified microbe or bacterial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “purified.”
  • purified EVs are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • EV compositions (or preparations) are, for example, purified from residual habitat products.
  • the term “purified EV composition” or “EV composition” refers to a preparation that includes EVs from bacteria 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 the EVs in any process used to produce the preparation. It also refers to a composition that has been significantly enriched or concentrated. In some embodiments, the EVs are concentrated by 2-fold, 3-fold, 4-fold, 5- fold, 10-fold, 100-fold, 1000-fold, 10,000-fold or more than 10,000-fold.
  • Strain refers to a member of a bacterial species with a genetic signature such that it may be differentiated from closely-related members of the same bacterial species.
  • the genetic signature may be the absence of all or part of at least one gene, the absence of all or part of at least on regulatory region (for example, a promoter, a terminator, a riboswitch, a ribosome binding site), the absence (“curing”) of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one foreign gene (a gene derived from another species), the presence at least one mutated regulatory region (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 antibiotic resistance cassette, or a combination thereof.
  • strains may be identified by PCR amplification optionally followed by DNA sequencing of the genomic region(s) of interest or of the whole genome.
  • strains may be differentiated by selection or counter-selection using an antibiotic or nutrient/metabolite, respectively.
  • Bacteria propagated as sources of EVs can be selected based on assays in the art that identify bacteria with properties of interest. For example, in some embodiments, bacteria are selected for the ability to modulate host immune response and/or affect cytokine levels.
  • EVs are selected from a bacterial strain that is associated with mucus.
  • the mucus is associated with the gut lumen.
  • the mucus is associated with the small intestine.
  • the mucus is associated with the respiratory tract.
  • EVs are selected from a bacterial strain that is associated with an epithelial tissue, such as oral cavity, lung, nose, or vagina.
  • the EVs are from bacteria that are human commensals.
  • the EVs are from human commensal bacteria that originate from the human small intestine.
  • the EVs are from human commensal bacteria that originate from the human small intestine and are associated there with the outer mucus layer.
  • taxonomic groups such as class, order, family, genus, species and/or strain
  • Examples of taxonomic groups (such as class, order, family, genus, species and/or strain) of bacteria that can be used as a source of EVs described herein are provided in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere throughout the specification.
  • the bacterial strain is a bacterial strain having 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 to a strain provided herein (for example, listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification).
  • the EVs are from an oncotrophic bacteria.
  • the EVs are from an immunostimulatory bacteria.
  • the EVs are from an immunosuppressive bacteria.
  • the EVs are from an immunomodulatory bacteria. In certain embodiments, EVs are generated from a combination of bacterial strains provided herein. 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.
  • the combination includes EVs from bacterial strains provided herein (for example, listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification and/or bacterial strains having 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 to a strain provided herein (for example, listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification.
  • bacteria from a taxonomic group for example, class, order, family, genus, species or strain
  • Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification can be used as a source of EVs.
  • the EVs are obtained from Gram negative bacteria.
  • the Gram negative bacteria belong to the class Negativicutes.
  • the Negativicutes represent a unique class of microorganisms as they are the only diderm members of the Firmicutes phylum. These anaerobic organisms can be found in the environment and are normal commensals of the oral cavity and GI tract of humans. Because these organisms have an outer membrane, the yields of EVs from this class were investigated. It was found that on a per cell basis these bacteria produce a high number of vesicles (10-150 EVs/cell). The EVs from these organisms are broadly stimulatory and highly potent in in vitro assays. Investigations into their therapeutic applications in several oncology and inflammation in vivo models have shown their therapeutic potential.
  • the Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidaminococcaceae. and Sporomusaceae .
  • the Negativicutes class includes the genera Megasphaera, Selenomonas, Propionospora, and Acidaminococcus.
  • Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestine, and Propionospora sp.
  • the EVs are obtained from Gram positive bacteria.
  • the EVs are from aerotol erant bacteria.
  • the EVs are from monoderm bacteria.
  • the EVs are from diderm bacteria.
  • the EVs are from bacteria of the family: Prevotellaceae; Veillonellaceae; Tannerellaceae; Rikenellaceae; Selenomonadaceae; Sporomusaceae; Synergistaceae; or Akkermaniaceae .
  • the EVs are from bacteria of the family Oscillospiraceae ; Clostridiaceae; Lachnospiraceae; or Christensenellaceae .
  • the EVs are from bacteria of the genus Prevotella.
  • the EVs are from bacteria of the genus Veillonella. [152] In some embodiments, the EVs are from bacteria of the mis Parabacteroides.
  • the EVs are from a bacterial strain of the Oscillospiraceae family.
  • the EVs are from a bacterial strain of the Tannerellaceae family.
  • the EVs are from a bacterial strain of the Prevotellaceae family.
  • the EVs are from a bacterial strain of the Veillonellaceae family.
  • the EVs are obtained from aerobic bacteria.
  • the EVs are obtained from anaerobic bacteria.
  • the anaerobic bacteria comprise obligate anaerobes.
  • the anaerobic bacteria comprise facultative anaerobes.
  • the EVs are obtained from acidophile bacteria.
  • the EVs are obtained from alkaliphile bacteria.
  • the EVs are obtained from neutral ophile bacteria.
  • the EVs are obtained from fastidious bacteria.
  • the EVs are obtained from nonfasti di ous bacteria.
  • bacteria from which EVs are obtained are lyophilized.
  • bacteria from which EVs are obtained are gamma irradiated (for example, at 17.5 or 25 kGy).
  • bacteria from which EVs are obtained are UV irradiated.
  • bacteria from which EVs are obtained are heat inactivated
  • bacteria from which EVs are obtained are acid treated.
  • bacteria from which EVs are obtained are oxygen sparged
  • the EVs are lyophilized.
  • the EVs are gamma irradiated (for example, at 17.5 or 25 kGy).
  • the EVs are UV irradiated.
