CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims the benefit of U.S. Provisional Application No. 63/416,277, filed on October 14, 2022, the content of which is hereby incorporated by reference in its entirety.

SUMMARY

[2] Provided herein are methods and compositions for assaying drug product and/or drug substance that comprise bacteria and/or microbial extracellular vesicles (mEVs). For example, in some embodiments, methods and compositions provided herein can be used as a quality control process to confirm the potency of such a drug product and/or drug substance, such as prior to final formulation as a solid dose form. In some embodiments, methods and compositions provided herein can be used to test whether drug product and/or drug substance maintained its potency after being stored for a period of time. In some embodiments, methods and compositions provided herein can be used to determine the potency of a drug product and/or drug substance, such as to ensure it possesses adequate potency prior to administration to a subject (e.g., a subject in need thereof).

[3] Provided herein are methods and compositions for assaying bacteria and/or microbial extracellular vesicles (mEVs) prior to preparation of a drug substance or drug product. For example, in some embodiments, methods and compositions provided herein can be used as a quality control process to assess the impact of changes in bioprocess (e.g., production parameter) development or downstream processing development. In some embodiments, methods and compositions provided herein can be used to test whether bacteria and/or microbial extracellular vesicles (mEVs) maintained their potency after a process parameter is altered. For example, the methods and compositions can be used to assay bacteria and/or microbial extracellular vesicles (mEVs) after one or more of the following process changes: change in a production parameter such as change in amount or presence of a growth media component, change in a growth condition (such as time, temperature, or optical density); or change in a downstream processing step, such as change in excipient, and change in formulating parameter (such as compression strength). [4] In certain aspects, provided herein are methods for assaying a drug product or drug substance that comprises bacteria and/or microbial extracellular vesicles (mEVs). In some embodiments, the method comprises contacting a cell of a mammalian immune cell line (e.g., a monocyte cell line, such as THP-1 cells, (e.g., PMA-differentiated THP-1 cells)) with the drug product or drug substance. The cell can comprise a nucleic acid sequence operatively linked to a NF-κB regulatory sequence (i.e., a sequence that is responsive to NF-κB, such as an NF-κB response element, or a sequence that is an NF-κB promoter). The nucleic acid sequence may encode a transcription product. The method may further comprise detecting the level of expression of a transcription product encoded by the nucleic acid sequence by the cell. In some embodiments, the drug product or drug substance comprises a strain of bacteria (e.g., Prevotella histicola, Veillonella parvula, Harryflintia acetispora, Foumierella massiliensis, Megasphaera Sp., Selenomonas felix, Acidaminococcus intestine, and/or Propionospora sp. bacteria) and/or microbial extracellular vesicles (mEVs) from a strain of bacteria (e.g, Prevotella histicola, Veillonella parvula, Harryflintia acetispora, Foumierella massiliensis, Megasphaera Sp., Selenomonas felix, Acidaminococcus intestine, and/ or Propionospora sp. microbial extracellular vesicles (mEVs)).

[5] In certain aspects, provided herein are methods for assaying bacteria and/or microbial extracellular vesicles (mEVs) prior to preparation of a drug substance or drug product. In some embodiments, the method comprises contacting a cell of a mammalian immune cell line (e.g., a monocyte cell line, such as THP-1 cells, (e.g., PMA-differentiated THP-1 cells)) with the bacteria and/or microbial extracellular vesicles (mEVs). The cell can comprise a nucleic acid sequence operatively linked to a NF-κB regulatory sequence (i.e., a sequence that is responsive to NF-κB, such as an NF-κB response element, or a sequence that is an NF-κB promoter). The nucleic acid sequence may encode a transcription product. The method may further comprise detecting the level of expression of a transcription product encoded by the nucleic acid sequence by the cell. In some embodiments, the bacteria are a strain of bacteria such as Prevotella histicola, Veillonella parvula, Harryflintia acetispora, Foumierella massiliensis, Megasphaera Sp., Selenomonas felix, Acidaminococcus intestine, and/ or Propionospora sp. bacteria; and/or the microbial extracellular vesicles (mEVs) are from a strain of bacteria such as Prevotella histicola, Veillonella parvula, Harryflintia acetispora, Foumierella massiliensis, Megasphaera Sp., Selenomonas felix, Acidaminococcus intestine, and/or Propionospora sp. microbial extracellular vesicles (mEVs).

[6] In some embodiments, the drug product or drug substance comprises bacteria of a strain of Prevotella histicola. In some embodiments, the bacteria (e.g, Prevotella histicola strain bacteria) comprise at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, 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 (e.g, Prevotella histicola strain bacteria) comprise 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 (e.g., Prevotella histicola strain bacteria) is the Prevotella Strain B (NRRL accession number B 50329).

[7] In some embodiments, a drug product or drug substance comprises mEVs of bacteria of a strain of Prevotella histicola. In some embodiments, mEVs can be mEVs of bacteria (e.g, Prevotella histicola strain bacteria) that comprise at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, 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, mEVs can be mEVs of bacteria (e.g., Prevotella histicola strain bacteria) that comprise 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 mEVs are of bacteria (e.g, Prevotella histicola strain bacteria) of the Prevotella Strain B (NRRL accession number B 50329).

[8] In some embodiments, a transcription product is a protein. In some embodiments, the expression of the protein is determined by an enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay, or immunocytostaining. In some embodiments, the protein is a luciferase protein, such as a firefly luciferase protein. In some embodiments, expression of the luciferase is determined by a luciferase assay. In some embodiments, the protein is a fluorescent protein, such as a green fluorescent protein, mCherry protein, a yellow fluorescent protein, a blue fluorescent protein, a cyan fluorescent protein, or a red fluorescent protein. In some embodiments, expression of the fluorescent protein is determined by detecting fluorescence. [9] In some embodiments, a transcription product is an RNA. In some embodiments, the expression of the RNA is determined by a northern hybridization method or using reverse transcription-polymerase chain reaction (RT-PCR).

[10] In some embodiments, a NF-κB regulatory sequence is responsive to NF-κB. In some embodiments, a nucleic acid sequence is operably linked to one or more NF-κB response elements (e.g., one or more copies of an NF-κB response element, e.g., one, two three, four, five, six or more copies). In various embodiments, the one or more NF-κB response elements have different sequences. In various embodiments, the one or more NF-κB response elements have the same sequence (and in various such embodiments can be referred to as “copies” of an NF-κB response element). In various embodiments, at least two of the one or more NF-κB response elements have the same sequence. In various embodiments, at least two of the one or more NF- κB response elements have different sequences. In some embodiments, the nucleic acid sequence is operably linked to four NF-κB response elements (e.g., four copies of an NF-κB response element), e.g., positioned upstream of a promoter such as a minimal TATA promoter. In some embodiments, the NF-κB regulatory sequence is an NF-κB promoter. In some embodiments, the mammalian immune cell line is a monocyte cell line, such as THP-1 cell. In some embodiments, the monocyte cell line is differentiated (such as with Phorbol 12-myristate 13 -acetate (PMA)), such as prior to contacting drug product or drug substance. In some embodiments, the method further comprises the step of contacting the cells with Phorbol 12- myristate 13-acetate (PMA), e.g., prior to contacting drug product or drug substance to the cells.

[11] In some embodiments, the method is a method of determining the potency of a drug product or drug substance, e.g., is a potency assay. In some embodiments, the method is a potency assay for batch release. In some embodiments, the method of determining the potency of a drug product or drug substance is stability indicating, e.g., the assay can be used to detect whether a drug product or drug substance (e.g., a batch thereof) has a potency that is different from (e.g., greater than or less than) a reference such as a reference standard.

[12] In some embodiments, a method further comprises serially diluting the drug product or drug substance prior to contacting the cell (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) with the drug product or drug substance.

[13] In some embodiments, a drug product or drug substance is contacted with the cells (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) for a time period sufficient for the drug product or drug substance to induce expression of the transcription product by the cell (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells). In some embodiments, the time period is at least 12 hours. In some embodiments, the time period is at least 24 hours. In some embodiments, the method further comprises determining a level of expression of the transcription product by the cell (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells). In some embodiments, the method further comprises comparing level of expression of the transcription product by the cell (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) after the drug product or drug substance is contacted with the cells to the level of expression of the transcription product by the cell (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) after a reference standard is contacted with the cells. In some embodiments, the method further comprises comparing the level of expression of the transcription product to a threshold level of expression of the transcription product. In some embodiments, the method further comprises determining the threshold level by contacting a cell of the mammalian immune cell line with a reference standard of the drug product or drug substance and detecting the level of expression of the transcription product by the cell contacted with the reference standard, wherein the level of expression by the cell contacted with the reference standard is the threshold level.

[14] In some embodiments, the method further comprises discarding the drug product or drug substance if the level of expression of the transcription product by the cell (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) is below a threshold level.

[15] In some embodiments, the method further comprises identifying the drug product or drug substance as a potent drug product or drug substance if the level of expression of the transcription product by the cell (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) is at or above a threshold level.

[16] In some embodiments, the method further comprises formulating the drug product or drug substance into a solid dosage form if it has been identified as a potent drug product or drug substance (e.g., a tablet, a minitablet, a capsule, or a powder).

[17] In some embodiments, a solid dosage form is a tablet or capsule.

[18] In some embodiments, the method further comprises packaging the drug product or drug substance if it has been identified as a potent drug product or drug substance. [19] In some embodiments, the method further comprises administering the drug product or drug substance to a subject in need thereof if it has been identified as a potent drug product or drug substance.

[20] In some embodiments, the method further comprises not discarding the drug product or drug substance if it has been identified as a potent drug product or drug substance.

[21] In some embodiments, the threshold level is about 50% to about 200%, such as 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% the level of expression of the transcription product by the cell (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) that have been contacted by a reference standard. In some embodiments, the threshold level is at least 70% the level of expression of the transcription product by cells (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) that have been contacted by a reference standard. In some embodiments, the threshold level is at most 135% the level of expression of the transcription product by cells (e.g., THP-1 cells, such as PMA- differentiated THP-1 cells) that have been contacted by a reference standard.

[22] In certain aspects, provided herein are methods of determining the potency of a drug product or drug substance, wherein potency is measured by expression of a coding nucleic acid sequence operatively linked to a regulatory sequence that is responsive to NF-κB or is an NF-κB promoter. Without wishing to be bound by any particular scientific theory, the present disclosure includes that expression of a coding nucleic acid sequence operatively linked, within a cell, to a regulatory sequence that is responsive to NF-κB or is an NF-κB promoter can correspond to and/or have a direct relationship with potency of a drug product or drug substance contacted with the cell. It will be appreciated from the present disclosure that the correspondence and/or relationship can be but need not be linear, and that in various embodiments expression can be measured by comparison to a reference such as a reference standard.

