PROCESSING EXTRACELLULAR VESICLES

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims the benefit of U.S. Provisional Application No. 63/414,284, filed on October 7, 2022, the content of which is hereby incorporated by reference in its entirety.

SUMMARY

[2] Extracellular vesicles (EVs) 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 (that is, the yields) 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.

[3] In some aspects, 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).

[4] In some embodiments, 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.

[5] In some embodiments, the perfusion culture increases EV yields after 24, 48, or 72 hours of culturing, as compared to a batch culture of the same bacteria.

[6] In some embodiments, EV production of the bacteria is coupled to growth in batch culture.

[7] In some embodiments, EV production of the bacteria is not coupled to growth in batch culture. [8] In some embodiments, 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).

[9] In some embodiments, a filter system (such as a one-filter system or a two-filter system) removes EVs, metabolites, and waste products of the culture media.

[10] In some embodiments, the filter system is a two-filter system.

[11] In some embodiments, the method further comprises filtering the culture media.

[12] In some embodiments, the method further comprises performing chromatography on the culture media.

[13] In some embodiments, the method further comprises performing tangential flow filtration on the culture media.

[14] In some embodiments, the method further comprises drying the culture media. In some embodiments, the culture media is dried after the growing step. In some embodiments, the culture media is dried after the filtering step. In some embodiments, the culture media is dried after the chromatography step. In some embodiments, the culture media is dried after the tangential flow filtration step.

[15] In some embodiments, the method further comprises milling the dried culture media.

[16] In some aspects, the disclosure provides a culture media produced by a perfusion culture method provided herein.

[17] In some aspects, the disclosure provides a culture media produced by a method of producing extracellular vesicles provided herein.

[18] In some aspects, 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.

[19] In some embodiments, the bacterial culture is a perfusion culture.

[20] In some embodiments, 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).

[21] In some embodiments, the product harvest filter and the medium exchange filter comprise the same material.

[22] In some embodiments, the product harvest filter comprises PES (polyethersulfone).

[23] In some embodiments, the medium exchange filter comprises PES (polyethersulfone). [24] In some embodiments, the product harvest filter and the medium exchange filter comprise PES (polyethersulfone).

[25] In some embodiments, EVs, media, waste and metabolites pass through the product harvest filter.

[26] In some embodiments, the pore size of the product harvest filter is about 0.5 micron.

[27] In some embodiments, media, waste and metabolites pass through the medium exchange filter.

[28] In some embodiments, the pore size of the medium exchange filter is less than about 0.5 micron.

[29] In some embodiments, the pore size of the medium exchange filter is about 0.05 micron.

[30] In some embodiments, the pore size of the medium exchange filter is about 0.02 micron.

[31] In some embodiments, the pore size of the medium exchange filter is about 0.01 micron.

[32] In some embodiments, the pore size of the medium exchange filter comprises a size cut off of 750kD (kilodalton).

[33] In some embodiments, the pore size of the medium exchange filter comprises a size cut off of 500kD.

[34] In some embodiments, the medium exchange filter runs at a higher flux than the product harvest filter.

[35] In some embodiments, the flux ratio (medium exchange filterproduct harvest filter) is about 5: 1.

[36] In some embodiments, the flux ratio (medium exchange filterproduct harvest filter) is about 9: 1.

[37] In some embodiments, the flux ratio (medium exchange filter product harvest filter) is about 10: 1.

[38] In some embodiments, the flux ratio (medium exchange filter product harvest filter) 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).

[39] In some embodiments, the volume of the output of the product harvest filter is about l/5x the volume than if a single-filter system was used.

[40] In some embodiments, 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.

[42] In some embodiments, the output of the product harvest filter comprises a higher concentration of EVs than if a single-filter system was used.

[43] In some embodiments, 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.

[44] In some embodiments, 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.

[45] In some embodiments, 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.

[46] In some embodiments, the method further comprises performing chromatography on the output of the product harvest filter.

[47] In some embodiments, the method further comprises performing tangential flow filtration on the output of the product harvest filter.

[48] In some embodiments, the method further comprises drying the output of the product harvest filter. In some embodiments, the output is dried after the filtering step. In some embodiments, the output is dried after the chromatography step. In some embodiments, the output is dried after the tangential flow filtration step.

[49] In some embodiments, the method further comprises milling the dried output of the product harvest filter.

[50] In some aspects, the disclosure provides an output of a product harvest filter produced by a method of processing bacterial culture media provided herein.

[51] In some aspects, 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.

[52] In some embodiments, the liquid comprises bacterial culture media.

[53] In some embodiments, the bacterial culture media is from a perfusion culture.

[54] In some embodiments, 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).

[55] In some embodiments, the liquid comprises a product-containing volume.

[56] In some embodiments, the product-containing volume is output from a single-filter system.

[57] In some embodiments, the product-containing volume is output from a two-filter system. [58] In some embodiments, the product-containing volume is output from a product harvest filter.

[59] In some embodiments, the product harvest filter comprises PES (polyethersulfone).

[60] In some embodiments, the pore size of the product harvest filter is about 0.5 micron.

[61] In some embodiments, the chromatography comprises a chromatography column.

[62] In some embodiments, the chromatography column comprises a monolith.

[63] In some embodiments, the chromatography column comprises a monolith ion exchange column.

[64] In some embodiments, the method comprises processing on one column.

[65] In some embodiments, the liquid is loaded onto the column and then EV-containing eluate is eluted.

[66] In some embodiments, the method comprises processing on two columns.

[67] In some embodiments, 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.

[68] In some embodiments, 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.

[69] In some embodiments, the loading and eluting steps on the first and second columns are alternated to process the liquid continuously.

[70] In some embodiments, bacterial culture media is filtered prior to performing the chromatography.

[71] In some embodiments, the chromatography enriches EV yield by greater than about 5-fold.

[72] In some embodiments, the chromatography enriches EV yield by about 6-fold.

[73] In some embodiments, the chromatography enriches EV yield by about 12-fold.

[74] In some embodiments, the yield of EVs from the chromatography is greater than about 50%.

[75] In some embodiments, the yield of EVs from the chromatography is greater than about 60%.

[76] In some embodiments, 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.

[78] In some embodiments, the method further comprises milling the dried EV eluate.

[79] In some aspects, the disclosure provides an EV eluate produced by a method of processing a liquid that comprises extracellular vesicles (EVs) provided herein.

[80] In some aspects, the disclosure provides 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 (e.g., as described herein); and

(ii) passing the bacterial culture media that comprises EVs through a two-filter system, wherein the first filter is a product harvest filter and the second filter is a medium exchange filter (e.g., as described herein), thereby preparing output of the product harvest filter, wherein the output comprises EVs.