  • the EVs are heat inactivated (for example, at 50°C for two hours or at 90°C for two hours).
  • the EVs are acid treated.
  • the EVs are oxygen sparged (for example, at 0.1 vvm for two hours).
  • the phase of growth can affect the amount or properties of bacteria and/or EVs produced by bacteria.
  • EVs can be isolated, for example, from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • EVs can be isolated from a batch culture of bacteria.
  • EVs can be isolated from a perfusion culture of bacteria.
  • the EVs described herein are obtained from obligate anaerobic bacteria.
  • obligate anaerobic bacteria include gram-negative rods (including the genera of Bacteroides, Prevotella, Porphyromonas, Fusobacterium, Bilophila and Sutterella sppf, gram -positive cocci (primarily Peptostreptococcus sppf, gram -positive spore-forming (Clostridium sppf, non-spore-forming bacilli (Actinomyces,
  • the obligate anaerobic bacteria are of a genus selected from the group consisting of Agathobaculum, Atopobium, Blautia, Burkholderia, Dielma, Longicatena, Paraclostridium, Turicibacter, and Tyzzerella.
  • the Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidaminococcaceae, and Sporomusaceae .
  • the Negativicutes class includes the genera Megasphaera, Selenomonas, Propionospora, mA Acidaminococcus.
  • Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestini, and Propionospora sp.
  • the EVs are from bacteria of the Negativicutes class.
  • the EVs are from bacteria of the Veillonellaceae family.
  • the EVs are from bacteria of the Selenomonadaceae family.
  • the EVs are from bacteria of the Acidaminococcaceae family.
  • the EVs are from bacteria of the Sporomusaceae family.
  • the EVs are from bacteria of the Megasphaera genus.
  • the EVs are from bacteria of the Selenomonas genus.
  • the EVs are from bacteria of the Propionospora genus.
  • the EVs are from bacteria of the Acidaminococcus genus.
  • the EVs are from Megasphaera sp. bacteria.
  • the EVs are from Selenomonas felix bacteria. [192] In some embodiments, the EVs are from Acidaminococcus intestini bacteria.
  • the EVs are from Propionospora sp. bacteria.
  • the EVs are from bacteria of the Clostridia class.
  • the EVs are from bacteria of the Oscillospriraceae family.
  • the EVs are from bacteria of the Faecalibacterium genus.
  • the EVs are from bacteria of the Fournierella genus.
  • the EVs are from bacteria of the Harryflintia genus.
  • the EVs are from bacteria of the Agathobaculum genus.
  • the EVs are from Faecalibacterium prausnitzii (for example, Faecalibacterium prausnitzii Strain A) bacteria.
  • the EVs are from Fournierella massiliensis (for example, Fournierella massiliensis Strain A) bacteria.
  • the EVs are from Harryflintia acetispora (for example, Harryflintia acetispora Strain A) bacteria.
  • the EVs are from Agathobaculum sp. (for example, Agathobaculum sp. Strain A) bacteria.
  • the EVs described herein are obtained from bacterium of a genus selected from the group consisting of Escherichia, Klebsiella, Lactobacillus, Shigella, and Staphylococcus.
  • the EVs described herein are obtained from a species selected from the group consisting of Blautia massiliensis, Paraclostridium benzoelyticum, Dielma fastidiosa, Longicatena caecimuris, Lactococcus lactis cremoris, Tyzzerella nexilis, Hungatella effluvia, Klebsiella quasipneumoniae subsp. Similipneumoniae, Klebsiella oxytoca, and Veillonella tobetsuensis.
  • the EVs described herein are obtained from a Prevotella bacteria.
  • the EVs described herein are obtained from a Prevotella bacteria selected from the group consisting of Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella bacteria.
  • the EVs described herein are obtained from a strain of bacteria comprising a genomic sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the genomic sequence of the strain of bacteria deposited with the ATCC Deposit number as provided in Table 3.
  • sequence identity for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity
  • the EVs described herein are obtained from a strain of bacteria comprising a 16S sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the 16S sequence of the strain of bacteria deposited with the ATCC Deposit number as provided in Table 3.
  • sequence identity for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity
  • the Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidaminococcaceae, and Sporomusaceae .
  • the Negativicutes class includes the genera Megasphaera, Selenomonas, Propionospora, mA Acidaminococcus.
  • Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestini, and Propionospora sp.
  • the Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidaminococcaceae, and Sporomusaceae .
  • the Negativicutes class includes the genera Megasphaera, Selenomonas, Propionospora, and Acidaminococcus.
  • Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestini, and Propionospora sp.
  • the bacteria from which the EVs are obtained are of the Negativicutes class.
  • the bacteria from which the EVs are obtained are of the Veillonellaceae family. [213] In some embodiments, the bacteria from which the EVs are obtained are of the Selenomonadaceae family.
  • the bacteria from which the EVs are obtained are of the Acidaminococcaceae family.
  • the bacteria from which the EVs are obtained are of the Sporomusaceae family.
  • the bacteria from which the EVs are obtained are of the Megasphaera genus.
  • the bacteria from which the EVs are obtained are of the Selenomonas genus.
  • the bacteria from which the EVs are obtained are of the Propionospora genus.
  • the bacteria from which the EVs are obtained are of the Acidaminococcus genus.
  • the bacteria from which the EVs are obtained are Megasphaera sp. bacteria.
  • the bacteria from which the EVs are obtained are Selenomonas felix bacteria.
  • the bacteria from which the EVs are obtained are Acidaminococcus intestini bacteria.
  • the bacteria from which the EVs are obtained are Propionospora sp. bacteria.
  • the bacteria from which the EVs are obtained are of the Clostridia class.
  • the bacteria from which the EVs are obtained are of the Oscillospriraceae family.
  • the bacteria from which the EVs are obtained are of the Faecalibacterium genus.
  • the bacteria from which the EVs are obtained are of the Fournierella genus.