[23] In various embodiments, a method of determining the potency of a drug product or drug substance comprises i) determining a threshold level of expression of a transcription product encoded by a nucleic acid sequence by contacting a cell of a mammalian immune cell line (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) with a reference standard, wherein the cell comprises the nucleic acid sequence operatively linked to a regulatory sequence, wherein the regulatory sequence is responsive to NF-κB or is an NF-κB promoter, and the reference standard is brought into contact with the cell (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) for a time period sufficient to allow the reference standard to induce expression of the transcription product by the cells (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells). The method may further comprise ii) determining a test level of expression the transcription product of by contacting cells (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) with the drug product or drug substance, wherein the drug product or drug substance is brought into contact with the cells (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) for a time period sufficient to allow the drug product or drug substance to induce expression of the transcription product by the cells (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells). The method my further comprise iii) comparing the test level of expression of the transcription product to the level of threshold level of expression of the transcription product to determine the potency of the drug product or drug substance; wherein the drug product or drug substance comprises bacteria and/or mEVs. In some embodiments, the drug product or drug substance comprises a strain of bacteria (e.g., Prevotella histicola, Veillonella parvula, Harryflintia acetispora, Foumierella massiliensis, Megasphaera Sp., Selenomonas felix, Acidaminococcus intestine, and/or Propionospora sp. strain bacteria) and/or microbial extracellular vesicles (mEVs) from a strain of bacteria (e.g., Prevotella histicola, Veillonella parvula, Harryflintia acetispora, Foumierella massiliensis, Megasphaera Sp., Selenomonas felix, Acidaminococcus intestine, and/ or Propionospora sp. strain microbial extracellular vesicles (mEVs)).

[24] In some embodiments, a drug product or drug substance comprises bacteria of a strain of Prevotella histicola. In some embodiments, the bacteria (e.g, Prevotella histicola strain bacteria) comprise at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, 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 (e.g, Prevotella histicola strain bacteria) comprise 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 (e.g, Prevotella histicola strain bacteria) is the Prevotella Strain B (NRRL accession number B 50329).

[25] In some embodiments, a drug product or drug substance comprises mEVs of bacteria of a strain of Prevotella histicola. In some embodiments, mEVs can be mEVs of bacteria (e.g, Prevotella histicola strain bacteria) that comprise at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, 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, mEVs can be mEVs of bacteria (e.g, Prevotella histicola strain bacteria) that comprise 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 mEVs are of bacteria (e.g, Prevotella histicola strain bacteria) of the Prevotella Strain B (NRRL accession number B 50329).

[26] In some embodiments, a transcription product is a protein. In some embodiments, the expression of the protein is determined by an enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay, or immunocytostaining. In some embodiments, the protein is a luciferase protein, such as a firefly luciferase protein. In some embodiments, expression of the luciferase is determined by a luciferase assay. In some embodiments, the protein is a fluorescent protein, such as a green fluorescent protein, mCherry protein, a yellow fluorescent protein, a blue fluorescent protein, a cyan fluorescent protein, or a red fluorescent protein. In some embodiments, expression of the fluorescent protein is determined by detecting fluorescence.

[27] In some embodiments, a transcription product is an RNA. In some embodiments, the expression of the RNA is determined by a northern hybridization method or using reverse transcription-polymerase chain reaction (RT-PCR).

[28] In some embodiments, a regulatory sequence is responsive to NF-κB. In some embodiments, the nucleic acid sequence is operably linked to one or more NF-κB response elements (e.g., one or more copies of an NF-κB response element). In some embodiments, the nucleic acid sequence is operably linked to four NF-κB response elements (e.g., four copies of an NF-κB response element) positioned upstream of a promoter such as a minimal TATA promoter. In some embodiments, a regulatory sequence is an NF-κB promoter. In some embodiments, the mammalian immune cell line is a monocyte cell line, such as THP-1 cell. In some embodiments, the monocyte cell line is differentiated (such as with Phorbol 12-myristate 13-acetate (PMA)), such as prior to contacting drug product or drug substance. In some embodiments, the method further comprises the step of contacting the cells with Phorbol 12-myristate 13-acetate (PMA), e.g., prior to contacting drug product or drug substance to the cells.

[29] In some embodiments, the method further comprises serially diluting the drug product or drug substance prior to bringing the drug product or drug substance into contact with the cells (e.g., THP-1 cells, such as PMA- differentiated THP-1 cells). In some embodiments, the method further comprises serially diluting the reference standard prior to bringing the reference standard into contact with the cells (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells).

[30] In some embodiments, a time period sufficient to allow a reference standard and/or a drug product or drug substance to induce expression of a transcription product encoded by cells (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) of an assay provided herein is at least 12 hours. In some embodiments, a time period sufficient to allow a reference standard and/or a drug product or drug substance to induce expression of a transcription product encoded by cells (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) of an assay disclosed herein is at least 24 hours.

[31] In some embodiments, a method of the present disclosure further comprises discarding the drug product or drug substance if the test level of expression of a transcription product encoded by cells of the assay is below a reference, e.g., below a threshold level of expression of the transcription product.

[32] In some embodiments, a method of the present disclosure further comprises identifying the drug product or drug substance as a potent drug product or drug substance if the test level of expression of the transcription product is at or above a threshold level of expression of the transcription product.

[33] In some embodiments, the method further comprises formulating the drug product or drug substance into a solid dosage form if it has been identified as a potent drug product or drug substance.

[34] In some embodiments, a solid dosage form is a tablet or a capsule.

[35] In some embodiments, the method further comprises packaging the drug product or drug substance if it has been identified as a potent drug product or drug substance.

[36] In some embodiments, the method further comprises administering the drug product or drug substance to a subject in need thereof it has been identified as a potent drug product or drug substance.

[37] In some embodiments, the method further comprises not discarding the drug product or drug substance if it has been identified as a potent drug product or drug substance.

[38] In some embodiments, a threshold level of expression of the transcription product is about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% of the level of expression of the transcription product by the cells (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) in step i). In some embodiments, the threshold level of expression of the transcription product is at least 70% of the level of expression of the transcription product by the cells (e.g., THP-1 cells, such as PMA- differentiated THP-1 cells) in step i). In some embodiments, the threshold level of expression of the transcription product is at most 135% of the level of expression of the transcription product by the cells (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) in step i).

[39] In some embodiments, a method further comprises the step of PMA-differentiating the cells (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells), e.g., prior to step i).

[40] In some embodiments of an aspect provided herein, the method of determining the potency of a drug product or drug substance is a potency assay for batch release.

[41] In some embodiments, of an aspect provided herein the method of determining the potency of a drug product or drug substance is stability indicating, e.g., the method can be used to detect whether a drug product or drug substance (e.g., a batch thereof) has potency that is different from (e.g., greater than or less than) a reference such as a reference standard.

[42] In some embodiments of an aspect provided herein, a drug product or drug substance comprises bacteria.

[43] In some embodiments of an aspect provided herein, a drug product or drug substance comprises isolated bacteria (e.g., from one or more strains of bacteria (e.g., bacteria of interest)) (e.g., a therapeutically effective amount thereof). In some embodiments, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the content of the drug product or drug substance (e.g., pharmaceutical agent) is isolated mEVs of bacteria (e.g., bacteria of interest).

[44] In some embodiments, of an aspect provided herein a drug product or drug substance comprises isolated bacteria (e.g., from one strain of bacteria (e.g., bacteria of interest)) (e.g., a therapeutically effective amount thereof). In some embodiments, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the content of the drug product or drug substance (e.g., pharmaceutical agent) is isolated mEVs of bacteria (e.g., bacteria of interest).

[45] In some embodiments of an aspect provided herein, a drug product or drug substance comprises bacteria and the bacteria are from one strain. [46] In some embodiments of an aspect provided herein, a drug product or drug substance comprises microbial extracellular vesicles (mEVs).

[47] In some embodiments of an aspect provided herein, a drug product or drug substance comprises bacteria and microbial extracellular vesicles (mEVs).

[48] In some embodiments of an aspect provided herein, a drug product or drug substance comprises isolated bacteria and/or isolated mEVs (e.g., from one or more strains of bacteria (e.g., bacteria of interest) (e.g., a therapeutically effective amount thereof)). E.g., wherein at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the content of the pharmaceutical agent is the isolated bacteria and/or isolated mEVs (e.g., bacteria of interest).

[49] In some embodiments of an aspect provided herein, a drug product or drug substance comprises isolated bacteria and/or isolated mEVs (e.g., from one strain of bacteria (e.g., bacteria of interest) (e.g., a therapeutically effective amount thereof)). In some embodiments, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the content of the pharmaceutical agent is the isolated bacteria and/or isolated mEVs (e.g., bacteria of interest).

[50] In some embodiments of an aspect provided herein, a drug product or drug substance comprises bacteria and/or mEVs that have been gamma irradiated, UV irradiated, heat inactivated, acid treated, or oxygen sparged.

[51] In some embodiments of an aspect provided herein, a drug product or drug substance comprises live bacteria.

[52] In some embodiments of an aspect provided herein, a drug product or drug substance comprises dead bacteria.

[53] In some embodiments of an aspect provided herein, a drug product or drug substance comprises non-replicating bacteria.

[54] In some embodiments of an aspect provided herein, a drug product or drug substance comprises one strain of bacteria or mEVs from one strain of bacteria.

[55] In some embodiments of an aspect provided herein, bacteria and/or mEVs are lyophilized (e.g., the lyophilized product further comprises a pharmaceutically acceptable excipient) (e.g., a powder form). [56] In some embodiments of an aspect provided herein, bacteria and/or mEVs are gamma irradiated.

[57] In some embodiments of an aspect provided herein, bacteria and/or mEVs are UV irradiated.

[58] In some embodiments of an aspect provided herein, bacteria and/or mEVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).

[59] In some embodiments of an aspect provided herein, bacteria and/or mEVs are acid treated.

[60] In some embodiments of an aspect provided herein, bacteria are and/or mEVs oxygen sparged (e.g., at 0.1 wm for two hours).

[61] In some embodiments of an aspect provided herein, a drug product or drug substance comprises isolated mEVs (e.g., from one or more strains of bacteria (e.g., bacteria of interest)) (e.g., a therapeutically effective amount thereof). E.g., wherein at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the content of the pharmaceutical agent is isolated mEVs of bacteria (e.g., bacteria of interest).

[62] In some embodiments, of an aspect provided herein a drug product or drug substance comprises isolated mEVs (e.g., from one strain of bacteria (e.g., bacteria of interest)) (e.g., a therapeutically effective amount thereof). In some embodiments, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the content of the pharmaceutical agent is isolated mEVs of bacteria (e.g., bacteria of interest).

[63] In some embodiments of an aspect provided herein, a drug product or drug substance comprises mEVs and the mEVs comprise secreted mEVs (smEVs).

[64] In some embodiments of an aspect provided herein, a drug product or drug substance comprises mEVs and the mEVs comprise processed mEVs (pmEVs).

[65] In some embodiments, of an aspect provided herein a drug product or drug substance comprises pmEVs and the pmEVs are produced from bacteria that have been gamma irradiated, UV irradiated, heat inactivated, acid treated, or oxygen sparged.

[66] In some embodiments of an aspect provided herein, a drug product or drug substance comprises pmEVs and the pmEVs are produced from live bacteria.

[67] In some embodiments of an aspect provided herein, a drug product or drug substance comprises pmEVs and the pmEVs are produced from dead bacteria. [68] In some embodiments of an aspect provided herein, a drug product or drug substance comprises pmEVs and the pmEVs are produced from non-replicating bacteria.

[69] In some embodiments of an aspect provided herein, a drug product or drug substance comprises mEVs and the mEVs are from one strain of bacteria.