[81] In some aspects, the disclosure provides an output of a product harvest filter produced by a method provided herein.

[82] In some aspects, the disclosure provides 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 (e.g., as described herein); and

(ii) performing chromatography on the bacterial culture media to prepare an eluate (e.g., as described herein), wherein the eluate comprises EVs.

[83] In some aspects, the disclosure provides an eluate produced by a method provided herein.

[84] In some aspects, the disclosure provides 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 (e.g., as described herein);

(ii) passing the bacterial culture media that comprises EVs through a two-filter system, wherein the first filter is a product harvest filter and the second filter is a medium exchange filter (e.g., as described herein), thereby preparing output of the product harvest filter, wherein the output comprises EVs; and

(iii) performing chromatography on the output of the product harvest filter to prepare an eluate (e.g., as described herein), wherein the eluate comprises EVs. [85] In some aspects, the disclosure provides an eluate produced by a method provided herein.

[86] In some embodiments of an aspect provided herein, the method comprises EVs from a bacterial strain that is associated with mucus.

[87] In some embodiments of an aspect provided herein, the method comprises EVs from anaerobic bacteria.

[88] In some embodiments of an aspect provided herein, the anaerobic bacteria are obligate (e.g., strict) anaerobes.

[89] In some embodiments of an aspect provided herein, the anaerobic bacteria are facultative anaerobes.

[90] In some embodiments of an aspect provided herein, the anaerobic bacteria are aerotolerant anaerobes.

[91] In some embodiments of an aspect provided herein, the EVs are from monoderm bacteria.

[92] In some embodiments of an aspect provided herein, the EVs are from diderm bacteria.

[93] In some embodiments of an aspect provided herein, the EVs are from Gram negative bacteria.

[94] In some embodiments of an aspect provided herein, the EVs are from bacteria of the Prevotellaceae; Veillonellaceae; Tannerellaceae; Rikenellaceae; Selenomonadaceae;

Sporomusaceae; Synergistaceae; Phrislensenellaceae or Akkermaniaceae family.

[95] In some embodiments of an aspect provided herein, the EVs are from Gram positive bacteria.

[96] In some embodiments of an aspect provided herein, the EVs are from bacteria of the Oscillospiraceae ; Clostridiaceae; or Lachnospiraceae family.

[97] In some embodiments of an aspect provided herein, the EVs are from bacteria of the genus Prevotella.

[98] In some embodiments of an aspect provided herein, the EVs are from bacteria of the genus Veillonella.

[99] In some embodiments of an aspect provided herein, the EVs are from bacteria of the genus Parabacteroides.

[100] In some embodiments of an aspect provided herein, 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.

[102] In some embodiments of an aspect provided herein, the EVs are from bacteria of the Prevotellaceae family.

[103] In some embodiments of an aspect provided herein, the EVs are from bacteria of the Veillonellaceae family.

[104] In some embodiments of an aspect provided herein, 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.

[105] In some aspects, the disclosure provides a product produced by a method provided herein.

BRIEF DESCRIPTION OF THE FIGURES

[106] Figure l is a schematic showing a process/manufacturing platform for EVs to improve productivity.

[107] 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. In both Figures 2A and 2B, the lower solid line with circles is the EV yield recovered from the perfusion culture (permeated product).

[108] Figure 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.

[109] 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

[HO] 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

[Hl] The use of perfusion culture systems offers advantages over batch process culture systems of bacteria for EV production. Batch anaerobic fermentation can result in poor substrate utilization and high inhibitor production. A perfusion process improves productivity through removal of inhibitory waste products and control of microbial metabolism. Perfusion provides a cell free product stream ready for downstream processing.

[112] For example, as compared to batch culture, 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.

[113] Such degrees of increased yields were unexpected. In perfusion, the growth rate of the bacterial cells is slower than the log phase growth rate of the cells in a batch culture. If a strain produces a large amount of EVs in log phase where growth rate is high (referred to as growth coupled), then less cell specific EV production would be expected in perfusion. If production of EVs is not tied to growth, then high or higher cell specific yields in perfusion may be produced where the growth rate is low. The high degree of increased yields using perfusion culture was not expected, and was seen for EV production by a strain in which EV production was coupled with growth, and also for a bacterial strain in which EV production was not coupled with growth.

[114] Considerations relating to perfusion culturing bacteria for EV production exist, 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). The considerations can include the following factors. For anaerobic bacteria (such as strict anaerobes), anaerobic conditions need to be maintained during the culture period. Larger amounts of raw materials and prepared media are needed for perfusion cultures than batch processes, for example, up to ten times more per day. [115] As media is flowed out of the perfusion culture (e.g., as the media is exchanged), a filter system (one-filter or two-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). Thus, in processing the media, 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. Also, flow rates and volumes need to be managed to minimize filter sieving. A two-filter system can address one or more of these considerations.

[116] Additional further processing of the output of the perfusion culture can be performed. Such 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.

Two-filter system

[117] In place of a single-filter system for processing 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))), 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. Media, metabolites, and waste products can also pass through the product harvest filter. 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. Rather than selecting by 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. Both the product harvest filter and the medium exchange filter can be made of the same material, such as PES (polyethersulfone).

[118] To reduce sieving of the product harvest filter, the medium exchange filter runs at a higher flux than the product harvest filter. For example, the flux ratio (medium exchange filterproduct harvest filter) can be 5: 1, or 9: 1, or 10: 1. In addition to reducing sieving of the product harvest filter, 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. For example, rather than further processing 120,000 liters, 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).

[119] As shown herein, a two-filter (dual-membrane) perfusion system in place of a single-filter perfusion system reduces downstream volume and increases product concentration.

[120] 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

[121] 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.

[122] 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. Rather than using a conventional resin, a monolith can be used (e.g., such as a monolith supplied by Sartorius). In contrast to a conventional resin, 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.

[123] Considerations relating to chromatography of culture media (e.g., from a perfusion culture) or a product-containing volume, such as after culture media containing product is filtered (such as through a single-filter system or a two-filter system) exist, particularly when 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). 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. [124] One or two columns (such as monoliths (such as monolith ion exchange 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. In a system in which one column is used, the culture media or product-containing volume is loaded onto the column and then the product is eluted. In a system in which two columns are used, 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. Once the second column has been loaded and product has been eluted from the first 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). As demonstrated herein for capture directly from perfusion culture media, 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%.

[125] 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.