  • the bacteria from which the EVs are obtained are of the Harryflintia genus.
  • the bacteria from which the EVs are obtained are of the Agathobaculum genus.
  • the bacteria from which the EVs are obtained are Faecalibacterium prausnitzii (for example, Faecalibacterium prausnitzii Strain A) bacteria.
  • the bacteria from which the EVs are obtained are Fournierella massiliensis (for example, Fournierella massiliensis Strain A) bacteria.
  • the bacteria from which the EVs are obtained are Harryflintia acetispora (for example, Harryflintia acetispora Strain A) bacteria.
  • the bacteria from which the EVs are obtained are Agathobaculum sp. (for example, Agathobaculum sp. Strain A) bacteria.
  • the bacteria from which the EVs are obtained are bacteria of a genus selected from the group consisting of Escherichia, Klebsiella, Lactobacillus, Shigella, and Staphylococcus.
  • the bacteria from which the EVs are obtained are a species selected from the group consisting of Blautia massiliensis, Paraclostridium benzoelyticum, Dielma fastidiosa, Longicatena caecimuris, Lactococcus lactis cremoris, Tyzzerella nexilis, Hungatella effluvia, Klebsiella quasipneumoniae subsp. Similipneumoniae, Klebsiella oxytoca, and Veillonella tobetsuensis.
  • the bacteria from which the EVs are obtained are a Prevotella bacteria selected from the group consisting of Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella t
  • the bacteria from which the EVs are obtained are a strain of bacteria comprising a genomic sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the genomic sequence of the strain of bacteria deposited with the ATCC Deposit number as provided in Table 3.
  • sequence identity for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity
  • the bacteria from which the EVs are obtained are a strain of bacteria comprising a 16S sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the 16S sequence of the strain of bacteria deposited with the ATCC Deposit number as provided in Table 3.
  • sequence identity for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity
  • the Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidaminococcaceae, and Sporomusaceae .
  • the Negativicutes class includes the genera Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestini, and Propionospora sp.
  • the bacteria from which the EVs are obtained are of the Negativicutes class.
  • the bacteria from which the EVs are obtained are of the Veillonellaceae family.
  • the bacteria from which the EVs are obtained are of the Selenomonadaceae family.
  • the bacteria from which the EVs are obtained are of the Acidaminococcaceae family.
  • the bacteria from which the EVs are obtained are of the Sporomusaceae family.
  • the bacteria from which the EVs are obtained are of the Prevotellaceae; Veillonellaceae; Tannerellaceae; Rikenellaceae; Selenomonadaceae; Sporomusaceae; Synergistaceae; Christensenellaceae; or Akkermaniaceae family.
  • the bacteria from which the EVs are obtained are of the Oscillospiraceae ; Clostridiaceae; or Lachnospiraceae family.
  • the bacteria from which the EVs are obtained are of the Megasphaera genus.
  • the bacteria from which the EVs are obtained are of the Selenomonas genus.
  • the bacteria from which the EVs are obtained are of the Propionospora genus.
  • the bacteria from which the EVs are obtained are of the Acidaminococcus genus.
  • the bacteria from which the EVs are obtained are Megasphaera sp. bacteria.
  • the bacteria from which the EVs are obtained are Selenomonas felix bacteria.
  • the bacteria from which the EVs are obtained are Acidaminococcus intestini bacteria.
  • the bacteria from which the EVs are obtained are Propionospora sp. bacteria.
  • the bacteria from which the EVs are obtained are of the Clostridia class.
  • the bacteria from which the EVs are obtained are of the Oscillospriraceae family.
  • the bacteria from which the EVs are obtained are of the Faecalibacterium genus.
  • the bacteria from which the EVs are obtained are of the Fournierella genus.
  • the bacteria from which the EVs are obtained are of the Harryflintia genus.
  • the bacteria from which the EVs are obtained are of the Agathobaculum genus.
  • the bacteria from which the EVs are obtained are Faecalibacterium prausnitzii (for example, Faecalibacterium prausnitzii Strain A) bacteria.
  • the bacteria from which the EVs are obtained are Fournierella massiliensis (for example, Fournierella massiliensis Strain A) bacteria.
  • the bacteria from which the EVs are obtained are Harryflintia acetispora (for example, Harryflintia acetispora Strain A) bacteria.
  • the bacteria from which the EVs are obtained are Agathobaculum sp. (for example, Agathobaculum sp. Strain A) bacteria.
  • the bacteria from which the EVs are obtained are a strain of Agathobaculum sp.
  • the Agathobaculum sp. strain is a strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (for example, genomic sequence, 16S sequence, CRISPR sequence) of the Agathobaculum sp.
  • Strain A ATCC Deposit Number PTA-125892
  • the Agathobaculum sp. strain is the Agathobaculum sp. Strain A (ATCC Deposit Number PTA- 125892).
  • the bacteria from which the EVs are obtained are of the class Bacteroidia [phylum Bacteroidota ⁇ . In some embodiments, the bacteria from which the EVs are obtained are bacteria of order Bacteroidales. In some embodiments, the bacteria from which the EVs are obtained are of the family Porphyromonoadaceae . In some embodiments, the bacteria from which the EVs are obtained are of the family Prevotellaceae . In some embodiments, the bacteria from which the EVs are obtained are bacteria of the class Bacteroidia wherein the cell envelope structure of the bacteria is diderm. In some embodiments, the bacteria from which the EVs are obtained are bacteria of the class Bacteroidia that stain Gram negative. In some embodiments, the bacteria from which the EVs are obtained are bacteria of the class Bacteroidia wherein the bacteria is diderm and the bacteria stain Gram negative.