DETAILED DESCRIPTION

General

This disclosure is based, in part, on the need to assess the potency of drug products and/or drug substances comprising bacteria and/or microbial extracellular vesicles (mEVs). Disclosed herein are assays to demonstrate bacterial test samples (e.g., drug products or drug substances comprising bacteria and/or microbial extracellular vesicles) induce expression of a transcription product in cells at levels similar to a reference standard. For example, as disclosed herein, potency of bacteria and/or microbial extracellular vesicles (e.g., Prevotella histicola bacteria and/or microbial extracellular vesicles) can be measured by incubating the bacteria and/or microbial extracellular vesicles with a population of one or more mammalian cells (e.g., THP-1 cells, e.g., PMA-differentiated THP-1 cells) and measuring expression of the transcription product. The expression of the transcription product can be measured by any of a variety of techniques disclosed herein or otherwise generally available to those of skill in the art. In various embodiments, expression of the transcription product can be measured by detecting expression of a nucleic acid encoding a detectable marker operably linked with a regulatory sequence, wherein the regulatory sequence is responsive to NF-κB or is an NF-κB promoter. In some embodiments, if a drug product or drug substance is determined to be not potent (e.g., to have a measured potency that is less than a potent reference or threshold of potency) according to an assay disclosed herein, the drug product or drug substance can be discarded from use. In some embodiments, if a drug product or drug substance is determined to be potent (e.g., to have a measured potency that is equal to and/or greater than a potent reference or threshold of potency) according to an assay disclosed herein, the drug product or drug substance is not discarded (e.g., the drug product or drug substance is formulated into a solid dosage form (e.g., capsules or tablets), packaged, and/or administered to a subject in need thereof). Definitions

[70] 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.

[71] “Administration” broadly refers to a route of administration of a composition (e.g., a drug substance or drug product described herein) to a subject. Examples of routes of administration include oral administration, rectal administration, topical administration, inhalation (nasal) or injection. Administration by injection includes intravenous (IV), intramuscular (IM), and subcutaneous (SC) administration. A drug substance or drug product described herein can be administered in any form by any effective route, including but not limited to oral, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal (e.g., using any standard patch), intradermal, ophthalmic, (intra)nasally, local, non-oral, such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, (trans)rectal, vaginal, intra-arterial, and intrathecal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), implanted, intravesical, intrapulmonary, intraduodenal, intragastrical, and intrabronchial. In preferred embodiments, a drug substance or drug product described herein is administered orally, rectally, topically, intravesically, by injection into or adjacent to a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously. In another preferred embodiment, a drug substance or drug product described herein is administered orally, or intravenously. In another embodiment, a drug substance or drug product described herein is administered orally.

[72] A “carbohydrate” refers to a sugar or polymer of sugars. The terms “saccharide,” “polysaccharide,” “carbohydrate,” and “oligosaccharide” may be used interchangeably. Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one on each carbon atom of the molecule. Carbohydrates generally have the molecular formula CnH2nOn. A carbohydrate may be a monosaccharide, a disaccharide, trisaccharide, oligosaccharide, or polysaccharide. The most basic carbohydrate is a monosaccharide, such as glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, and fructose. Disaccharides are two joined monosaccharides. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose. Typically, an oligosaccharide includes between three and six monosaccharide units (e.g., raffinose, stachyose), and polysaccharides include six or more monosaccharide units. Exemplary polysaccharides include starch, glycogen, and cellulose. Carbohydrates may contain modified saccharide units such as 2’ -deoxyribose wherein a hydroxyl group is removed, 2’ -fluororibose wherein a hydroxyl group is replaced with a fluorine, or N-acetylglucosamine, a nitrogen- containing form of glucose (e.g., 2’ -fluororibose, deoxyribose, and hexose). Carbohydrates may exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.

[73] “Cellular augmentation” broadly refers to the influx of cells or expansion of cells in an environment that are not substantially present in the environment prior to administration of a composition and not present in the composition itself. Cells that augment the environment include immune cells, stromal cells, bacterial and fungal cells.

[74] A “combination” of bacteria from two or more strains includes the physical co-existence of the bacteria, either in the same material or product or in physically connected products, as well as the temporal co-administration or co-localization of the bacteria from the two or more strains.

[75] A “combination” of mEVs (such as smEVs and/or pmEVs) from two or more microbial (such as bacteria) strains includes the physical co-existence of the microbes from which the mEVs (such as smEVs and/or pmEVs) are obtained, either in the same material or product or in physically connected products, as well as the temporal co-administration or co-localization of the mEVs (such as smEVs and/or pmEVs) from the two or more strains.

[76] The term “decrease” or “deplete,” and grammatical equivalents thereof, indicate qualitative or quantitative difference from a reference. For example, a decrease can refer to a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000 or undetectable after treatment when compared to a pre-treatment state. Properties that may be decreased include the number of immune cells, bacterial cells, stromal cells, myeloid derived suppressor cells, fibroblasts, metabolites; the level of a cytokine; expression of a protein, or another physical parameter (such as ear thickness (e.g., in a DTH animal model) or tumor size).

[77] The term “drug substance” or “therapeutic agent” refers to an agent for therapeutic use and comprising bacteria and/or microbial extracellular vesicles (mEVs) (such as smEVs and/or pmEVs), e.g., that can be used to treat and/or prevent a disease and/or condition. In some embodiments, the therapeutic agent is a pharmaceutical agent. In some embodiments, a medicinal product, medical food, a food product, or a dietary supplement comprises a therapeutic agent. For example, the therapeutic agent disclosed herein may be a powder comprising bacteria and/or microbial extracellular vesicles (mEVs) (such as smEVs and/or pmEVs). In some embodiments, the therapeutic agent may further comprise an excipient.

[78] The term “drug product” or “therapeutic composition” refers to a composition that comprises a therapeutically effective amount of a therapeutic agent. In some embodiments, the therapeutic composition is (or is present in) a medicinal product, medical food, a food product, or a dietary supplement. For example, the therapeutic composition may be a tablet or capsule comprising the therapeutic agent. In some embodiments, the therapeutic composition may be a powder comprising the therapeutic agent and additional excipients. In some embodiments, a therapeutic compositioncomprises a therapeutic agent and an additional excipient.

[79] “Dysbiosis” refers to a state of the microbiota or microbiome of the gut or other body area, including, e.g., mucosal or skin surfaces (or any other microbiome niche) in which the normal diversity and/or function of the host gut microbiome ecological networks “microbiome”) are disrupted. A state of dysbiosis may result in a diseased state, or it may be unhealthy under only certain conditions or only if present for a prolonged period. Dysbiosis may be due to a variety of factors, including, environmental factors, infectious agents, host genotype, host diet and/or stress. A dysbiosis may result in: a change (e.g., increase or decrease) in the prevalence of one or more bacteria types (e.g., anaerobic), species and/or strains, change (e.g., increase or decrease) in diversity of the host microbiome population composition; a change (e.g., increase or reduction) of one or more populations of symbiont organisms resulting in a reduction or loss of one or more beneficial effects; overgrowth of one or more populations of pathogens (e.g., pathogenic bacteria); and/or the presence of, and/or overgrowth of, symbiotic organisms that cause disease only when certain conditions are present.

[80] As used herein, “engineered bacteria” are any bacteria that have been genetically altered from their natural state by human activities, and the progeny of any such bacteria. Engineered bacteria include, for example, the products of targeted genetic modification, the products of random mutagenesis screens and the products of directed evolution.

[81] The term “gene” refers to a DNA sequence that is or includes coding sequence (i.e., a DNA sequence that encodes an expression product, such as an RNA product and/or a polypeptide product), optionally together with some or all of regulatory sequences that control expression of the coding sequence. In some embodiments, a gene includes non-coding sequence such as, without limitation, introns. In some embodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequences. In some embodiments, a gene includes a regulatory sequence that is a promoter or a transcription factor response element. In some embodiments, a gene includes one or both of a (i) DNA nucleotides extending a predetermined number of nucleotides upstream of the coding sequence in a reference context, such as a source genome, and (ii) DNA nucleotides extending a predetermined number of nucleotides downstream of the coding sequence in a reference context, such as a source genome. In various embodiments, the predetermined number of nucleotides can be 500 bp, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 75 kb, or 100 kb. As used herein, a “transgene” refers to a gene that is not endogenous or native to a reference context in which the gene is present or into which the gene may be placed by engineering.

[82] “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(1): 387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., et al., J Molec Biol 215:403 (1990); Guide to Huge Computers, Mrtin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al. (1988) SIAM J Applied Math 48: 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.)).

[83] As used herein, the term “immune disorder” refers to any disease, disorder or disease symptom caused by an activity of the immune system, including autoimmune diseases, inflammatory diseases and allergies. Immune disorders include, but are not limited to, autoimmune diseases (e.g., psoriasis, atopic dermatitis, lupus, scleroderma, hemolytic anemia, vasculitis, type one diabetes, Grave’s disease, rheumatoid arthritis, multiple sclerosis, Goodpasture’s syndrome, pernicious anemia and/or myopathy), inflammatory diseases (e.g., acne vulgaris, asthma, celiac disease, chronic prostatitis, glomerulonephritis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplant rejection, vasculitis and/or interstitial cystitis), and/or an allergies (e.g., food allergies, drug allergies and/or environmental allergies). [84] The term “increase,” and grammatical equivalents thereof, indicates qualitative or quantitative difference from a reference. For example, an increase can refer to a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 4-fold, 10-fold, 100-fold, 10 ^A 3 fold, 10 ^A 4 fold, 10 ^A 5 fold, 10 ^A 6 fold, and/or 10 ^A 7 fold greater after treatment when compared to a pre-treatment state. Properties that may be increased include the number of immune cells, bacterial cells, stromal cells, myeloid derived suppressor cells, fibroblasts, metabolites; the level of a cytokine; expression of a protein, or another physical parameter (such as ear thickness (e.g., in a DTH animal model) or tumor size).

[85] ‘ ‘Innate immune agonists” or “immuno-adjuvants” are small molecules, proteins, or other agents that specifically target innate immune receptors including Toll-Like Receptors (TLR), NOD receptors, RLRs, C-type lectin receptors, STING-cGAS Pathway components, inflammasome complexes. For example, LPS is a TLR-4 agonist that is bacterially derived or synthesized and aluminum can be used as an immune stimulating adjuvant, immuno-adjuvants are a specific class of broader adjuvant or adjuvant therapy. Examples of STING agonists include, but are not limited to, 2'3'- cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, 2'2'-cGAMP, and 2'3'-cGAM(PS)2 (Rp/Sp) (Rp, Sp-isomers of the bis-phosphorothioate analog of 2'3'- cGAMP). Examples of TLR agonists include, but are not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10 and TLR11. Examples of NOD agonists include, but are not limited to, N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyldipeptide (MDP)), gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP), and desmuramylpeptides (DMP).

[86] The “internal transcribed spacer” or “ITS” is a piece of non-functional RNA positioned between structural ribosomal RNAs (rRNA) on a common precursor transcript often used for identification of eukaryotic species in particular fungi. The rRNA of fungi that forms the core of the ribosome is transcribed as a signal gene and consists of the 8S, 5.8S and 28S regions with ITS4 and 5 between the 8S and 5.8S and 5.8S and 28S regions, respectively. These two intercistronic segments between the 18S and 5.8S and 5.8S and 28S regions are removed by splicing and contain significant variation between species for barcoding purposes as previously described (Schoch et al Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. PNAS 109:6241-6246. 2012). 18S rDNA is traditionally used for phylogenetic reconstruction however the ITS can serve this function as it is generally highly conserved but contains hypervariable regions that harbor sufficient nucleotide diversity to differentiate genera and species of most fungus.