Definitions

[126] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a," "an," and "the" are understood to be singular or plural.

[127] The term “about” when used before a numerical value indicates that the value may vary within a reasonable range, such as within ±10%, ±5% or ±1% of the stated value.

[128] “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). Further, 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). As used herein, the term “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.

[129] “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. Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al. (1988) SIAM J Applied Math 48: 1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar “MegAlign” program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison Wis.)).

[130] The term “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. In some embodiments, 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.

[131] “Metabolite” as used herein refers to any and all molecular compounds, compositions, molecules, ions, co-factors, catalysts or nutrients used as substrates in any cellular or bacterial metabolic reaction or resulting as product compounds, compositions, molecules, ions, co-factors, catalysts or nutrients from any cellular or bacterial metabolic reaction.

[132] As used herein, a substance is “pure” if it is substantially free of other components. The terms “purify,” “purifying,” and “purified” refer to an EV (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.” In some embodiments, purified EVs are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. EV compositions (or preparations) are, for example, purified from residual habitat products.

[133] As used herein, 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.

[134] “ 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. Genetic signatures between different strains may be identified by PCR amplification optionally followed by DNA sequencing of the genomic region(s) of interest or of the whole genome. In the case in which one strain (compared with another of the same species) has gained or lost antibiotic resistance or gained or lost a biosynthetic capability (such as an auxotrophic strain), strains may be differentiated by selection or counter-selection using an antibiotic or nutrient/metabolite, respectively.

Bacterial Extracellular Vesicles

[135] 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.

[136] In some embodiments, EVs are selected from a bacterial strain that is associated with mucus. In some embodiments, the mucus is associated with the gut lumen. In some embodiments, the mucus is associated with the small intestine. In some embodiments, the mucus is associated with the respiratory tract.

[137] In some embodiments, EVs are selected from a bacterial strain that is associated with an epithelial tissue, such as oral cavity, lung, nose, or vagina.

[138] In some embodiments, the EVs are from bacteria that are human commensals.

[139] In some embodiments, the EVs are from human commensal bacteria that originate from the human small intestine.

[140] In some embodiments, the EVs are from human commensal bacteria that originate from the human small intestine and are associated there with the outer mucus layer.

[141] 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. In some embodiments, 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). In some embodiments, the EVs are from an oncotrophic bacteria. In some embodiments, the EVs are from an immunostimulatory bacteria. In some embodiments, the EVs are from an immunosuppressive bacteria. In some embodiments, 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. In some embodiments, 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. In some embodiments, bacteria from a taxonomic group (for example, class, order, family, genus, species or strain)) listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification can be used as a source of EVs.

[142] In some embodiments, the EVs are obtained from Gram negative bacteria.

[143] In some embodiments, 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.

[144] In some embodiments, the EVs are obtained from Gram positive bacteria.

[145] In some embodiments, the EVs are from aerotol erant bacteria.

[146] In some embodiments, the EVs are from monoderm bacteria.

[147] In some embodiments, the EVs are from diderm bacteria.

[148] In some, the EVs are from bacteria of the family: Prevotellaceae; Veillonellaceae; Tannerellaceae; Rikenellaceae; Selenomonadaceae; Sporomusaceae; Synergistaceae; or Akkermaniaceae .

[149] In some embodiments, the EVs are from bacteria of the family Oscillospiraceae ; Clostridiaceae; Lachnospiraceae; or Christensenellaceae .

[150] In some embodiments, the EVs are from bacteria of the genus Prevotella.

[151] In some embodiments, the EVs are from bacteria of the genus Veillonella. [152] In some embodiments, the EVs are from bacteria of the mis Parabacteroides.

[153] In some embodiments, the EVs are from a bacterial strain of the Oscillospiraceae family.

[154] In some embodiments, the EVs are from a bacterial strain of the Tannerellaceae family.

[155] In some embodiments, the EVs are from a bacterial strain of the Prevotellaceae family.

[156] In some embodiments, the EVs are from a bacterial strain of the Veillonellaceae family.

[157] In some embodiments, the EVs are obtained from aerobic bacteria.

[158] In some embodiments, the EVs are obtained from anaerobic bacteria. In some embodiments, the anaerobic bacteria comprise obligate anaerobes. In some embodiments, the anaerobic bacteria comprise facultative anaerobes.

[159] In some embodiments, the EVs are obtained from acidophile bacteria.

[160] In some embodiments, the EVs are obtained from alkaliphile bacteria.

[161] In some embodiments, the EVs are obtained from neutral ophile bacteria.

[162] In some embodiments, the EVs are obtained from fastidious bacteria.

[163] In some embodiments, the EVs are obtained from nonfasti di ous bacteria.

[164] In some embodiments, bacteria from which EVs are obtained are lyophilized.

[165] In some embodiments, bacteria from which EVs are obtained are gamma irradiated (for example, at 17.5 or 25 kGy).

[166] In some embodiments, bacteria from which EVs are obtained are UV irradiated.

[167] In some embodiments, bacteria from which EVs are obtained are heat inactivated

(for example, at 50°C for two hours or at 90°C for two hours).

[168] In some embodiments, bacteria from which EVs are obtained are acid treated.

[169] In some embodiments, bacteria from which EVs are obtained are oxygen sparged

(for example, at 0.1 vvm for two hours).

[170] In some embodiments, the EVs are lyophilized.

[171] In some embodiments, the EVs are gamma irradiated (for example, at 17.5 or 25 kGy).

[172] In some embodiments, the EVs are UV irradiated.

[173] In some embodiments, the EVs are heat inactivated (for example, at 50°C for two hours or at 90°C for two hours).

[174] In some embodiments, the EVs are acid treated. [175] In some embodiments, the EVs are oxygen sparged (for example, at 0.1 vvm for two hours).

[176] The phase of growth can affect the amount or properties of bacteria and/or EVs produced by bacteria. For example, in the methods of EVs preparation provided herein, 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.

[177] EVs can be isolated from a batch culture of bacteria.

[178] EVs can be isolated from a perfusion culture of bacteria.

[179] In certain embodiments, the EVs described herein are obtained from obligate anaerobic bacteria. Examples of 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,

Propioni bacterium, Eubacterium, Lactobacillus and Bifidobacterium sppf, and gramnegative cocci (mainly Veillonella spp. ). In some embodiments, the obligate anaerobic bacteria are of a genus selected from the group consisting of Agathobaculum, Atopobium, Blautia, Burkholderia, Dielma, Longicatena, Paraclostridium, Turicibacter, and Tyzzerella.

[180] 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.