  • the bacteria from which the EVs are obtained are bacteria of the class Clostridia [phylum Firmicutes ⁇ . In some embodiments, the bacteria from which the EVs are obtained are of the order Eubacteriales. In some embodiments, the bacteria from which the EVs are obtained are of the family Oscillispiraceae . In some embodiments, the bacteria from which the EVs are obtained are of the family Lachnospiraceae . In some embodiments, the bacteria from which the EVs are obtained are of the family Peptostreptococcaceae . In some embodiments, the bacteria from which the EVs are obtained are of the family Clostridiales family XIII/ Incertae sedis 41.
  • the bacteria from which the EVs are obtained are of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm. In some embodiments, the bacteria from which the EVs are obtained are of the class Clostridia that stain Gram negative. In some embodiments, the bacteria from which the EVs are obtained are of the class Clostridia that stain Gram positive. In some embodiments, the bacteria from which the EVs are obtained are of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm and the bacteria stain Gram negative. In some embodiments, the bacteria from which the EVs are obtained are of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm and the bacteria stain Gram positive.
  • the bacteria from which the EVs are obtained are of the class Negativicutes [phylum Firmicutes ⁇ . In some embodiments, the bacteria from which the EVs are obtained are of the order Veillonellales. In some embodiments, the bacteria from which the EVs are obtained are of the family Veillonelloceae. In some embodiments, the bacteria from which the EVs are obtained are of the order Selenomonadales. In some embodiments, the bacteria from which the EVs are obtained are bacteria of the family Selenomonadaceae . In some embodiments, the bacteria from which the EVs are obtained are of the family Sporomusaceae .
  • t the bacteria from which the EVs are obtained are of the class Negativicutes wherein the cell envelope structure of the bacteria is diderm. In some embodiments, the bacteria from which the EVs are obtained are of the bacteria from which the EVs are obtained are the EVs are from bacteria of the class Negativicutes wherein the cell envelope structure of the bacteria is diderm and the bacteria stain Gram negative.
  • the bacteria from which the EVs are obtained are of the class Synergistia [phylum Synergistota ⁇ . In some embodiments, the bacteria from which the EVs are obtained are of the order Synergistales. In some embodiments, the bacteria from which the EVs are obtained are of the family Synergistaceae . In some embodiments, the bacteria from which the EVs are obtained are of the class Synergistia wherein the cell envelope structure of the bacteria is diderm. In some embodiments, the bacteria from which the EVs are obtained are of the class Synergistia that stain Gram negative. In some embodiments, the bacteria from which the EVs are obtained are of the class Synergistia wherein the cell envelope structure of the bacteria is diderm and the bacteria stain Gram negative.
  • the bacteria from which the EVs are obtained are from one strain of bacteria, for example, a strain provided herein.
  • the bacteria from which the EVs are obtained are from one strain of bacteria (for example, a strain provided herein) or from more than one strain provided herein.
  • the bacteria from which the EVs are obtained are Lactococcus lactis cremoris bacteria, for example, a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Lactococcus lactis cremoris Strain A (ATCC designation number PTA-125368).
  • the bacteria from which the EVs are obtained are Lactococcus bacteria, for example, Lactococcus lactis cremoris Strain A (ATCC designation number PTA-125368).
  • the bacteria from which the EVs are obtained are of the Prevotella genus. In some embodiments, the bacteria from which the EVs are obtained are Prevotella histicola bacteria.
  • the bacteria from which the EVs are obtained are Prevotella bacteria, for example, a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
  • the bacteria from which the EVs are obtained are Prevotella bacteria, for example, Prevotella Strain B 50329 (NRRL accession number B 50329).
  • the bacteria from which the EVs are obtained are Prevotella histicola bacteria, for example, a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella histicola Strain C deposited as ATCC designation number PTA-126140.
  • the bacteria from which the EVs are obtained are Prevotella histicola bacteria, for example Prevotella histicola Strain C deposited as ATCC designation number PTA-126140).
  • the bacteria from which the EVs are obtained are Bifidobacterium bacteria, for example, a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Bifidobacterium bacteria deposited as ATCC designation number PTA-125097.
  • the bacteria from which the EVs are obtained are Bifidobacterium bacteria, for example, Bifidobacterium bacteria deposited as ATCC designation number PTA-125097.
  • the bacteria from which the EVs are obtained are Veillonella bacteria, for example, a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Veillonella bacteria deposited as ATCC designation number PTA-125691.
  • the bacteria from which the EVs are obtained are Veillonella bacteria, for example, Veillonella bacteria deposited as ATCC designation number PTA-125691.
  • the bacteria from which the EVs are obtained are Ruminococcus gnavus bacteria.
  • the Ruminococcus gnavus bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Ruminococcus gnavus bacteria deposited as ATCC designation number PTA-126695.
  • the Ruminococcus gnavus bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Ruminococcus gnavus bacteria deposited as ATCC designation number PTA-126695.
  • the Ruminococcus gnavus bacteria are Ruminococcus gnavus bacteria deposited as ATCC designation number PTA-126695.
  • the bacteria from which the EVs are obtained are Megasphaera sp. bacteria.
  • the Megasphaera sp. bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Megasphaera sp. bacteria deposited as ATCC designation number PTA-126770.
  • the Megasphaera sp. bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Megasphaera .s/ bacteria deposited as ATCC designation number PTA- 126770.
  • the Megasphaera sp. bacteria are Megasphaera sp. bacteria deposited as ATCC designation number PTA-126770.
  • the bacteria from which the EVs are obtained are Fournierella massiliensis bacteria.
  • the Fournierella massiliensis bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Fournierella massiliensis bacteria deposited as ATCC designation number PTA-126696.
  • the Fournierella massiliensis bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Fournierella massiliensis bacteria deposited as ATCC designation number PTA-126696.
  • the Fournierella massiliensis bacteria are Fournierella massiliensis bacteria deposited as ATCC designation number PTA-126696.
  • the bacteria from which the EVs are obtained are Harryflintia acetispora bacteria.
  • the Harryflintia acetispora bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694.