[87] The term “isolated” or “enriched” encompasses a microbe (such as a bacterium), mEV (such as an smEV and/or pmEV), or other entity or substance that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by the hand of man. Isolated microbes or mEVs 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 microbes or mEVs 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. As used herein, a substance is “pure” if it is substantially free of other components. The terms “purify,” “purifying” and “purified” refer to a microbe or mEV or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production. A microbe or a microbial population or mEVs may be considered purified if it is isolated at or after production, such as from a material or environment containing the microbe or microbial population, and a purified microbe or microbial population or mEVs may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “isolated.” In some embodiments, purified microbes or microbial population or mEVs 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. In the instance of microbial compositions provided herein, the one or more microbial or mEV types present in the composition can be independently purified from one or more other microbes produced and/or present in the material or environment containing the microbial type. Microbial compositions and the microbial components (such as mEVs) thereof are generally purified from residual habitat products.

[88] As used herein a “lipid” includes fats, oils, triglycerides, cholesterol, phospholipids, fatty acids in any form including free fatty acids. Fats, oils and fatty acids can be saturated, unsaturated (cis or trans) or partially unsaturated (cis or trans). [89] The term “LPS mutant or lipopolysaccharide mutant” broadly refers to selected bacteria that comprises loss of LPS. Loss of LPS might be due to mutations or disruption to genes involved in lipid A biosynthesis, such as IpxA, IpxC, and IpxD. Bacteria comprising LPS mutants can be resistant to aminoglycosides and polymyxins (polymyxin B and colistin).

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

[91] “Microbial extracellular vesicles” (mEVs) can be obtained from microbes such as bacteria, archaea, fungi, microscopic algae, protozoans, and parasites. In some embodiments, the mEVs are obtained from bacteria. mEVs include secreted microbial extracellular vesicles (smEVs) and processed microbial extracellular vesicles (pmEVs). “Secreted microbial extracellular vesicles” (smEVs) are naturally-produced vesicles derived from microbes. smEVs are comprised of microbial lipids and/or microbial proteins and/or microbial nucleic acids and/or microbial carbohydrate moieties, and are isolated from culture supernatant. The natural production of these vesicles can be artificially enhanced (e.g., increased) or decreased through manipulation of the environment in which the bacterial cells are being cultured (e.g., by media or temperature alterations). Further, smEV compositions may be modified to reduce, increase, add, or remove microbial components or foreign substances to alter efficacy, immune stimulation, stability, immune stimulatory capacity, stability, organ targeting (e.g., lymph node), absorption (e.g., gastrointestinal), and/or yield (e.g., thereby altering the efficacy). As used herein, the term “purified smEV composition” or “smEV composition” refers to a preparation of smEVs that have been separated from at least one associated substance found in a source material (e.g., separated from at least one other microbial component) or any material associated with the smEVs in any process used to produce the preparation. It can also refer to a composition that has been significantly enriched for specific components. “Processed microbial extracellular vesicles” (pmEVs) are a non-naturally- occurring collection of microbial membrane components that have been purified from artificially lysed microbes (e.g., bacteria) (e.g., microbial membrane components that have been separated from other, intracellular microbial cell components), and which may comprise particles of a varied or a selected size range, depending on the method of purification. A pool of pmEVs is obtained by chemically disrupting (e.g., by lysozyme and/or lysostaphin) and/or physically disrupting (e.g., by mechanical force) microbial cells and separating the microbial membrane components from the intracellular components through centrifugation and/or ultracentrifugation, or other methods. The resulting pmEV mixture contains an enrichment of the microbial membranes and the components thereof (e.g., peripherally associated or integral membrane proteins, lipids, glycans, polysaccharides, carbohydrates, other polymers), such that there is an increased concentration of microbial membrane components, and a decreased concentration (e.g., dilution) of intracellular contents, relative to whole microbes. For gram-positive bacteria, pmEVs may include cell or cytoplasmic membranes. For gramnegative bacteria, a pmEV may include inner and outer membranes. pmEVs may be modified to increase purity, to adjust the size of particles in the composition, and/or modified to reduce, increase, add or remove, microbial components or foreign substances to alter efficacy, immune stimulation, stability, immune stimulatory capacity, stability, organ targeting (e.g., lymph node), absorption (e.g., gastrointestinal), and/or yield (e.g., thereby altering the efficacy). pmEVs can be modified by adding, removing, enriching for, or diluting specific components, including intracellular components from the same or other microbes. As used herein, the term “purified pmEV composition” or “pmEV composition” refers to a preparation of pmEVs that have been separated from at least one associated substance found in a source material (e.g., separated from at least one other microbial component) or any material associated with the pmEVs in any process used to produce the preparation. It can also refer to a composition that has been significantly enriched for specific components.

[92] “Microbe” refers to any natural or engineered organism characterized as a archaeaon, parasite, bacterium, fungus, microscopic alga, protozoan, and the stages of development or life cycle stages (e.g., vegetative, spore (including sporulation, dormancy, and germination), latent, biofilm) associated with the organism. Examples of gut microbes include: Actinomyces graevenitzii, Actinomyces odontolyticus, Akkermansia muciniphila, Bacteroides caccae, Bacteroides fragilis, Bacteroides putredinis, Bacteroides thetaiotaomicron, Bacteroides vultagus, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bilophila wadsworthia, Blautia, Butyrivibrio, Campylobacter gracilis, Clostridia cluster III, Clostridia cluster IV, Clostridia cluster IX (Acidaminococcaceae group), Clostridia cluster XI, Clostridia cluster XIII (Peptostreptococcus group), Clostridia cluster XIV, Clostridia cluster XV, Collinsella aerofaciens, Coprococcus, Corynebacterium sunsvallense, Desulfomonas pigra, Dorea formicigenerans, Dorea longicatena, Escherichia coli, Eubacterium hadrum, Eubacterium rectale, Faecalibacteria prausnitzii, Gemella, Lactococcus, Lanchnospira, Mollicutes cluster XVI, Mollicutes cluster XVIII, Prevotella, Rothia mucilaginosa, Ruminococcus callidus, Ruminococcus gnavus, Ruminococcus torques, and Streptococcus.

[93] “Microbiome” broadly refers to the microbes residing on or in body site of a subject or patient. Microbes in a microbiome may include bacteria, viruses, eukaryotic microorganisms, and/or viruses. Individual microbes in a microbiome may be metabolically active, dormant, latent, or exist as spores, may exist planktonically or in biofilms, or may be present in the microbiome in sustainable or transient manner. The microbiome may be a commensal or healthy- state microbiome or a disease-state microbiome. The microbiome may be native to the subject or patient, or components of the microbiome may be modulated, introduced, or depleted due to changes in health state or treatment conditions (e.g, antibiotic treatment, exposure to different microbes). In some embodiments, the microbiome occurs at a mucosal surface. In some embodiments, the microbiome is a gut microbiome.

[94] “Modified” in reference to a bacteria broadly refers to a bacteria that has undergone a change from its wild-type form. Bacterial modification can result from engineering bacteria. Examples of bacterial modifications include genetic modification, gene expression modification, phenotype modification, formulation modification, chemical modification, and dose or concentration. Examples of improved properties are described throughout this specification and include, e.g., attenuation, auxotrophy, homing, or antigenicity. Phenotype modification might include, by way of example, bacteria growth in media that modify the phenotype of a bacterium such that it increases or decreases virulence. mEVs can be modified, e.g., as described herein.

[95] “Operably linked” refers to the association of at least a first element and a second element such that the component elements are in a relationship permitting them to function in their intended manner. For example, a nucleic acid sequence or amino acid sequence is operably linked with another sequence if it modifies the expression, structure, or activity of the linked sequence, e.g., in an intended manner. In many cases, two nucleic acid sequences are operably linked if they contribute to the expression, structure, or activity of a gene or encoded polypeptide. For example, a nucleic acid regulatory sequence is "operably linked" to a nucleic acid coding sequence if the regulatory sequence and coding sequence are associated in a manner that permits control of expression of the coding sequence by the regulatory sequence. In some embodiments, an "operably linked" regulatory sequence is directly or indirectly covalently associated with a coding sequence (e.g., in a single nucleic acid). In some embodiments, a regulatory sequence controls expression of a coding sequence in trans and inclusion of the regulatory sequence in the same nucleic acid as the coding sequence is not a requirement of operable linkage.

[96] As used herein, a gene is “overexpressed” in a bacterium if it is expressed at a higher level in an engineered bacterium under at least some conditions than it is expressed by a wild- type bacterium of the same species under the same conditions. Similarly, a gene is “underexpressed” in a bacterium if it is expressed at a lower level in an engineered bacterium under at least some conditions than it is expressed by a wild-type bacterium of the same species under the same conditions.

[97] The terms “polynucleotide,” and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), micro RNA (miRNA), silencing RNA (siRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide may be further modified, such as by conjugation with a labeling component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.

[98] As used herein, the term “preventing” a disease or condition in a subject refers to administering to the subject to a pharmaceutical treatment, e.g., the administration of one or more agents (e.g., pharmaceutical agent), such that onset of at least one symptom of the disease or condition is delayed or prevented.

[0001] As used herein, a “promoter” or “promoter sequence” can be a DNA regulatory sequence that directly or indirectly (e.g., through promoter-bound proteins or substances) participates in initiation and/or processivity of transcription of a coding sequence. A promoter may, under suitable conditions, initiate transcription of a coding sequence upon binding of one or more transcription factors and/or regulatory moieties with the promoter. A promoter that participates in initiation of transcription of a coding sequence can be “operably linked” to the coding sequence. Promoters can be positioned in a nucleic acid near to and upstream of the transcription start sites of genes (5' of a transcription start site). In certain instances, a promoter can be or include a DNA regulatory sequence that extends from a transcription start site (at its 3’ terminus) to an upstream (5’ direction) position such that the sequence so designated includes one or both of a minimum number of bases or elements necessary to initiate a transcription event. A promoter can be about 100-3000 or about 100-1000 base pairs in length. A promoter may be, include, or be operably associated with or operably linked to, expression control sequences such as enhancer and repressor sequences.

[99] As used herein, “protein expression” and grammatical equivalents thereof refers individually and/or cumulatively to one or more biological process that result in production from a nucleic acid sequence of an encoded agent, such as a polypeptide. Expression specifically includes either or both of transcription and translation. Expression of a protein can be detected directly (i.e., by measuring the level of the protein in a relevant context) or indirectly (e.g., by measuring an activity of the protein in a relevant context, or by measuring a surrogate for expression of the protein such as expression of a transgene that includes a nucleic acid sequence encoding a detectable marker operably linked with a promoter native to the protein of interest). Accordingly, expression of a protein can refer to, and/or be detected by measuring, the level or production of the protein, level or production of mRNA encoding the protein, or activity of a promoter that controls expression of the protein.

[100] As used herein, a substance is “pure” if it is substantially free of other components. The terms “purify,” “purifying” and “purified” refer to an mEV (such as an smEV and/or a pmEV) 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 (e.g., whether in nature or in an experimental setting), or during any time after its initial production. An mEV (such as an smEV and/or a pmEV) 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 microbial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “purified.” In some embodiments, purified mEVs (such as smEVs and/or pmEVs) 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. mEV (such as a smEV and/or a pmEV) compositions (or preparations) are, e.g., purified from residual habitat products.

[101] As used herein, the term “purified mEV composition” or “mEV composition” refers to a preparation that includes mEVs (such as smEVs and/or pmEVs) that have been separated from at least one associated substance found in a source material (e.g., separated from at least one other bacterial component) or any material associated with the mEVs (such as smEVs and/or pmEVs) 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 mEVs (such as smEVs and/or pmEVs) 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.