[181] In some embodiments, the EVs are from bacteria of the Negativicutes class.

[182] In some embodiments, the EVs are from bacteria of the Veillonellaceae family.

[183] In some embodiments, the EVs are from bacteria of the Selenomonadaceae family.

[184] In some embodiments, the EVs are from bacteria of the Acidaminococcaceae family.

[185] In some embodiments, the EVs are from bacteria of the Sporomusaceae family.

[186] In some embodiments, the EVs are from bacteria of the Megasphaera genus.

[187] In some embodiments, the EVs are from bacteria of the Selenomonas genus.

[188] In some embodiments, the EVs are from bacteria of the Propionospora genus.

[189] In some embodiments, the EVs are from bacteria of the Acidaminococcus genus.

[190] In some embodiments, the EVs are from Megasphaera sp. bacteria.

[191] In some embodiments, the EVs are from Selenomonas felix bacteria. [192] In some embodiments, the EVs are from Acidaminococcus intestini bacteria.

[193] In some embodiments, the EVs are from Propionospora sp. bacteria.

[194] The Oscillospriraceae family within the Clostridia class of microorganisms are common commensal organisms of vertebrates.

[195] In some embodiments, the EVs are from bacteria of the Clostridia class.

[196] In some embodiments, the EVs are from bacteria of the Oscillospriraceae family.

[197] In some embodiments, the EVs are from bacteria of the Faecalibacterium genus.

[198] In some embodiments, the EVs are from bacteria of the Fournierella genus.

[199] In some embodiments, the EVs are from bacteria of the Harryflintia genus.

[200] In some embodiments, the EVs are from bacteria of the Agathobaculum genus.

[201] In some embodiments, the EVs are from Faecalibacterium prausnitzii (for example, Faecalibacterium prausnitzii Strain A) bacteria.

[202] In some embodiments, the EVs are from Fournierella massiliensis (for example, Fournierella massiliensis Strain A) bacteria.

[203] In some embodiments, the EVs are from Harryflintia acetispora (for example, Harryflintia acetispora Strain A) bacteria.

[204] In some embodiments, the EVs are from Agathobaculum sp. (for example, Agathobaculum sp. Strain A) bacteria.

[205] In some embodiments, the EVs described herein are obtained from bacterium of a genus selected from the group consisting of Escherichia, Klebsiella, Lactobacillus, Shigella, and Staphylococcus.

[206] In some embodiments, 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.

[207] In some embodiments, the EVs described herein are obtained from a Prevotella bacteria. In some embodiments, 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 salivae, Prevotella stercorea, Prevotella tannerae, Prevotella timonensis, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis, Prevotella dentasini, Prevotella enoeca, Prevotella falsenii, Prevotella fusca, Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella saccharolytica, Prevotella scopos, Prevotella shahii, Prevotella zoogleoformans, and Prevotella veroralis.

[208] In some embodiments, 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. In some embodiments, 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.

[209] 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.

[210] 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.

[211] In some embodiments, the bacteria from which the EVs are obtained are of the Negativicutes class.

[212] In some embodiments, 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.

[214] In some embodiments, the bacteria from which the EVs are obtained are of the Acidaminococcaceae family.

[215] In some embodiments, the bacteria from which the EVs are obtained are of the Sporomusaceae family.

[216] In some embodiments, the bacteria from which the EVs are obtained are of the Megasphaera genus.

[217] In some embodiments, the bacteria from which the EVs are obtained are of the Selenomonas genus.

[218] In some embodiments, the bacteria from which the EVs are obtained are of the Propionospora genus.

[219] In some embodiments, the bacteria from which the EVs are obtained are of the Acidaminococcus genus.

[220] In some embodiments, the bacteria from which the EVs are obtained are Megasphaera sp. bacteria.

[221] In some embodiments, the bacteria from which the EVs are obtained are Selenomonas felix bacteria.

[222] In some embodiments, the bacteria from which the EVs are obtained are Acidaminococcus intestini bacteria.

[223] In some embodiments, the bacteria from which the EVs are obtained are Propionospora sp. bacteria.

[224] The Oscillospriraceae family within the Clostridia class of microorganisms are common commensal organisms of vertebrates.

[225] In some embodiments, the bacteria from which the EVs are obtained are of the Clostridia class.

[226] In some embodiments, the bacteria from which the EVs are obtained are of the Oscillospriraceae family.

[227] In some embodiments, the bacteria from which the EVs are obtained are of the Faecalibacterium genus.

[228] In some embodiments, the bacteria from which the EVs are obtained are of the Fournierella genus.

[229] In some embodiments, the bacteria from which the EVs are obtained are of the Harryflintia genus. [230] In some embodiments, the bacteria from which the EVs are obtained are of the Agathobaculum genus.

[231] In some embodiments, the bacteria from which the EVs are obtained are Faecalibacterium prausnitzii (for example, Faecalibacterium prausnitzii Strain A) bacteria.

[232] In some embodiments, the bacteria from which the EVs are obtained are Fournierella massiliensis (for example, Fournierella massiliensis Strain A) bacteria.

[233] In some embodiments, the bacteria from which the EVs are obtained are Harryflintia acetispora (for example, Harryflintia acetispora Strain A) bacteria.

[234] In some embodiments, the bacteria from which the EVs are obtained are Agathobaculum sp. (for example, Agathobaculum sp. Strain A) bacteria.

[235] In some embodiments, 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.

[236] In some embodiments, 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.

[237] In some embodiments, 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 tannerae, Prevotella timonensis, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis, Prevotella dentasini, Prevotella enoeca, Prevotella falsenii, Prevotella fusca, Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella saccharolytica, Prevotella scopos, Prevotella shahii, Prevotella zoogleoformans, and Prevotella veroralis.

[238] In some embodiments, 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. In some embodiments, 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.

[239] 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.

[240] In some embodiments, the bacteria from which the EVs are obtained are of the Negativicutes class.

[241] In some embodiments, the bacteria from which the EVs are obtained are of the Veillonellaceae family.

[242] In some embodiments, the bacteria from which the EVs are obtained are of the Selenomonadaceae family.

[243] In some embodiments, the bacteria from which the EVs are obtained are of the Acidaminococcaceae family.

[244] In some embodiments, the bacteria from which the EVs are obtained are of the Sporomusaceae family.

[245] In some embodiments, the bacteria from which the EVs are obtained are of the Prevotellaceae; Veillonellaceae; Tannerellaceae; Rikenellaceae; Selenomonadaceae; Sporomusaceae; Synergistaceae; Christensenellaceae; or Akkermaniaceae family.