  • the Harryflintia acetispora bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694.
  • the Harryflintia acetispora bacteria are Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694.
  • the bacteria from which the EVs are obtained are bacteria that produce metabolites, for example, the bacteria produce butyrate, iosine, proprionate, or tryptophan metabolites.
  • the bacteria from which the EVs are obtained are bacteria that produce butyrate. In some embodiments, the bacteria are from the genus Blautia;
  • the bacteria from which the EVs are obtained are bacteria that produce iosine.
  • the bacteria are from the genus Bifidobacterium; Lactobacillus; or Olsenella.
  • the bacteria from which the EVs are obtained are bacteria that produce proprionate.
  • the bacteria are from the genus Akkermansia; Bacteriodes; Dialister; Eubacterium; Megasphaera; Parabacteriodes;
  • the bacteria from which the EVs are obtained are bacteria that produce tryptophan metabolites.
  • the bacteria are from the genus Lactobacillus or Peptostreptococcus.
  • the bacteria from which the EVs are obtained are bacteria that produce inhibitors of histone deacetylase 3 (HDAC3).
  • the bacteria are from the species Bariatricus massiliensis, Faecalibacterium prausnitzii, Megasphaera massiliensis or Roseburia intestinalis.
  • the bacteria are from the genus Alloiococcus; Bacillus; Catenibacterium; Corynebacterium; Cupriavidus; Enhydrobacter; Exiguobacterium; Faecalibacterium; Geobacillus; Methylobacterium; Micrococcus; Morganella; Proteus;
  • the bacteria are from the genus Cutibacterium. In some embodiments, the bacteria are from the species Cutibacterium avidum. In some embodiments, the bacteria are from the genus Lactobacillus. In some embodiments, the bacteria are from the species Lactobacillus gasseri. In some embodiments, the bacteria are from the genus Dysosmobacter . In some embodiments, the bacteria are from the species Dysosmobacter welbionis.
  • the bacteria from which the EVs are obtained are of the genus Alloiococcus; Bacillus; Catenibacterium; Corynebacterium; Cupriavidus;
  • the bacteria from which the EVs are obtained are of the Cutibacterium genus. In some embodiments, the bacteria from which the EVs are obtained are Cutibacterium avidum bacteria.
  • the bacteria from which the EVs are obtained are of the genus Leuconostoc.
  • the bacteria from which the EVs are obtained are of the genus Lactobacillus.
  • the bacteria from which the EVs are obtained are of the genus Akkermansia; Bacillus; Blautia; Cupriavidus; Enhydrobacter; Faecalibacterium; Lactobacillus; Lactococcus; Micrococcus; Morganella; Propionibacterium; Proteus; Rhizobium; or Streptococcus.
  • the bacteria from which the EVs are obtained are Leuconostoc holzapfelii bacteria.
  • the bacteria from which the EVs are obtained are Akkermansia muciniphila; Cupriavidus metallidurans; Faecalibacterium prausnitzii; Lactobacillus casei; Lactobacillus plantarum; Lactobacillus paracasei; Lactobacillus plantarum; Lactobacillus rhamnosus; Lactobacillus sakei; or Streptococcus pyogenes bacteria.
  • the bacteria from which the EVs are obtained are Lactobacillus casei; Lactobacillus plantarum; Lactobacillus paracasei; Lactobacillus plantarum; Lactobacillus rhamnosus; or Lactobacillus sakei bacteria.
  • the EVs described herein are obtained from a genus selected from the group consisting of Acinetobacter; Deinococcus; Helicobacter; Rhodococcus;
  • the EVs described herein are obtained from a species selected from the group consisting of Acinetobacter baumanii; Deinococcus radiodurans; Helicobacter pylori; Rhodococcus equi; Weissella cibaria; Alloiococcus otitis; Atopobium vaginae; Catenibacterium mituokai; Corynebacterium glutamicum; Exiguobacterium aurantiacum; Geobacillus stearothermophilus; Methylobacterium jeotgali; Micrococcus luteus; Morganella morganii; Proteus mirabilis; Rhizobium leguminosarum; Rothia amarae; Sphingomonas paucimobilis; and Sphingomonas koreens.
  • the EVs are from Leuconostoc holzapfelii bacteria. In some embodiments, the EVs are from Leuconostoc holzapfelii Ceb-kc-003 (KCCM11830P) bacteria.
  • the bacteria from which the EVs are obtained are Megasphaera sp. bacteria (for example, from the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387).
  • the bacteria from which the EVs are obtained are Megasphaera massiliensis bacteria (for example, from the strain with accession number NCIMB 42787, NCIMB 43388 or NCIMB 43389).
  • the bacteria from which the EVs are obtained are Megasphaera massiliensis bacteria (for example, from the strain with accession number DSM 26228).
  • the bacteria from which the EVs are obtained are Parabacteroides distasonis bacteria (for example, from the strain with accession number NCIMB 42382).
  • the bacteria from which the EVs are obtained are Megasphaera massiliensis bacteria (for example, from the strain with accession number NCIMB 43388 or NCIMB 43389), or a derivative thereof. See, for example, WO 2020/120714.
  • the Megasphaera massiliensis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (for example, genomic sequence, 16S sequence, and/or CRISPR sequence) of Megasphaera massiliensis bacteria from the strain with accession number NCIMB 43388 or NCIMB 43389.
  • the Megasphaera massiliensis bacteria is the strain with accession number NCIMB 43388 or NCIMB 43389.
  • the bacteria from which the EVs are obtained are Megasphaera massiliensis bacteria strain deposited under accession number NCIMB 42787, or a derivative thereof. See, for example, WO 2018/229216.
  • the Megasphaera massiliensis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (for example, genomic sequence, 16S sequence, and/or CRISPR sequence) of the Megasphaera massiliensis bacteria strain deposited under accession number NCIMB 42787.
  • the Megasphaera massiliensis bacteria is the strain deposited under accession number NCIMB 42787.