[102] ‘Reference” refers to a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, sample, sequence, cell, subject, animal, or individual, or population thereof, or a measure or characteristic representative thereof, is compared with a reference, an agent, sample, sequence, cell, subject, animal, or individual, or population thereof, or a measure or characteristic representative thereof. In some embodiments, a reference is a measured value. In some embodiments, a reference is an established standard or expected value. In some embodiments, a reference is a historical reference. A reference can be quantitative of qualitative. Typically, as would be understood by those of skill in the art, a reference and the value to which it is compared represent assessments under comparable conditions. Those of skill in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison. In some embodiments, an appropriate reference may be an agent, sample, sequence, cell, subject, animal, or individual, or population thereof, under conditions those of skill in the art will recognize as comparable, e.g., for the purpose of assessing one or more particular variables (e.g., potency), or a measure or characteristic representative thereof.

[103] As used herein, “reference standard” means a normalized value obtained from a standardized sample, and in the case of analyzing the potency of a drug product or drug substance, can mean normalized expression of a transcription product (e.g., a reporter mRNA or protein) encoded by a nucleic acid sequence operatively linked to a regualtory sequence measured in a reference sample of known potency (e.g., as measured in parallel with measurement of the same parameter in a test sample to which the reference is compared, with same or comparable steps and conditions applied to both the reference sample and the test sample). In some embodiments, a reference standard is a sample of a drug product or drug substance that has previously been confirmed to be potent. For example, a reference standard can be a preparation of the same bacteria or mEV as in a drug product or drug substance being tested, wherein the reference standard causes a certain level of transcription product expression in the assay.

[104] As used herein in the context of expression of a nucleic acid coding sequence, a “regulatory sequence” refers to a nucleic acid sequence that controls expression of a coding sequence. In some embodiments, a regulatory sequence can control or impact one or more aspects of gene expression (e.g., cell-type-specific expression, inducible expression, etc.).

[105] ‘Residual habitat products” refers to material derived from the habitat for microbiota within or on a subject. For example, fermentation cultures of microbes can contain contaminants, e.g., other microbe strains or forms (e.g., bacteria, virus, mycoplasm, and/or fungus). For example, microbes live in feces in the gastrointestinal tract, on the skin itself, in saliva, mucus of the respiratory tract, or secretions of the genitourinary tract (i.e., biological matter associated with the microbial community). Substantially free of residual habitat products means that the microbial composition no longer contains the biological matter associated with the microbial environment on or in the culture or human or animal subject and is 100% free, 99% free, 98% free, 97% free, 96% free, or 95% free of any contaminating biological matter associated with the microbial community. Residual habitat products can include abiotic materials (including undigested food) or it can include unwanted microorganisms. Substantially free of residual habitat products may also mean that the microbial composition contains no detectable cells from a culture contaminant or a human or animal and that only microbial cells are detectable. In one embodiment, substantially free of residual habitat products may also mean that the microbial composition contains no detectable viral (including bacteria, viruses (e.g., phage)), fungal, mycoplasmal contaminants. In another embodiment, it means that fewer than 1x10 ^-2 %, 1x10 ^-3 %, 1x10 ^-4 %, 1x10 ^-5 %, 1x10 ^-6 %, 1x10 ^-7 %, 1x10 ^-8 % of the viable cells in the microbial composition are human or animal, as compared to microbial cells. There are multiple ways to accomplish this degree of purity, none of which are limiting. Thus, contamination may be reduced by isolating desired constituents through multiple steps of streaking to single colonies on solid media until replicate (such as, but not limited to, two) streaks from serial single colonies have shown only a single colony morphology. Alternatively, reduction of contamination can be accomplished by multiple rounds of serial dilutions to single desired cells (e.g., a dilution of 10 ^-8 or 10 ^-9 ), such as through multiple 10-fold serial dilutions. This can further be confirmed by showing that multiple isolated colonies have similar cell shapes and Gram staining behavior. Other methods for confirming adequate purity include genetic analysis (e.g., PCR, DNA sequencing), serology and antigen analysis, enzymatic and metabolic analysis, and methods using instrumentation such as flow cytometry with reagents that distinguish desired constituents from contaminants.

[106] 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 one regulatory sequence (e.g., a promoter, a response element, 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 sequence (e.g., a promoter, a response element, 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.

[107] The terms “subject” or “patient” refers to any mammal. A subject or a patient described as “in need thereof’ refers to one in need of a treatment (or prevention) for a disease. Mammals (i.e., mammalian animals) include humans, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows, sheep, goats, pigs), and household pets (e.g., dogs, cats, rodents). The subject may be a human. The subject may be a non-human mammal including but not limited to of a dog, a cat, a cow, a horse, a pig, a donkey, a goat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, a gorilla or a chimpanzee. The subject may be healthy, or may be suffering from a cancer at any developmental stage, wherein any of the stages are either caused by or opportunistically supported of a cancer associated or causative pathogen, or may be at risk of developing a cancer, or transmitting to others a cancer associated or cancer causative pathogen. In some embodiments, a subject has lung cancer, bladder cancer, prostate cancer, plasmacytoma, colorectal cancer, rectal cancer, Merkel Cell carcinoma, salivary gland carcinoma, ovarian cancer, and/or melanoma. The subject may have a tumor. The subject may have a tumor that shows enhanced macropinocytosis with the underlying genomics of this process including Ras activation. In other embodiments, the subject has another cancer. In some embodiments, the subject has undergone a cancer therapy.

[108] As used herein, a “systemic effect” in a subject treated with a pharmaceutical composition containing bacteria or mEVs (e.g., a pharmaceutical agent comprising bacteria or mEVs) provided herein means a physiological effect occurring at one or more sites outside the gastrointestinal tract. Systemic effect(s) can result from immune modulation (e.g., via an increase and/or a reduction of one or more immune cell types or subtypes (e.g., CD8+ T cells) and/or one or more cytokines). Such systemic effect(s) may be the result of the modulation by bacteria or mEVs provided herein on immune or other cells (such as epithelial cells) in the gastrointestinal tract which then, directly or indirectly, result in the alteration of activity (activation and/or deactivation) of one or more biochemical pathways outside the gastrointestinal tract. The systemic effect may include treating or preventing a disease or condition in a subject.

[109] As used herein, the term “treating” a disease in a subject or “treating” a subject having or suspected of having a disease refers to administering to the subject to a pharmaceutical treatment, e.g., the administration of one or more agents, such that at least one symptom of the disease is decreased or prevented from worsening. Thus, in one embodiment, “treating” refers inter aha to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof. Bacteria

[110] The drug products and drug substances disclosed herein can comprise a pharmaceutical agent comprising bacteria and/or microbial extracellular vesicles (mEVs) (such as smEVs and/or pmEVs). For example, the drug products and drug substances disclosed herein can comprise a powder comprising bacteria and/or microbial extracellular vesicles (mEVs) (such as smEVs and/or pmEVs) (e.g., the powder can be resuspended prior to use in a method described herein). Within a pharmaceutical agent that contains bacteria and mEVs, the mEVs can be from the same bacterial origin (e.g., same strain) as the bacteria of the pharmaceutical agent (e.g., drug substance). Drug products (e.g., pharmaceutical compositions) and drug substances (e.g., pharmaceutical agents) can contain bacteria and/or mEVs from one or more strains. Drug products (e.g., pharmaceutical compositions) and drug substances (e.g., pharmaceutical agents) can contain bacteria and/or mEVs from one strain.

[111] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are modified to reduce toxicity or other adverse effects, to enhance delivery) (e.g., oral delivery) (e.g., by improving acid resistance, muco- adherence and/or penetration and/or resistance to bile acids, digestive enzymes, resistance to anti-microbial peptides and/or antibody neutralization), to target desired cell types (e.g., M-cells, goblet cells, enterocytes, dendritic cells, macrophages), to enhance their immunomodulatory and/or therapeutic effect of the bacteria and/or mEVs (e.g., either alone or in combination with another therapeutic agent), and/or to enhance immune activation or suppression by the bacteria and/or mEVs (such as smEVs and/or pmEVs) (e.g., through modified production of polysaccharides, pili, fimbriae, adhesins). In some embodiments, the engineered bacteria described herein are modified to improve bacteria and/or mEV (such as smEV and/or pmEV) manufacturing (e.g., 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 result 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.

[112] Examples of taxonomic groups (e.g., class, order, family, genus, species or strain) of bacteria that can be used as a source of bacteria and/or mEVs (such as smEVs and/or pmEVs) for a pharmaceutical agent described herein are provided herein (e.g., listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in 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 listed herein. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are oncotrophic bacteria. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are immunomodulatory bacteria. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are immunostimulatory bacteria. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are immunosuppressive bacteria. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are generated from one of the bacterial strains provided herein. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are generated from a combination of the 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 the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are from bacterial strains listed herein 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 listed herein (e.g., listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification). In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are generated from a bacterial strain provided herein. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are from a bacterial strain listed herein (e.g., listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification) and/or 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 listed herein (e.g., listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification).

[113] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Gram negative bacteria.

[114] 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.

[115] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Gram positive bacteria.

[116] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are aerobic bacteria.

[117] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are anaerobic bacteria. In some embodiments, the anaerobic bacteria comprise obligate anaerobes. In some embodiments, the anaerobic bacteria comprise facultative anaerobes.

[118] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are acidophile bacteria. [119] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are alkaliphile bacteria.

[120] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are neutral ophile bacteria.

[121] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are fastidious bacteria.

[122] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are nonfasti dious bacteria.

[123] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained or the mEVs themselves are lyophilized.

[124] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained or the mEVs themselves are gamma irradiated (e.g., at 17.5 or 25 kGy).

[125] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained or the mEVs themselves are UV irradiated.

[126] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained or the mEVs themselves are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).

[127] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained or the mEVs themselves are acid treated.

[128] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained or the mEVs themselves are oxygen sparged (e.g., at 0.1 wm for two hours).

[129] The phase of growth can affect the amount or properties of bacteria and/or mEVs produced by bacteria. For example, in the methods of mEVs preparation provided herein, mEVs can be isolated, e.g., 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.

[130] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent 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 spp.), gram-positive cocci (primarily Pepto streptococcus spp.), gram-positive spore-forming (Clostridium spp.), non-sporeforming bacilli (Actinomyces, Propionibacterium, Eubacterium, Lactobacillus and Bifidobacterium spp.), and gram-negative 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.

[131] 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.

[132] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Negativicutes class.

[133] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Veillonellaceae family.

[134] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Selenomonadaceae family.

[135] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Acidaminococcaceae family.

[136] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Sporomusaceae family.

[137] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Megasphaera genus.

[138] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Selenomonas genus.

[139] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Propionospora genus.

[140] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Acidaminococcus genus.

[141] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained axe Megasphaera sp. bacteria. [142] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Selenomonas felix bacteria.

[143] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Acidaminococcus intestini bacteria.

[144] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Propionospora sp. bacteria.

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

[146] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Clostridia class.

[147] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Oscillospriraceae family.

[148] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Faecalibacterium genus.

[149] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Fournierella genus.

[150] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Harryflintia genus.

[151] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Agathobaculum genus.

[152] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Faecalibacterium prausnitzii

(e.g., Faecalibacterium prausnitzii Strain A) bacteria.

[153] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Fournierella massiliensis (e.g., Fournierella massiliensis Strain A) bacteria.