[246] In some embodiments, the bacteria from which the EVs are obtained are of the Oscillospiraceae ; Clostridiaceae; or Lachnospiraceae family.

[247] In some embodiments, the bacteria from which the EVs are obtained are of the Megasphaera genus. [248] In some embodiments, the bacteria from which the EVs are obtained are of the Selenomonas genus.

[249] In some embodiments, the bacteria from which the EVs are obtained are of the Propionospora genus.

[250] In some embodiments, the bacteria from which the EVs are obtained are of the Acidaminococcus genus.

[251] In some embodiments, the bacteria from which the EVs are obtained are Megasphaera sp. bacteria.

[252] In some embodiments, the bacteria from which the EVs are obtained are Selenomonas felix bacteria.

[253] In some embodiments, the bacteria from which the EVs are obtained are Acidaminococcus intestini bacteria.

[254] In some embodiments, the bacteria from which the EVs are obtained are Propionospora sp. bacteria.

[255] The Oscillospriraceae family within the Clostridia class of microorganisms are common commensal organisms of vertebrates.

[256] In some embodiments, the bacteria from which the EVs are obtained are of the Clostridia class.

[257] In some embodiments, the bacteria from which the EVs are obtained are of the Oscillospriraceae family.

[258] In some embodiments, the bacteria from which the EVs are obtained are of the Faecalibacterium genus.

[259] In some embodiments, the bacteria from which the EVs are obtained are of the Fournierella genus.

[260] In some embodiments, the bacteria from which the EVs are obtained are of the Harryflintia genus.

[261] In some embodiments, the bacteria from which the EVs are obtained are of the Agathobaculum genus.

[262] In some embodiments, the bacteria from which the EVs are obtained are Faecalibacterium prausnitzii (for example, Faecalibacterium prausnitzii Strain A) bacteria.

[263] In some embodiments, the bacteria from which the EVs are obtained are Fournierella massiliensis (for example, Fournierella massiliensis Strain A) bacteria.

[264] In some embodiments, the bacteria from which the EVs are obtained are Harryflintia acetispora (for example, Harryflintia acetispora Strain A) bacteria. [265] In some embodiments, the bacteria from which the EVs are obtained are Agathobaculum sp. (for example, Agathobaculum sp. Strain A) bacteria.

[266] In some embodiments, the bacteria from which the EVs are obtained are a strain of Agathobaculum sp. In some embodiments, 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). In some embodiments, the Agathobaculum sp. strain is the Agathobaculum sp. Strain A (ATCC Deposit Number PTA- 125892).

[267] In some embodiments, 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.

[268] In some embodiments, 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. 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. 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.

[269] In some embodiments, 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 . In some embodiments, 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.

[270] In some embodiments, 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.

[271] In some embodiments, the bacteria from which the EVs are obtained are from one strain of bacteria, for example, a strain provided herein.

[272] In some embodiments, 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.

[273] In some embodiments, 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). In some embodiments, the bacteria from which the EVs are obtained are Lactococcus bacteria, for example, Lactococcus lactis cremoris Strain A (ATCC designation number PTA-125368).

[274] In some embodiments, 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.

[275] In some embodiments, 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). In some embodiments, the bacteria from which the EVs are obtained are Prevotella bacteria, for example, Prevotella Strain B 50329 (NRRL accession number B 50329).

[276] In some embodiments, 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. In some embodiments, 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).

[277] In some embodiments, 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. In some embodiments, the bacteria from which the EVs are obtained are Bifidobacterium bacteria, for example, Bifidobacterium bacteria deposited as ATCC designation number PTA-125097.

[278] In some embodiments, 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. In some embodiments, the bacteria from which the EVs are obtained are Veillonella bacteria, for example, Veillonella bacteria deposited as ATCC designation number PTA-125691.

[279] In some embodiments, the bacteria from which the EVs are obtained are Ruminococcus gnavus bacteria. In some embodiments, 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. In some embodiments, 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. In some embodiments, the Ruminococcus gnavus bacteria are Ruminococcus gnavus bacteria deposited as ATCC designation number PTA-126695.

[280] In some embodiments, the bacteria from which the EVs are obtained are Megasphaera sp. bacteria. In some embodiments, 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. In some embodiments, 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. In some embodiments, the Megasphaera sp. bacteria are Megasphaera sp. bacteria deposited as ATCC designation number PTA-126770.

[281] In some embodiments, the bacteria from which the EVs are obtained are Fournierella massiliensis bacteria. In some embodiments, 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. In some embodiments, 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. In some embodiments, the Fournierella massiliensis bacteria are Fournierella massiliensis bacteria deposited as ATCC designation number PTA-126696.

[282] In some embodiments, the bacteria from which the EVs are obtained are Harryflintia acetispora bacteria. In some embodiments, 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. In some embodiments, 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. In some embodiments, the Harryflintia acetispora bacteria are Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694. [283] In some embodiments, the bacteria from which the EVs are obtained are bacteria that produce metabolites, for example, the bacteria produce butyrate, iosine, proprionate, or tryptophan metabolites.

[284] In some embodiments, the bacteria from which the EVs are obtained are bacteria that produce butyrate. In some embodiments, the bacteria are from the genus Blautia;

Christensella; Copracoccus; Eubacterium; Lachnosperacea; Megasphaera; or Roseburia.

[285] In some embodiments, the bacteria from which the EVs are obtained are bacteria that produce iosine. In some embodiments, the bacteria are from the genus Bifidobacterium; Lactobacillus; or Olsenella.

[286] In some embodiments, the bacteria from which the EVs are obtained are bacteria that produce proprionate. In some embodiments, the bacteria are from the genus Akkermansia; Bacteriodes; Dialister; Eubacterium; Megasphaera; Parabacteriodes;

Prevotella; Ruminococcus; or Veillonella.

[287] In some embodiments, the bacteria from which the EVs are obtained are bacteria that produce tryptophan metabolites. In some embodiments, the bacteria are from the genus Lactobacillus or Peptostreptococcus.

[288] In some embodiments, the bacteria from which the EVs are obtained are bacteria that produce inhibitors of histone deacetylase 3 (HDAC3). In some embodiments, the bacteria are from the species Bariatricus massiliensis, Faecalibacterium prausnitzii, Megasphaera massiliensis or Roseburia intestinalis.

[289] In some embodiments, the bacteria are from the genus Alloiococcus; Bacillus; Catenibacterium; Corynebacterium; Cupriavidus; Enhydrobacter; Exiguobacterium; Faecalibacterium; Geobacillus; Methylobacterium; Micrococcus; Morganella; Proteus;

Pseudomonas; Rhizobium; or Sphingomonas. In some embodiments, 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.