  • the bacteria from which the EVs are obtained are Megasphaera spp. bacteria from the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387, or a derivative thereof. See, for example, WO 2020/120714. In some embodiments, the Megasphaera sp.
  • bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (for example, genomic sequence, 16S sequence, and/or CRISPR sequence) of the Megasphaera sp. from a strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387.
  • the Megasphaera sp. bacteria is the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387.
  • the bacteria from which the EVs are obtained are Parabacteroides distasonis bacteria deposited under accession number NCIMB 42382, or a derivative thereof. See, for example, WO 2018/229216.
  • the Parabacteroides distasonis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (for example, genomic sequence, 16S sequence, and/or CRISPR sequence) of the Parabacteroides distasonis bacteria deposited under accession number NCIMB 42382.
  • the Parabacteroides distasonis bacteria is the strain deposited under accession number NCIMB 42382.
  • the bacteria from which the EVs are obtained are Megasphaera massiliensis bacteria deposited under accession number DSM 26228, or a derivative thereof. See, for example, WO 2018/229216.
  • the Megasphaera massiliensis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (for example, genomic sequence, 16S sequence, and/or CRISPR sequence) of Megasphaera massiliensis bacteria deposited under accession number DSM 26228.
  • the Megasphaera massiliensis bacteria is the strain deposited under accession number DSM 26228.
  • the bacteria from which the EVs are obtained are modified (for example, engineered) to reduce toxicity or other adverse effects, to enhance delivery) (for example, oral delivery) of the EVs (for example, by improving acid resistance, muco- adherence and/or penetration and/or resistance to bile acids, digestive enzymes, resistance to anti-microbial peptides and/or antibody neutralization), to target desired cell types (for example, M-cells, goblet cells, enterocytes, dendritic cells, macrophages), to enhance their immunomodulatory and/or therapeutic effect of the EVs (for example, either alone or in combination with another therapeutic agent), and/or to enhance immune activation or suppression by the EVs (for example, through modified production of polysaccharides, pili, fimbriae, adhesins).
  • the engineered bacteria described herein are modified to improve EV manufacturing (for example, higher oxygen tolerance, stability, improved freeze-thaw tolerance, shorter generation times).
  • the engineered bacteria described include bacteria harboring one or more genetic changes, such change being an insertion, deletion, translocation, or substitution, or any combination thereof, of one or more nucleotides contained on the bacterial chromosome or endogenous plasmid and/or one or more foreign plasmids, wherein the genetic change may results in the overexpression and/or underexpression of one or more genes.
  • the engineered bacteria may be produced using any technique known in the art, including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, or any combination thereof.
  • the EVs described herein are modified such that they comprise, are linked to, and/or are bound by a therapeutic moiety.
  • the therapeutic moiety is a cancer-specific moiety.
  • the cancer-specific moiety has binding specificity for a cancer cell (for example, has binding specificity for a cancer-specific antigen).
  • the cancer-specific moiety comprises an antibody or antigen binding fragment thereof.
  • the cancer-specific moiety comprises a T cell receptor or a chimeric antigen receptor (CAR).
  • the cancer-specific moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof.
  • the cancer-specific moiety is a bipartite fusion protein that has two parts: a first part that binds to and/or is linked to the bacterium and a second part that is capable of binding to a cancer cell (for example, by having binding specificity for a cancer-specific antigen).
  • the first part is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP.
  • the first part has binding specificity for the EV (for example, by having binding specificity for a bacterial antigen).
  • the first and/or second part comprises an antibody or antigen binding fragment thereof.
  • the first and/or second part comprises a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the first and/or second part comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptorbinding fragment thereof. In certain embodiments, co-administration of the cancer-specific moiety with the EVs (either in combination or in separate administrations) increases the targeting of the EVs to the cancer cells.
  • the EVs described herein are engineered such that they comprise, are linked to, and/or are bound by a magnetic and/or paramagnetic moiety (for example, a magnetic bead).
  • the magnetic and/or paramagnetic moiety is comprised by and/or directly linked to the bacteria. In some embodiments, the magnetic and/or paramagnetic moiety is linked to and/or a part of an EV-binding moiety that that binds to the EV. In some embodiments, the EV-binding moiety is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP. In some embodiments the EV-binding moiety has binding specificity for the EV (for example, by having binding specificity for a bacterial antigen). In some embodiments, the EV-binding moiety comprises an antibody or antigen binding fragment thereof.
  • the EV-binding moiety comprises a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the EV-binding moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof. In certain embodiments, co-administration of the magnetic and/or paramagnetic moiety with the EVs (either together or in separate administrations) can be used to increase the targeting of the EVs (for example, to cancer cells and/or a part of a subject where cancer cells are present.
  • the EVs from bacteria described herein are prepared using any method known in the art.
  • the EVs are prepared without an EV purification step.
  • bacteria described herein are killed using a method that leaves the EVs intact and the resulting bacterial components, including the EVs, are used in the methods and compositions described herein.
  • the bacteria are killed using an antibiotic (for example, using an antibiotic described herein).
  • the bacteria are killed using UV irradiation.
  • the bacteria are heat- killed.
  • the EVs described herein are purified from one or more other bacterial components.
  • Methods for purifying EVs from bacteria are known in the art.
  • EVs are prepared from bacterial cultures using methods described in S. Bin Park, et al. PLoS ONE. 6(3):el7629 (2011) or G. Norheim, et al. PLoS ONE. 10(9): eO 134353 (2015) or Jeppesen, et al. Cell 177:428 (2019), each of which is hereby incorporated by reference in its entirety.
  • the bacteria are cultured to high optical density and then centrifuged to pellet bacteria (for example, at 10,000 x g for 30 min at 4°C, at 15,500 x g for 15 min at 4°C).
  • the culture supernatants are then passed through filters to exclude intact bacterial cells (for example, a 0.22 pm filter).