[154] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Harryflintia acetispora (e.g., Harryflintia acetispora Strain A) bacteria. [155] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Agathobaculum sp. (e.g., Agathobaculum sp. Strain A) bacteria.

[156] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are bacteria of a genus selected from the group consisting of Escherichia, Klebsiella, Lactobacillus, Shigella, and Staphylococcus.

[157] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent 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 .

[158] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent 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.

[159] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent 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 (e.g, 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 of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent 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 (e.g., 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.

[160] 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.

[161] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Negativicutes class.

[162] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Veillonellaceae family.

[163] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Selenomonadaceae family.

[164] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Acidaminococcaceae family.

[165] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Sporomusaceae family.

[166] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Megasphaera genus.

[167] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Selenomonas genus.

[168] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Propionospora genus.

[169] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Acidaminococcus genus. [170] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Megasphaera sp. bacteria.

[171] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Selenomonas felix bacteria.

[172] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Acidaminococcus intestini bacteria.

[173] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Propionospora sp. bacteria.

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

[175] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Clostridia class.

[176] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Oscillospriraceae family.

[177] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Faecalibacterium genus.

[178] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Fournierella genus.

[179] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Harryflintia genus.

[180] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Agathobaculum genus.

[181] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Faecalibacterium prausnitzii (e.g., Faecalibacterium prausnitzii Strain A) bacteria.

[182] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Fournierella massiliensis (e.g., Fournierella massiliensis Strain A) bacteria.

[183] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Harryflintia acetispora (e.g., Harryflintia acetispora Strain A) bacteria. [184] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Agathobaculum sp. (e.g., Agathobaculum sp. Strain A) bacteria.

[185] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent 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 (e.g., 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 (e.g., 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).

[186] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the class Bacteroidia [phylum Bacteroidota], In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are bacteria of order Bacteroidales. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the family Porphyromonoadaceae . In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the family Prevotellaceae. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are bacteria of the class Bacteroidia wherein the cell envelope structure of the bacteria is diderm. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are bacteria of the class Bacteroidia that stain Gram negative. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are bacteria of the class Bacteroidia wherein the bacteria is diderm and the bacteria stain Gram negative.

[187] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are bacteria of the class Clostridia [phylum Firmicutes], In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the order Eubacteriales. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the family Oscillispiraceae . In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the family Lachnospiraceae . In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the family Peptostreptococcaceae . In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the family Clostridiales family XIII/ Incertae sedis 41. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the class Clostridia that stain Gram negative. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the class Clostridia that stain Gram positive. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent 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 of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm and the bacteria stain Gram positive.

[188] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the class Negativicutes [phylum Firmicutes], In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the order Veillonellales. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the family Veillonelloceae. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the order Selenomonadales. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are bacteria of the family Selenomonadaceae . In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the family Sporomusaceae. In some embodiments, t the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the class Negativicutes wherein the cell envelope structure of the bacteria is diderm. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent 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.

[189] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the class Synergistia [phylum Synergistota}. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the order Synergistales. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the family Synergistaceae. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the class Synergistia wherein the cell envelope structure of the bacteria is diderm. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the class Synergistia that stain Gram negative. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the class Synergistia wherein the cell envelope structure of the bacteria is diderm and the bacteria stain Gram negative.

[190] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are from one strain of bacteria, e.g., a strain provided herein.

[191] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are from one strain of bacteria (e.g., a strain provided herein) or from more than one strain provided herein.

[192] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Lactococcus lactis cremoris bacteria, e.g., 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 of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Lactococcus bacteria, e.g., Lactococcus lactis cremoris Strain A (ATCC designation number PTA-125368).

[193] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Prevotella bacteria, e.g., 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 of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Prevotella bacteria, e.g., Prevotella Strain B 50329 (NRRL accession number B 50329).

[194] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Prevotella histicola bacteria, e.g., 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 ATCC designation number PTA-126140. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Prevotella histicola bacteria, e.g., Prevotella histicola ATCC designation number PTA-126140.

[195] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Bifidobacterium bacteria, e.g., 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 of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Bifidobacterium bacteria, e.g., Bifidobacterium bacteria deposited as ATCC designation number PTA-125097.

[196] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Veillonella bacteria, e.g., 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 of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Veillonella bacteria, e.g., Veillonella bacteria deposited as ATCC designation number PTA-125691. [197] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent 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.

[198] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent 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 sp. 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.

[199] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Foumierella massiliensis bacteria. In some embodiments, the Foumierella 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 Foumierella massiliensis bacteria deposited as ATCC designation number PTA- 126696. In some embodiments, the Foumierella massiliensis bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Foumierella massiliensis bacteria deposited as ATCC designation number PTA- 126696. In some embodiments, the Foumierella massiliensis bacteria are Foumierella massiliensis bacteria deposited as ATCC designation number PTA- 126696.

[200] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent 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.

[201] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are bacteria that produce metabolites, e.g., the bacteria produce butyrate, iosine, proprionate, or tryptophan metabolites.

[202] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are bacteria that produce butyrate. In some embodiments, the bacteria are from the genus Blautia; Christensella; Copracoccus;

Eubacterium; Lachnosperacea; Megasphaera; or Roseburia.

[203] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are bacteria that produce iosine. In some embodiments, the bacteria are from the genus Bifidobacterium; Lactobacillus; or Olsenella.

[204] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent 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.

[205] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are bacteria that produce tryptophan metabolites. In some embodiments, the bacteria are from the genus Lactobacillus or Peptostreptococcus.

[206] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent 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.

[207] In some embodiments, the bacteria are from the genus Alloiococcus; Bacillus;

Catenibacterium; Corynebacterium; Cupriavidus; Enhydrobacter; Exiguobacterium; Faecalibacterium; Geobacillus,' Methylobacterium; Micrococcus,' Morganelkr, 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.

[208] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the genus Alloiococcus; Bacillus,' Catenibacterium; Corynebacterium; Cupriavidus; Enhydrobacter; Exiguobacterium;

Faecalibacterium,' Geobacillus,' Methylobacterium,' Micrococcus,' Morganelkr, Proteus,' Pseudomonas,' Rhizobium; or Sphingomonas.

[209] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the Cutibacterium genus. In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Cutibacterium avidum bacteria.

[210] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the genus Leuconostoc.

[211] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the genus Lactobacillus.

[212] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are of the genus Akkermanskr, Bacillus,' Blautia; Cupriavidus,' Enhydrobacter, Faecalibacterium, ' Lactobacillus,' Lactococcus,' Micrococcus,' Morganelkr, Propionibacterium; Proteus,' Rhizobium; or Streptococcus.

[213] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Leuconostoc holzapfelii bacteria.

[214] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent 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. [215] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Lactobacillus casei; Lactobacillus plantarum; Lactobacillus paracasei; Lactobacillus plantarum; Lactobacillus rhamnosus; or Lactobacillus sakei bacteria.

[216] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Megasphaera sp. bacteria (e.g., from the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387).

[217] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Megasphaera massiliensis bacteria (e.g., from the strain with accession number NCIMB 42787, NCIMB 43388 or NCIMB 43389).

[218] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained axe Megasphaera massiliensis bacteria (e.g., from the strain with accession number DSM 26228).

[219] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Bacillus amyloliquefaciens bacteria (e.g., from the strain with accession number NCIMB 43088, NCIMB 43087, or NCIMB 43086).

[220] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Parabacteroides distasonis bacteria (e.g., from the strain with accession number NCIMB 42382).

[221] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained axe Megasphaera massiliensis bacteria (e.g., from the strain with accession number NCIMB 43388 or NCIMB 43389), or a derivative thereof. See, e.g., 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 (e.g., 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 (e.g., 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. [222] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Megasphaera massiliensis bacteria strain deposited under accession number NCIMB 42787, or a derivative thereof. See, e.g., 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 (e.g., 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 (e.g., 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.

[223] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained axe Megasphaera sp. bacteria from the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387, or a derivative thereof. See, e.g., 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 (e.g., 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 (e.g., 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.

[224] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Parabacteroides distasonis bacteria deposited under accession number NCIMB 42382, or a derivative thereof. See, e.g., 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 (e.g., 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 (e.g., 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.

[225] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Megasphaera massiliensis bacteria deposited under accession number DSM 26228, or a derivative thereof. See, e.g., 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 (e.g., 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 (e.g., 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.

[226] In some embodiments, the bacteria of the pharmaceutical agent or from which the mEVs of the pharmaceutical agent are obtained are Bacillus amyloliquefaciens bacteria (e.g., from the strain with accession number NCIMB 43088, NCIMB 43087, or NCIMB 43086, or a derivative thereof. See, e.g., WO 2019/236806. In some embodiments, the Bacillus amyloliquefaciens 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 (e.g., 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 (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of Bacillus amyloliquefaciens bacteria from the strain with accession number NCIMB 43088, NCIMB 43087, or NCIMB 43086. In some embodiments, the Bacillus amyloliquefaciens bacteria is the strain with accession number NCIMB 43088, NCIMB 43087, or NCIMB 43086. In some embodiments, the Bacillus amyloliquefaciens bacteria is the strain with accession number NCIMB 43088.

Table 1: Bacteria by Class

48

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

Table 2: Exemplary Bacterial Strains

Table 3: Exemplary Bacterial Strains Table 4. Exemplary Bacterial Strains

Modified Bacteria and mEVs

[227] In certain aspects, the bacteria and/or mEVs (such as smEVs and/or pmEVs) described herein are modified such that they comprise, are linked to, and/or are bound by a therapeutic moiety.

[228] 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 (e.g., 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 (e.g., 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 mEV (e.g., 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 receptor-binding fragment thereof. In some embodiments, co-administration of the cancer-specific moiety with the pharmaceutical agent (either in combination or in separate administrations) increases the targeting of the pharmaceutical agent to the cancer cells.

[229] In some embodiments, the bacteria and/or mEVs described herein can be modified such that they comprise, are linked to, and/or are bound by a magnetic and/or paramagnetic moiety (e.g., 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 a bacteria- or a mEV-binding moiety that binds to the bacteria or mEV. In some embodiments, the bacteria- or mEV-binding moiety is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP. In some embodiments the bacteria- or mEV-binding moiety has binding specificity for the bacteria or mEV (e.g., by having binding specificity for a bacterial antigen). In some embodiments, the bacteria- or mEV-binding moiety comprises an antibody or antigen binding fragment thereof. In some embodiments, the bacteria- or mEV-binding moiety comprises a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the bacteria- or mEV- binding moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof. In some embodiments, co-administration of the magnetic and/or paramagnetic moiety with the bacteria or mEVs (either together or in separate administrations) can be used to increase the targeting of the mEVs (e.g., to cancer cells and/or a part of a subject where cancer cells are present. Production of Processed Microbial Extracellular Vesicles (pmEVs)

[230] In certain aspects, the pmEVs described herein can be prepared using any method known in the art.

[231] In some embodiments, the pmEVs are prepared without a pmEV purification step. For example, in some embodiments, bacteria from which the pmEVs described herein are released are killed using a method that leaves the bacterial pmEVs intact, and the resulting bacterial components, including the pmEVs, are used in the methods and compositions described herein. In some embodiments, the bacteria are killed using an antibiotic (e.g., using an antibiotic described herein). In some embodiments, the bacteria are killed using UV irradiation.