[290] In some embodiments, the bacteria from which the EVs are obtained are of the genus Alloiococcus; Bacillus; Catenibacterium; Corynebacterium; Cupriavidus;

Enhydrobacter; Exiguobacterium; Faecalibacterium; Geobacillus; Methylobacterium; Micrococcus; Morganella; Proteus; Pseudomonas; Rhizobium; or Sphingomonas. [291] In some embodiments, 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.

[292] In some embodiments, the bacteria from which the EVs are obtained are of the genus Leuconostoc.

[293] In some embodiments, the bacteria from which the EVs are obtained are of the genus Lactobacillus.

[294] In some embodiments, 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.

[295] In some embodiments, the bacteria from which the EVs are obtained are Leuconostoc holzapfelii bacteria.

[296] In some embodiments, 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.

[297] In some embodiments, the bacteria from which the EVs are obtained are Lactobacillus casei; Lactobacillus plantarum; Lactobacillus paracasei; Lactobacillus plantarum; Lactobacillus rhamnosus; or Lactobacillus sakei bacteria.

[298] In some embodiments, the EVs described herein are obtained from a genus selected from the group consisting of Acinetobacter; Deinococcus; Helicobacter; Rhodococcus;

Weissella cibaria; Alloiococcus; Atopobium; Catenibacterium; Corynebacterium; Exiguobacterium; Geobacillus; Methylobacterium; Micrococcus; Morganella; Proteus; Rhizobium; Rothia; Sphingomonas; Sphingomonas; and Leuconostoc.

[299] In some embodiments, 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. [300] In some embodiments, the EVs are from Leuconostoc holzapfelii bacteria. In some embodiments, the EVs are from Leuconostoc holzapfelii Ceb-kc-003 (KCCM11830P) bacteria.

[301] In some embodiments, 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).

[302] In some embodiments, 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).

[303] In some embodiments, the bacteria from which the EVs are obtained are Megasphaera massiliensis bacteria (for example, from the strain with accession number DSM 26228).

[304] In some embodiments, the bacteria from which the EVs are obtained are Parabacteroides distasonis bacteria (for example, from the strain with accession number NCIMB 42382).

[305] In some embodiments, 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. In some embodiments, 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. In some embodiments, the Megasphaera massiliensis bacteria is the strain with accession number NCIMB 43388 or NCIMB 43389.

[306] In some embodiments, 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. In some embodiments, 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. In some embodiments, the Megasphaera massiliensis bacteria is the strain deposited under accession number NCIMB 42787.

[307] In some embodiments, 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. In some embodiments, the Megasphaera sp. bacteria is the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387.

[308] In some embodiments, 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. In some embodiments, 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. In some embodiments, the Parabacteroides distasonis bacteria is the strain deposited under accession number NCIMB 42382.

[309] In some embodiments, 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. In some embodiments, 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. In some embodiments, the Megasphaera massiliensis bacteria is the strain deposited under accession number DSM 26228.

[310] In some embodiments, 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). In some embodiments, the engineered bacteria described herein are modified to improve EV manufacturing (for example, higher oxygen tolerance, stability, improved freeze-thaw tolerance, shorter generation times). For example, in some embodiments, 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.

[311] Table 1. Bacteria by Class

* The abbreviation given in the parenthetical is for the species in the row in which it is listed.

[312] Table 2. Exemplary Bacterial Strains

[313] Table 3. Exemplary Bacterial Strains

[314] Table 4. Exemplary Bacterial Strains

Modified EVs

[315] In some aspects, the EVs described herein are modified such that they comprise, are linked to, and/or are bound by a therapeutic moiety.

[316] In some embodiments, the therapeutic moiety is a cancer-specific moiety. In some embodiments, the cancer-specific moiety has binding specificity for a cancer cell (for example, has binding specificity for a cancer-specific antigen). In some embodiments, the cancer-specific moiety comprises an antibody or antigen binding fragment thereof. In some embodiments, the cancer-specific moiety comprises a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the cancer-specific moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof. In some embodiments, the cancer-specific 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). In some embodiments, the first part is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP. In some embodiments the first part has binding specificity for the EV (for example, by having binding specificity for a bacterial antigen). In some embodiments, the first and/or second part comprises an antibody or antigen binding fragment thereof. In some embodiments, 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. [317] In some embodiments, 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). In some embodiments, 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. In some embodiments, 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.

Production of Bacterial Extracellular Vesicles (EVs)

[318] In certain aspects, the EVs from bacteria described herein are prepared using any method known in the art.

[319] In some embodiments, the EVs are prepared without an EV purification step. For example, in some embodiments, 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. In some embodiments, the bacteria are killed using an antibiotic (for example, using an antibiotic described herein). In some embodiments, the bacteria are killed using UV irradiation. In some embodiments, the bacteria are heat- killed.

[320] In some embodiments, the EVs described herein are purified from one or more other bacterial components. Methods for purifying EVs from bacteria are known in the art. In some embodiments, 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. In some embodiments, 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). In some embodiments, the culture supernatants are then passed through filters to exclude intact bacterial cells (for example, a 0.22 pm filter). In some embodiments, 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. In some embodiments, 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). In some embodiments, 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. In some embodiments, the EVs are further purified by treatment with DNase and/or proteinase K.

[321] For example, in some embodiments, 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. For example, for 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. Using ultracentrifugation, filtered supernatants can be centrifuged at 100,000-200,000 x g for 1-16 hours at 4°C. The pellet of this centrifugation contains bacterial EVs and other debris such as large protein complexes. In some embodiments, using a filtration technique, such as through the use of an Amicon Ultra spin filter or by tangential flow filtration, supernatants can be filtered so as to retain species of molecular weight > 50 or 100 kDa.

[322] Alternatively, 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). 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. For example, 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. Alternatively, 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.

[323] 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.

[324] In some embodiments, to confirm sterility and isolation of the EV preparations, 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.

[325] In some embodiments, for preparation of 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). In some embodiments, for preparation of EVs used for in vivo injections, EVs in PBS are sterile- filtered to < 0.22 pm.

[326] In certain embodiments, to make samples compatible with further testing (for example, to remove sucrose prior to TEM imaging or in vitro assays), 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.

[327] In some embodiments, 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.

[328] In some embodiments, select EVs are isolated and enriched by chromatography and binding surface moieties on EVs. In some embodiments, 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.

[329] In some embodiments, EVs are analyzed, for example, as described in Jeppesen, et al. Cell 177:428 (2019).