  • the supernatants are then subjected to tangential flow filtration, during which the supernatant is concentrated, species smaller than 100 kDa are removed, and the media is partially exchanged with PBS.
  • filtered supernatants are centrifuged to pellet bacterial EVs (for example, at 100,000-150,000 x g for 1-3 hours at 4°C, at 200,000 x g for 1-3 hours at 4°C).
  • the EVs are further purified by resuspending the resulting EV pellets (for example, in PBS), and applying the resuspended EVs to an Optiprep (iodixanol) gradient or gradient (for example, a 30-60% discontinuous gradient, a 0-45% discontinuous gradient), followed by centrifugation (for example, at 200,000 x g for 4-20 hours at 4°C).
  • EV bands can be collected, diluted with PBS, and centrifuged to pellet the EVs (for example, at 150,000 x g for 3 hours at 4°C, at 200,000 x g for 1 hour at 4°C).
  • the purified EVs can be stored, for example, at -80°C or -20°C until use.
  • the EVs are further purified by treatment with DNase and/or proteinase K.
  • cultures of bacteria can be centrifuged at 11,000 x g for 20-40 min at 4°C to pellet bacteria.
  • Culture supernatants may be passed through a 0.22 pm filter to exclude intact bacterial cells.
  • Filtered supernatants may then be concentrated using methods that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration.
  • ammonium sulfate precipitation 1.5-3 M ammonium sulfate can be added to filtered supernatant slowly, while stirring at 4°C.
  • Precipitations can be incubated at 4°C for 8-48 hours and then centrifuged at 11,000 x g for 20-40 min at 4°C.
  • the resulting pellets contain bacteria EVs and other debris.
  • filtered supernatants can be centrifuged at 100,000-200,000 x g for 1-16 hours at 4°C.
  • the pellet of this centrifugation contains bacterial EVs and other debris such as large protein complexes.
  • supernatants can be filtered so as to retain species of molecular weight > 50 or 100 kDa.
  • EVs can be obtained from bacteria cultures continuously during growth, or at selected time points during growth, for example, by connecting a bioreactor to an alternating tangential flow (ATF) system (for example, XCell ATF from Repligen).
  • ATF alternating tangential flow
  • the ATF system retains intact cells (>0.22 pm) in the bioreactor, and allows smaller components (for example, EVs, free proteins) to pass through a filter for collection.
  • the system may be configured so that the ⁇ 0.22 pm filtrate is then passed through a second filter of 100 kDa, allowing species such as EVs between 0.22 pm and 100 kDa to be collected, and species smaller than 100 kDa to be pumped back into the bioreactor.
  • the system may be configured to allow for medium in the bioreactor to be replenished and/or modified during growth of the culture.
  • EVs collected by this method may be further purified and/or concentrated by ultracentrifugation or filtration as described above for filtered supernatants.
  • EVs obtained by methods provided herein may be further purified by size-based column chromatography, by affinity chromatography, by ion-exchange chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column.
  • Samples are applied to a 35- 60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in PBS and 3 volumes of 60% Optiprep are added to the sample. In some embodiments, if filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep. Samples are applied to a 0-45% discontinuous Optiprep gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C, for example, 4-24 hours at 4°C.
  • EVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 um filter to exclude intact cells. To further increase purity, isolated EVs may be DNase or proteinase K treated.
  • EVs used for in vivo injections purified EVs are processed as described previously (G. Norheim, et al. PLoS ONE. 10(9): e0134353 (2015)). Briefly, after sucrose gradient centrifugation, bands containing EVs are resuspended to a final concentration of 50 pg/mL in a solution containing 3% sucrose or other solution suitable for in vivo injection known to one skilled in the art. This solution may also contain adjuvant, for example aluminum hydroxide at a concentration of 0-0.5% (w/v).
  • EVs in PBS are sterile- filtered to ⁇ 0.22 pm.
  • samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 using filtration (for example, Amicon Ultra columns), dialysis, or ultracentrifugation (200,000 x g, > 3 hours, 4°C) and resuspension.
  • filtration for example, Amicon Ultra columns
  • dialysis for example, dialysis
  • ultracentrifugation 200,000 x g, > 3 hours, 4°C
  • the sterility of the EV preparations can be confirmed by plating a portion of the EVs onto agar medium used for standard culture of the bacteria used in the generation of the EVs and incubating using standard conditions.
  • select EVs are isolated and enriched by chromatography and binding surface moieties on EVs.
  • select EVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.
  • EVs are analyzed, for example, as described in Jeppesen, et al. Cell 177:428 (2019).
  • EVs are lyophilized.
  • EVs are gamma irradiated (for example, at 17.5 or 25 kGy).
  • EVs are UV irradiated.
  • EVs are heat inactivated (for example, at 50°C for two hours or at 90°C for two hours).
  • EVs are acid treated.
  • EVs are oxygen sparged (for example, at 0.1 vvm for two hours).
  • the phase of growth can affect the amount or properties of bacteria and/or EVs produced by bacteria.
  • EVs can be isolated, for example, from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • the growth environment can affect the amount of EVs produced by bacteria.
  • the yield of EVs can be increased by an EV inducer, as provided in Table 5.
  • Table 5 Culture Techniques to Increase EV Production
  • the method can optionally include exposing a culture of bacteria to an EV inducer prior to isolating EVs from the bacterial culture.
  • the culture of bacteria can be exposed to an EV inducer at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • engineered bacteria for the production of the EVs described herein.
  • the engineered bacteria are modified to enhance certain desirable properties.
  • the engineered bacteria are modified to enhance the immunomodulatory and/or therapeutic effect of the EVs (for example, either alone or in combination with another therapeutic agent), to reduce toxicity and/or to improve bacterial and/or EV manufacturing (for example, higher oxygen tolerance, improved freeze-thaw tolerance, shorter generation times).