[232] In some embodiments, the pmEVs described herein are purified from one or more other bacterial components. Methods for purifying pmEVs from bacteria (and optionally, other bacterial components) are known in the art. In some embodiments, pmEVs are prepared from bacterial cultures using methods described in Thein, et al. (J. Proteome Res. 9(12):6135-6147 (2010)) or Sandrini, et al. (Bio-protocol 4(21): el287 (2014)), 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 (e.g., at 10,000- 15,000 x g for 10- 15 min at room temperature or 4°C). In some embodiments, the supernatants are discarded and cell pellets are frozen at -80°C. In some embodiments, cell pellets are thawed on ice and resuspended in 100 mM Tris-HCl, pH 7.5 supplemented with 1 mg/mL DNase I. In some embodiments, cells are lysed using an Emulsiflex C-3 (Avestin, Inc.) under conditions recommended by the manufacturer. In some embodiments, debris and unlysed cells are pelleted by centrifugation at 10,000 x g for 15 min at 4°C. In some embodiments, supernatants are then centrifuged at 120,000 x g for 1 hour at 4°C. In some embodiments, pellets are resuspended in ice-cold 100 mM sodium carbonate, pH 11, incubated with agitation for 1 hr at 4°C, and then centrifuged at 120,000 x g for 1 hour at 4°C. In some embodiments, pellets are resuspended in 100 mM Tris-HCl, pH 7.5, re-centrifuged at 120,000 x g for 20 min at 4°C, and then resuspended in 0.1 M Tris-HCl, pH 7.5 or in PBS. In some embodiments, samples are stored at -20°C.

[233] In certain aspects, pmEVs are obtained by methods adapted from Sandrini et al, 2014. In some embodiments, bacterial cultures are centrifuged at 10,000-15,500 x g for 10-15 min at room temp or at 4°C. In some embodiments, cell pellets are frozen at -80°C and supernatants are discarded. In some embodiments, cell pellets are thawed on ice and resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA supplemented with 0.1 mg/mL lysozyme. In some embodiments, samples are incubated with mixing at room temp or at 37°C for 30 min. In some embodiments, samples are re-frozen at -80°C and thawed again on ice. In some embodiments, DNase I is added to a final concentration of 1.6 mg/mL and MgC12 to a final concentration of 100 mM. In some embodiments, samples are sonicated using a QSonica Q500 sonicator with 7 cycles of 30 sec on and 30 sec off. In some embodiments, debris and unlysed cells are pelleted by centrifugation at 10,000 x g for 15 min. at 4°C. In some embodiments, supernatants are then centrifuged at 110,000 x g for 15 min at 4°C. In some embodiments, pellets are resuspended in 10 mM Tris-HCl, pH 8.0, 2% Triton X-100 and incubated 30-60 min with mixing at room temperature. In some embodiments, samples are centrifuged at 110,000 x g for 15 min at 4°C. In some embodiments, pellets are resuspended in PBS and stored at -20°C.

[234] In certain aspects, a method of forming (e.g., preparing) isolated bacterial pmEVs, described herein, comprises the steps of: (a) centrifuging a bacterial culture, thereby forming a first pellet and a first supernatant, wherein the first pellet comprises cells; (b) discarding the first supernatant; (c) resuspending the first pellet in a solution; (d) lysing the cells; (e) centrifuging the lysed cells, thereby forming a second pellet and a second supernatant; (f) discarding the second pellet and centrifuging the second supernatant, thereby forming a third pellet and a third supernatant; (g) discarding the third supernatant and resuspending the third pellet in a second solution, thereby forming the isolated bacterial pmEVs.

[235] In some embodiments, the method further comprises the steps of: (h) centrifuging the solution of step (g), thereby forming a fourth pellet and a fourth supernatant; (i) discarding the fourth supernatant and resuspending the fourth pellet in a third solution. In some embodiments, the method further comprises the steps of: (j) centrifuging the solution of step (i), thereby forming a fifth pellet and a fifth supernatant; and (k) discarding the fifth supernatant and resuspending the fifth pellet in a fourth solution.

[236] In some embodiments, the centrifugation of step (a) is at 10,000 x g. In some embodiments the centrifugation of step (a) is for 10-15 minutes. In some embodiments, the centrifugation of step (a) is at 4 °C or room temperature. In some embodiments, step (b) further comprises freezing the first pellet at -80 °C. In some embodiments, the solution in step (c) is 100mM Tris-HCl, pH 7.5 supplemented with Img/ml DNasel. In some embodiments, the solution in step (c) is lOmM Tris-HCl, pH 8.0, ImMEDTA, supplemented with 0.1 mg/ml lysozyme. In some embodiments, step (c) further comprises incubating for 30 minutes at 37 °C or room temperature. In some embodiments, step (c) further comprises freezing the first pellet at - 80 °C. In some embodiments, step (c) further comprises adding DNase I to a final concentration of 1.6mg/ml. In some embodiments, step (c) further comprises adding MgChto a final concentration of lOOmM. In some embodiments, the cells are lysed in step (d) via homogenization. In some embodiments, the cells are lysed in step (d) via emulsiflex C3. In some embodiments, the cells are lysed in step (d) via sonication. In some embodiments, the cells are sonicated in 7 cycles, wherein each cycle comprises 30 seconds of sonication and 30 seconds without sonication. In some embodiments, the centrifugation of step (e) is at 10,000 x g. In some embodiments, the centrifugation of step (e) is for 15 minutes. In some embodiments, the centrifugation of step (e) is at 4 °C or room temperature.

[237] In some embodiments, the centrifugation of step (f) is at 120,000 x g. In some embodiments, the centrifugation of step (f) is at 110,000 x g. In some embodiments, the centrifugation of step (f) is for 1 hour. In some embodiments, the centrifugation of step (f) is for 15 minutes. In some embodiments, the centrifugation of step (f) is at 4 °C or room temperature. In some embodiments, the second solution in step (g) is 100 mM sodium carbonate, pH 11. In some embodiments, the second solution in step (g) is 10mM Tris-HCl pH 8.0, 2% triton X-100. In some embodiments, step (g) further comprises incubating the solution for 1 hour at 4 °C. In some embodiments, step (g) further comprises incubating the solution for 30-60 minutes at room temperature. In some embodiments, the centrifugation of step (h) is at 120,000 x g. In some embodiments, the centrifugation of step (h) is at 110,000 x g. In some embodiments, the centrifugation of step (h) is for 1 hour. In some embodiments, the centrifugation of step (h) is for 15 minutes. In some embodiments, the centrifugation of step (h) is at 4 °C or room temperature. In some embodiments, the third solution in step (i) is 100mM Tris-HCl, pH 7.5. In some embodiments, the third solution in step (i) is PBS. In some embodiments, the centrifugation of step (j) is at 120,000 x g. In some embodiments, the centrifugation of step (j) is for 20 minutes. In some embodiments, the centrifugation of step (j) is at 4 °C or room temperature. In some embodiments, the fourth solution in step (k) is 100mM Tris-HCl, pH 7.5 or PBS.

[238] pmEVs obtained by methods provided herein may be further purified by size based column chromatography, by affinity chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 35% Optiprep in PBS. In some embodiments, if filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C.

[239] In some embodiments, to confirm sterility and isolation of the pmEV preparations, pmEVs 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 pmEVs may be DNase or proteinase K treated.

[240] In some embodiments, the sterility of the pmEV preparations can be confirmed by plating a portion of the pmEVs onto agar medium used for standard culture of the bacteria used in the generation of the pmEVs and incubating using standard conditions.

[241] In some embodiments select pmEVs are isolated and enriched by chromatography and binding surface moieties on pmEVs. In other embodiments, select pmEVs 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.

[242] The pmEVs can be analyzed, e.g., as described in Jeppesen, et al. Cell 177:428 (2019).

[243] In some embodiments, pmEVs are lyophilized.

[244] In some embodiments, pmEVs are gamma irradiated (e.g., at 17.5 or 25 kGy).

[245] In some embodiments, pmEVs are UV irradiated.

[246] In some embodiments, pmEVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours). [247] In some embodiments, pmEVs are acid treated.

[248] In some embodiments, pmEVs are oxygen sparged (e.g., at 0.1 wm for two hours).

[249] The phase of growth can affect the amount or properties of bacteria. In the methods of pmEV preparation provided herein, pmEVs can be isolated, e.g., 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.

Production of Secreted Microbial Extracellular Vesicles (smEVs)

[250] In certain aspects, the smEVs described herein can be prepared using any method known in the art.

[251] In some embodiments, the smEVs are prepared without a smEV purification step. For example, in some embodiments, bacteria described herein are killed using a method that leaves the smEVs intact and the resulting bacterial components, including the smEVs, are used in the methods and compositions described herein. In some embodiments, the bacteria are killed using an antibiotic (e.g., using an antibiotic described herein). In some embodiments, the bacteria are killed using UV irradiation. In some embodiments, the bacteria are heat-killed.

[252] In some embodiments, the smEVs described herein are purified from one or more other bacterial components. Methods for purifying smEVs from bacteria are known in the art. In some embodiments, smEVs 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): e0134353 (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 (e.g., 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 (e.g., 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 smEVs (e.g., 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 smEVs are further purified by resuspending the resulting smEV pellets (e.g., in PBS), and applying the resuspended smEVs to an Optiprep (iodixanol) gradient or gradient (e.g., a 30-60% discontinuous gradient, a 0-45% discontinuous gradient), followed by centrifugation (e.g., at 200,000 x g for 4-20 hours at 4°C). smEV bands can be collected, diluted with PBS, and centrifuged to pellet the smEVs (e.g., at 150,000 x g for 3 hours at 4°C, at 200,000 x g for 1 hour at 4°C). The purified smEVs can be stored, for example, at -80°C or - 20°C until use. In some embodiments, the smEVs are further purified by treatment with DNase and/or proteinase K.

[253] 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 smEVs 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 bacteria smEVs 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.

[254] Alternatively, smEVs 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 (e.g., XCell ATF from Repligen). The ATF system retains intact cells (>0.22 um) in the bioreactor, and allows smaller components (e.g., smEVs, free proteins) to pass through a filter for collection. For example, the system may be configured so that the <0.22 um filtrate is then passed through a second filter of 100 kDa, allowing species such as smEVs between 0.22 um 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. smEVs collected by this method may be further purified and/or concentrated by ultracentrifugation or filtration as described above for filtered supernatants. [255] smEVs obtained by methods provided herein may be further purified by sizebased 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, e.g., 4-24 hours at 4°C.

[256] In some embodiments, to confirm sterility and isolation of the smEV preparations, smEVs 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 smEVs may be DNase or proteinase K treated.

[257] In some embodiments, for preparation of smEVs used for in vivo injections, purified smEVs are processed as described previously (G. Norheim, et al. PLoS ONE. 10(9): e0134353 (2015)). Briefly, after sucrose gradient centrifugation, bands containing smEVs 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 smEVs used for in vivo injections, smEVs in PBS are sterile- filter ed to < 0.22 um.

[258] In some embodiments, to make samples compatible with further testing (e.g., 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 (e.g., Amicon Ultra columns), dialysis, or ultracentrifugation (200,000 x g, ≥ 3 hours, 4°C) and resuspension.

[259] In some embodiments, the sterility of the smEV preparations can be confirmed by plating a portion of the smEVs onto agar medium used for standard culture of the bacteria used in the generation of the smEVs and incubating using standard conditions.

[260] In some embodiments, select smEVs are isolated and enriched by chromatography and binding surface moieties on smEVs. In other embodiments, select smEVs 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.

[261] The smEVs can be analyzed, e.g., as described in Jeppesen, et al. Cell 177:428 (2019).