[330] In some embodiments, EVs are lyophilized.

[331] In some embodiments, EVs are gamma irradiated (for example, at 17.5 or 25 kGy).

[332] In some embodiments, EVs are UV irradiated.

[333] In some embodiments, EVs are heat inactivated (for example, at 50°C for two hours or at 90°C for two hours).

[334] In some embodiments, EVs are acid treated.

[335] In some embodiments, EVs are oxygen sparged (for example, at 0.1 vvm for two hours).

[336] The phase of growth can affect the amount or properties of bacteria and/or EVs produced by bacteria. For example, in the methods of EV preparation provided herein, 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.

[337] The growth environment (for example, culture conditions) can affect the amount of EVs produced by bacteria. For example, the yield of EVs can be increased by an EV inducer, as provided in Table 5. Table 5: Culture Techniques to Increase EV Production [338] In the methods of EVs preparation provided herein, 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.

Methods of Making Enhanced Bacteria

[339] In certain aspects, provided herein are methods of making engineered bacteria for the production of the EVs described herein. In some embodiments, the engineered bacteria are modified to enhance certain desirable properties. For example, in some embodiments, 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.

[340] In some embodiments of the methods provided herein, the bacterium is modified by directed evolution. In some embodiments, 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. In some embodiments, the method comprises a screen of mutagenized bacteria using an assay that identifies enhanced bacterium. In some embodiments, 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).

Exemplary Embodiments

1. 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).

2. The method of embodiment 1, wherein 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.

3. The method of embodiment 1 or 2, wherein the perfusion culture increases EV yields after 24, 48, or 72 hours of culturing, as compared to a batch culture of the same bacteria.

4. The method of any one of embodiments 1 to 3, wherein EV production of the bacteria is coupled to growth in batch culture.

5. The method of any one of embodiments 1 to 4, wherein EV production of the bacteria is not coupled to growth in batch culture.

6. The method of any one of embodiments 1 to 5, wherein 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).

7. The method of any one of embodiments 1 to 6, wherein a filter system (one-filter or two- filter system) removes EVs, metabolites, and waste products of (e.g., from) the culture media.

8. The method of embodiment 7, wherein the filter system is a two-filter system.

9. The method of any one of embodiments 1 to 6, wherein the method further comprises filtering the culture media.

10. The method of any one of embodiments 1 to 9, wherein the method further comprises performing chromatography on the culture media.

11. The method of any one of embodiments 1 to 10, wherein the method further comprises performing tangential flow filtration on the culture media.

12. The method of any one of embodiments 1 to 11, wherein the method further comprises drying the culture media.

13. The method of embodiment 12, wherein the method further comprises milling the dried culture media.

14. 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., 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.

15. The method of embodiment 14, wherein the bacterial culture media is from a perfusion culture. 16. The method of embodiment 15, wherein 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).

17. The method of any one of embodiments 14 to 16, wherein the product harvest filter and the medium exchange filter comprise the same material.

18. The method of any one of embodiments 14 to 17, wherein the product harvest filter comprises PES (polyethersulfone).

19. The method of any one of embodiments 14 to 18, wherein the medium exchange filter comprises PES (polyethersulfone).

20. The method of any one of embodiments 14 to 19, wherein the product harvest filter and the medium exchange filter comprise PES (polyethersulfone).

21. The method of any one of embodiments 14 to 20, wherein EVs, media, waste and metabolites pass through the product harvest filter.

22. The method of any one of embodiments 14 to 21, wherein the pore size of the product harvest filter is about 0.5 micron.

23. The method of any one of embodiments 14 to 22, wherein media, waste and metabolites pass through the medium exchange filter.

24. The method of any one of embodiments 14 to 23, wherein the pore size of the medium exchange filter is less than about 0.5 micron.

25. The method of any one of embodiments 14 to 24, wherein the pore size of the medium exchange filter is about 0.05 micron.

26. The method of any one of embodiments 14 to 24, wherein the pore size of the medium exchange filter is about 0.02 micron.

27. The method of any one of embodiments 14 to 24, wherein the pore size of the medium exchange filter is about 0.01 micron.

28. The method of any one of embodiments 14 to 24, wherein the pore size of the medium exchange filter comprises a size cut off of 750kD (kilodalton).

29. The method of any one of embodiments 14 to 24, wherein the pore size of the medium exchange filter comprises a size cut off of 500kD.

30. The method of any one of embodiments 14 to 29, wherein the medium exchange filter runs at a higher flux than the product harvest filter.

31. The method of any one of embodiments 14 to 30, wherein the flux ratio (medium exchange filterproduct harvest filter) is about 5: 1.

32. The method of any one of embodiments 14 to 30, wherein the flux ratio (medium exchange filterproduct harvest filter) is about 9: 1. 33. The method of any one of embodiments 14 to 30, wherein the flux ratio (medium exchange filterproduct harvest filter) is about 10: 1.

34. The method of any one of embodiments 14 to 33, wherein the flux ratio (medium exchange filterproduct harvest filter) 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).

35. The method of any one of embodiments 14 to 34, wherein the 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).

36. The method of any one of embodiments 14 to 34, wherein the volume of the output of the product harvest filter is about l/9x the volume than if a single-filter system was used.

37. The method any one of embodiments 14 to 34, wherein the volume of the output of the product harvest filter is about l/10x the volume than if a single-filter system was used.

38. The method of any one of embodiments 14 to 37, wherein the output of the product harvest filter comprises a higher concentration of EVs than if a single-filter system was used.

39. The method of any one of embodiments 14 to 38, wherein the output of the product harvest filter comprises a concentration of EVs that is at least about 5x higher than if a singlefilter system was used.

40. The method of any one of embodiments 14 to 38, wherein the output of the product harvest filter comprises a concentration of EVs that is at least about 9x higher than if a singlefilter system was used.

41. The method of any one of embodiments 14 to 38, wherein 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.

42. The method of any one of embodiments 14 to 41, wherein the method further comprises performing chromatography on the output of the product harvest filter.

43. The method of any one of embodiments 14 to 42, wherein the method further comprises performing tangential flow filtration on the output of the product harvest filter.

44. The method of any one of embodiments 14 to 43, wherein the method further comprises drying the output of the product harvest filter.

45. The method of embodiment 44, wherein the method further comprises milling the dried output of the product harvest filter. 46. A method of processing a liquid that comprises extracellular vesicles (EVs) to prepare an EV eluate, the method comprising performing chromatography on the liquid.