  • the engineered bacteria may be produced using any technique known in the art, including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, CRISPR/Cas9, or any combination thereof.
  • the bacterium is modified by directed evolution.
  • the directed evolution comprises exposure of the bacterium to an environmental condition and selection of bacterium with improved survival and/or growth under the environmental condition.
  • the method comprises a screen of mutagenized bacteria using an assay that identifies enhanced bacterium.
  • the method further comprises mutagenizing the bacteria (for example, by exposure to chemical mutagens and/or UV radiation) or exposing them to a therapeutic agent (for example, antibiotic) followed by an assay to detect bacteria having the desired phenotype (for example, an in vivo assay, an ex vivo assay, or an in vitro assay).
  • a method of producing extracellular vesicles comprising growing EV- producing bacteria in a perfusion culture (e.g., wherein the perfusion culture comprises culture media that comprises EVs) (e.g., under conditions that support bacteria growth and EV production).
  • a filter system removes EVs, metabolites, and waste products of (e.g., from) the culture media.
  • the first filter is a product harvest filter (e.g., and passing the bacterial culture media that comprises EVs through the product harvest filter produces an output of the product harvest filter) and the second filter is a medium exchange filter.
  • the bacterial culture media is from a perfusion culture.
  • the perfusion culture is at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter scale or 20,000 liter scale or greater).
  • the medium exchange filter comprises PES (polyethersulfone).
  • volume of the output of the product harvest filter is about l/5x the volume than if a single-filter system was used (e.g., the volume of the output of the product harvest filter is about l/5x the volume as compared to the volume that would result from a single-filter system).
  • a method comprising:
  • a method comprising: (i) growing EV-producing bacteria in a perfusion culture (e.g., under conditions that support bacteria growth and EV production), wherein the perfusion culture comprises bacterial culture media that comprises EVs; and
  • a method comprising:
  • EVs are from bacteria of the Prevotellaceae; Veillonellaceae; Tannerellaceae; Rikenellaceae; Selenomonadaceae; Sporomusaceae; Synergistaceae; Phrislensenellaceae or Akkermaniaceae family.
  • EVs are from Gram positive bacteria.
  • EVs are from bacteria of the Oscillospiraceae ; Clostridiaceae; or Lachnospiraceae family.
  • Example 1 EV manufacturing platform
  • Figure l is a schematic showing a process/manufacturing platform for EVs to improve productivity.
  • a system provides culture intensification and clarification, using a high density perfusion process at 20,000L-scale process, and could increase manufacturing plant output >50-fold and includes using a two-filter system (such as hollow fiber product separation).
  • purification and concentration provide continuous capture to achieve pure and highly concentrated process intermediates that contain EVs, and can include continuous chromatography capture and tangential flow filtration.
  • the output can be further processed by drying (such as spray drying or lyophilization) of EVs, and can undergo post-processing, such as milling.
  • Example 2 Perfusion culture yields
  • Figures 2A and 2B are graphs showing comparisons of EV yields (EV product batches (-fold)) from batch culture versus perfusion culture yields over time (hours).
  • Figure 2A shows the batch yields (batch yield; dashed line) and perfusion EV yield (total product; upper solid line with circles) for a first strain of bacteria for which EV production is coupled with growth.
  • Figure 2B shows the batch yields (batch yield; dashed line) and perfusion EV yield (total product; upper solid line with circles) for a first strain of bacteria for which EV production is not coupled with growth.
  • the lower solid line with circles is the EV yield recovered from the perfusion culture (permeated product).
  • FIG. 3 is a schematic showing a set up for a two-filter system.
  • Harvest volume transfers from the fermenter to the two filters: product harvest filter and medium exchange filter.
  • Captured product from the product harvest filter transfers to a product reservoir and can be further processed, such as through a capture step(s). Metabolites, and waste products that passed through the medium exchange filter transfer to a waste reservoir.
  • This two-filter system can extend operating time. In addition to reducing sieving of the product harvest filter, this system allows the product harvest filter to collect higher concentration product (EVs) in a smaller volume than if a single filter system was used.
  • EVs concentration product
  • Figure 4 is a graph showing a theoretical result of using a two-filter (dual membrane perfusion) system. Overall perfusion rate remains constant at 6 volumes/day.
  • Shifting flux from the “Product Harvest” filter to the “Medium Exchange” filter increases the Flux Ratio, and results in product concentration (-fold; upward sloping line) increasing and permeate volume (downward sloping line; volume (vol)/day) decreasing with increasing ratio.
  • pH also affects and can improve capacity (column volume), enrichment, and yield. See Table 6 where the performance of two pH conditions (pH A and pH B) were evaluated.

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Abstract

L'invention concerne des procédés pour récolter des vésicules extracellulaires (tels que des rendements accrus de vésicules extracellulaires) à partir de cultures bactériennes.
PCT/US2023/034623 2022-10-07 2023-10-06 Traitement de vésicules extracellulaires WO2024076723A1 (fr)

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WO2018229216A1 (fr) 2017-06-14 2018-12-20 4D Pharma Research Limited Compositions comprenant une souche bactérienne du genre megasphera et leurs utilisations
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WO2020120714A1 (fr) 2018-12-12 2020-06-18 4D Pharma Research Limited Compositions comprenant des souches bactériennes
WO2020191369A1 (fr) * 2019-03-21 2020-09-24 Codiak Biosciences, Inc. Procédé de préparation de vésicules extracellulaires
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WO2018229216A1 (fr) 2017-06-14 2018-12-20 4D Pharma Research Limited Compositions comprenant une souche bactérienne du genre megasphera et leurs utilisations
WO2019060629A1 (fr) * 2017-09-21 2019-03-28 Codiak Biosciences, Inc. Production de vésicules extracellulaires dans une suspension de cellules isolées à l'aide de milieux de culture cellulaire chimiquement définis
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