47. The method of embodiment 46, wherein the liquid comprises bacterial culture media.

48. The method of embodiment 47, wherein the bacterial culture media is from a perfusion culture.

49. The method of embodiment 48, wherein 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).

50. The method of embodiment 46, wherein the liquid comprises a product-containing volume.

51. The method of embodiment 50, wherein the product-containing volume is output from a single-filter system.

52. The method of embodiment 50, wherein the product-containing volume is output from a two-filter system.

53. The method of embodiment 50, wherein the product-containing volume is output from a product harvest filter.

54. The method of embodiment 53, wherein the product harvest filter comprises PES (polyethersulfone).

55. The method of embodiment 53 or 54, wherein the pore size of the product harvest filter is about 0.5 micron.

56. The method of any one of embodiments 46 to 55, wherein the chromatography comprises a chromatography column.

57. The method of embodiment 57, wherein the chromatography column comprises a monolith.

58. The method of embodiment 56 or 57, wherein the chromatography column comprises a monolith ion exchange column.

59. The method of any one of embodiments 56 to 58, wherein the method comprises processing on one column.

60. The method of any one of embodiments 56 to 59, wherein the liquid is loaded onto the column and an EV-containing eluate is eluted.

61. The method any one of embodiments 56 to 58, wherein the method comprises processing on two columns.

62. The method of any one of embodiments 56 to 58 or 61, wherein the liquid is loaded onto a first column, the EV-containing eluate is eluted from the first column, wherein while EV-containing eluate is eluted from the first column, liquid is loaded onto the second column. 63. The method of any one of embodiments 56 to 58, 61, or 62, wherein EV-containing eluate is eluted from the second column, wherein while EV-containing eluate is eluted from the second column, liquid is loaded onto the first column.

64. The method of any one of embodiments 56 to 58 or 61 to 63, wherein the loading and eluting steps on the first and second columns are alternated to process the liquid continuously.

65. The method of any one of embodiments 46 to 64, wherein bacterial culture media is filtered prior to performing the chromatography.

66. The method of any one of embodiments 46 to 65, wherein the chromatography enriches EV yield by greater than about 5-fold (e.g., as compared to EV yield from the liquid in the absence of chromatography).

67. The method of any one of embodiments 46 to 66, wherein the chromatography enriches EV yield by about 6-fold.

68. The method of any one of embodiments 46 to 66, wherein the chromatography enriches EV yield by about 12-fold.

69. The method of any one of embodiments 46 to 68, wherein the yield of EVs from the chromatography is greater than about 50%.

70. The method of any one of embodiments 46 to 69, wherein the yield of EVs from the chromatography is greater than about 60%.

72. The method of any one of embodiments 46 to 70, wherein the method further comprises performing tangential flow filtration on the EV eluate.

73. The method of any one of embodiments 46 to 72, wherein the method further comprises drying the EV eluate.

74. The method of embodiment 73, wherein the method further comprises milling the dried EV eluate.

75. 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

(ii) passing the bacterial culture media that comprises EVs through a two-filter system, wherein the first filter is a product harvest filter and the second filter is a medium exchange filter, thereby preparing output of the product harvest filter, wherein the output comprises EVs.

76. 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

(ii) performing chromatography on the bacterial culture media to prepare an eluate, wherein the eluate comprises EVs.

77. 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;

(ii) passing the bacterial culture media that comprises EVs through a two-filter system, wherein the first filter is a product harvest filter and the second filter is a medium exchange filter, thereby preparing output of the product harvest filter, wherein the output comprises EVs; and

(iii) performing chromatography on the output of the product harvest filter to prepare an eluate, wherein the eluate comprises EVs.

78. The method of any one of embodiments 1 to 77, wherein the method comprises EVs from a bacterial strain that is associated with mucus.

79. The method of any one of embodiments 1 to 77, wherein the method comprises EVs from anaerobic bacteria.

80. The method of embodiment 79, wherein the anaerobic bacteria are obligate (e.g., strict) anaerobes.

81. The method of embodiment 79, wherein the anaerobic bacteria are facultative anaerobes.

82. The method of embodiment 79, wherein the anaerobic bacteria are aerotolerant anaerobes.

83. The method of any one of embodiments 1 to 77, wherein the EVs are from monoderm bacteria.

84. The method of any one of embodiments 1 to 77, wherein the EVs are from diderm bacteria.

85. The method of any one of embodiments 1 to 77, wherein the EVs are from Gram negative bacteria.

86. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of the Prevotellaceae; Veillonellaceae; Tannerellaceae; Rikenellaceae; Selenomonadaceae; Sporomusaceae; Synergistaceae; Phrislensenellaceae or Akkermaniaceae family. 87. The method of any one of embodiments 1 to 77, wherein the EVs are from Gram positive bacteria.

88. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of the Oscillospiraceae ; Clostridiaceae; or Lachnospiraceae family.

89. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of the genus Prevotella.

90. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of the genus Veillonella.

91. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of the genus Parabacteroides.

92. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of the Oscillospiraceae family.

93. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of the Tannerellaceae family.

94. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of the Prevotellaceae family.

95. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of the Veillonellaceae family.

96. The method of any one of embodiments 1 to 77, wherein 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.

EXAMPLES

Example 1: EV manufacturing platform

[341] Figure l is a schematic showing a process/manufacturing platform for EVs to improve productivity. Such 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). Next, 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

[342] 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. In both Figures 2A and 2B, the lower solid line with circles is the EV yield recovered from the perfusion culture (permeated product).

[343] Total EV productivity was significantly improved (total product; upper solid line with circles in both graphs).

[344] Media exchange rate was high.

[345] Current filter sieving limited EV recovered in the filtrate, this provides high potential for improvement. See lower solid line with circles in both graphs.

Example 3: Two-filter system

[346] Figure 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.

[347] As shown in the figure, a 9: 1 flux ratio (Medium Exchange: Product Harvest) yields lOx the product in 1/1 Oth the volume.

[348] “Medium Exchange” filter handles the bulk of perfusion and is impermeable to product.

[349] “Product Harvest” filter operates at low flux to reduce sieving.

[350] This two-filter system (dual-membrane perfusion) 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.

Example 4: Dual membrane perfusion results

[351] 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. [352] 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.

[353] Dual-membrane perfusion reduces downstream volume and increases product concentration.

Example 5: Monolith ion exchange chromatography

[354] Harvest volume directly from a perfusion culture showed that a multi-column capture directly from perfusion had acceptable capacity.

[355] 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.

[356] Table 6. Effects of pH on capacity, enrichment, and yield.

Incorporation by Reference

[357] All publications patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Equivalents

[358] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.