[262] In some embodiments, smEVs are lyophilized.

[263] In some embodiments, smEVs are gamma irradiated (e.g., at 17.5 or 25 kGy).

[264] In some embodiments, smEVs are UV irradiated.

[265] In some embodiments, smEVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).

[266] In some embodiments, smEVs s are acid treated.

[267] In some embodiments, smEVs are oxygen sparged (e.g., at 0.1 wm for two hours).

[268] The phase of growth can affect the amount or properties of bacteria and/or smEVs produced by bacteria. For example, in the methods of smEV preparation provided herein, smEVs can be isolated, e.g., 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.

[269] The growth environment (e.g., culture conditions) can affect the amount of smEVs produced by bacteria. For example, the yield of smEVs can be increased by an smEV inducer, as provided in Table 5.

Table 5: Culture Techniques to Increase smEV Production

[270] In the methods of smEVs preparation provided herein, the method can optionally include exposing a culture of bacteria to a smEV inducer prior to isolating smEVs from the bacterial culture. The culture of bacteria can be exposed to a smEV 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

[271] In certain aspects, provided herein are methods of making engineered bacteria for the production of the bacteria and/or mEVs (such as smEVs and/or pmEVs) 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 bacteria and/or mEVs (such as smEVs and/or pmEVs) (e.g., either alone or in combination with another therapeutic agent), to reduce toxicity and/or to improve bacterial and/or mEV (such as smEV and/or pmEV) manufacturing (e.g., 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.

[272] 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 (e.g., by exposure to chemical mutagens and/or UV radiation) or exposing them to a therapeutic agent (e.g., antibiotic) followed by an assay to detect bacteria having the desired phenotype (e.g., an in vivo assay, an ex vivo assay, or an in vitro assay).

Expression of a Transcription Product: Potency Assay

[273] The present disclosure includes, among other things, assays useful for determining the potency of a drug product or drug substance, based at least in part on measuring expression of a transcription product in cells contacted with the drug product or drug substance. In various embodiments, the drug product or drug substance can be bacteria and/or microbial extracellular vesicles (e.g., Prevotella histicola bacteria and/or microbial extracellular vesicles). Without wishing to be bound by any particular scientific theory, expression of the transcription product by cells contacted with the drug product or drug substance (e.g., with bacteria and/or microbial extracellular vesicles) is representative of biological activity thereof. Without wishing to be bound by any particular scientific theory, expression of the transcription product by cells contacted with the drug product or drug substance (e.g., with bacteria and/or microbial extracellular vesicles) is relevant to and/or indicative of a desired physiological effect to be achieved by delivery and/or administration of the drug product or drug substance, e.g., to a subject to benefit therefrom.

[274] In certain aspects, provided herein are methods for assaying a drug product or drug substance (e.g., a drug product or drug substance that is or includes bacteria and/or microbial extracellular vesicles) that includes contacting (e.g., incubating) cells (e.g., THP-1 cells) with the drug product or drug substance and detecting expression of the transcription product by the cells, wherein the drug product or drug substance comprises a strain of bacteria (e.g., Prevotella histicola strain bacteria) and/or microbial extracellular vesicles (mEVs) from a strain of bacteria (e.g, Prevotella histicola strain microbial extracellular vesicles (mEVs)). In some embodiments, the bacterial strain is a strain of Prevotella histicola having 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 Prevotella histicola bacteria are of Prevotella Strain B (NRRL accession number B 50329). In some embodiments, the bacterial strain is a strain of Prevotella histicola having at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of Prevotella histicola ATCC designation number PTA-126140. In some embodiments, the Prevotella histicola bacteria are of Prevotella histicola ATCC designation number PTA-126140.

[275] The drug product or drug substance may comprise bacteria and/or microbial extracellular vesicles (mEVs). In some embodiments, the drug product or drug substance comprises a strain of bacteria (e.g, Prevotella histicola, Veillonella parvula, Harryflintia acetispora, Foumierella massiliensis, Megasphaera Sp., Selenomonas felix, Acidaminococcus intestine, and/or Propionospora sp. bacteria) and/or microbial extracellular vesicles (mEVs) from a strain of bacteria (e.g, Prevotella histicola, Veillonella parvula, Harryflintia acetispora, Fournierella massiliensis, Megasphaera Sp., Selenomonas felix, Acidaminococcus intestine, and/or Propionospora sp. microbial extracellular vesicles (mEVs)).

[276] In certain aspects, provided herein are methods for assaying a drug product or drug substance comprising contacting a cell of a mammalian immune cell line (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) with the drug product or drug substance. The cell may comprise a nucleic acid sequence operatively linked to a regulatory sequence, wherein the regulatory sequence is responsive to NF-κB or is an NF-κB promoter. The method may further comprise detecting the level of expression of a transcription product encoded by the nucleic acid sequence by the cell.

[277] In various embodiments, a regulatory sequence that is responsive to NF-κB is or includes an NF-κB response element. An NF-κB response element, as used herein, can refer to a sequence that is bound by NF-κB (e.g., a sequence that is in a promoter or other regulatory sequence of a coding sequence, e.g., where expression of the coding sequences is regulated and/or controlled by NF-κB), and/or to an NF-κB response element sequence as disclosed herein or known in the art. The consensus sequence for NF-κB response element is known in art (for example Mulero, M.C., et al. Nucleic Acids Res. 2019 Nov 4;47(19):9967-9989). In various embodiments, an NF-κB response element can have the sequence TGGGGACTTTCCGC (SEQ ID NO: 1). In various embodiments, an NF-κB response element can have a sequence according to GGGRNWYYCC (where R is a purine, Y is a pyrimidine, W is an adenine or thymine and N is an unspecified base (SEQ ID NO: 2), an example of which is the NF-κB response element GGGAATTCCC (SEQ ID NO: 3)). In various embodiments, an NF-κB response element can have a sequence that is a portion of any one of SEQ ID NOs: 1-3, e.g., that comprises at least 6, 7, 8, 9, 10, 11, 12, 13, or 14 contiguous nucleotides of any of SEQ ID NOs: 1-3. In various embodiments, an NF-κB response element can have a sequence that differs from SEQ ID NO: 1, 2 or 3 in that it includes at least 1, 2, 3, 4, or 5 nucleotide sequence differences, where the difference can be, e.g., an insertion, deletion or substitution of a nucleotide to or of SEQ ID NO: 1, 2 or 3. In various embodiments, an NF-κB response element can have a sequence that is identical to a portion of any of SEQ ID NOs: 1-3, e.g., that comprises at least 6, 7, 8, 9, 10, 11, 12, 13, or 14 contiguous nucleotides of any of SEQ ID NOs: 1-3, except in that the portion includes at least 1, 2, 3, 4, or 5 nucleotide sequence differences from a corresponding portion of any of SEQ ID NOs: 1-3. [278] In various embodiments in which a regulatory sequence includes a plurality of NF-κB response elements, the NF-κB response elements can be positioned contiguously or such that one or more of the plurality of NF-κB response elements are separated by one or more intervening nucleotides, the number of intervening nucleotides between any two response elements in each case independently selected from, e.g., at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides. In various embodiments in which a regulatory sequence includes one or more NF-κB response elements and a promoter, the NF-κB response element nearest to the promoter can be adjacent to the promoter or separated from the promoter by one or more intervening nucleotides (e.g., by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides). In various embodiments, the regulatory sequence includes 1-30 NF-κB response elements. In various embodiments, the regulatory sequence includes 1, 2, 3, 4, 5, 6, 7,

8, 9, or 10 NF-κB response elements. In various embodiments, the regulatory sequence includes four NF-κB response elements.

[279] In various embodiments, an NF-κB promoter can refer to a sequence that has at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with a sequence that is positioned near to and upstream of the transcription start site of an NF-κB coding sequence (e.g., in the genome of a wild type mammalian cell). In various embodiments, a sequence that is is positioned near to and upstream of the transcription start site of an NF-κB coding sequence extends from a position that is immediately adjacent to or not more than 500 basepairs upstream (5’) from the NF-κB transcription start site to a further upstream position. In various embodiments, a sequence that is is positioned near to and upstream of the transcription start site of an NF-κB coding sequence has a length of, e.g, about 100-3000 nucleotides, e.g., about 100-1000 nucleotides.

[280] Bacteria and/or microbial extracellular vesicles assayed for potency using methods and compositions disclosed herein can be, or be present in, a drug product or drug substance. Bacteria and/or microbial extracellular vesicles assayed for potency using methods and compositions disclosed herein can be any bacteria and/or microbial extracellular vesicles disclosed herein, and/or any bacteria and/or microbial extracellular vesicles produced according to methods disclosed herein. In various embodiments, bacteria and/or microbial extracellular vesicles assayed for potency using methods and compositions disclosed herein can be Prevotella histicola bacteria and/or microbial extracellular vesicles. [281] In various embodiments, a potency assay of the present disclosure includes contacting cells with a drug product or drug substance. In various embodiments, the cells are or include a population of cells, such as a culture of cells. In various embodiments, the cells are or include mammalian cells.

[282] In various embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with one or more cells that are or include white blood cells and/or cells derived (e.g., differentiated) therefrom. In various embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with one or more cells that are or include immune cells and/or cells derived (e.g., differentiated) therefrom. In some embodiments, cells derived from a lineage, or from cells, comprise an immortalized cell line.

[283] In various embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with one or more cells that are or include cells of a monocyte lineage and/or cells derived (e.g., differentiated) therefrom (such as monocytes, macrophages, or dendritic cells). In various embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with one or more cells that are or include cells differentiated from a monocyte lineage, such as monocytes, macrophages, or dendritic cells. In some embodiments, cells derived from a lineage, or from cells, comprise an immortalized cell line. Monocyte- derived macrophages can be polarized into Ml, M2a, M2b, and M2c cells.

[284] In various embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with one or more cells that are or include immortalized cells and/or cells derived (e.g., differentiated) therefrom. In various embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with one or more cells that are or include cancer or cancer-derived cells, and/or cells derived (e.g., differentiated) therefrom. In various embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with one or more cells that are or include cells that are, or are derived from, cells of a human leukemia, and/or cells derived (e.g., differentiated) therefrom.

[285] In various embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with one or more cells that are or include immortalized monocytes and/or cells derived (e.g., differentiated) therefrom. In various embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with one or more cells that are or include cancer or cancer- derived monocytes and/or cells derived (e.g., differentiated) therefrom. In various embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with one or more cells that are or include monocytes that are, or are derived from, cells of a human leukemia or lymphoma, and/or cells derived (e.g., differentiated) therefrom.

[286] In various embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with one or more cells that are or include myeloid cells, cells of myeloid lineage, and/or cells derived from myeloid cells, and/or cells derived (e.g., differentiated) therefrom. In various embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with one or more cells that are or include T cells and/or cells derived (e.g., differentiated) therefrom. In various embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with one or more cells that are or include Jurkat cells and/or cells derived (e.g., differentiated) therefrom. In various embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with one or more cells that are or include B cells and/or cells derived (e.g., differentiated) therefrom. In various embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with one or more cells that are or include Raji cells and/or cells derived (e.g., differentiated) therefrom. In any of the various embodiments of a potency assay of the present disclosure, cells with which a drug product or drug substance are contacted can be or include human cells.

[287] In various embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with one or more cells that are or include U937 cells and/or cells derived (e.g., differentiated) therefrom. In various embodiments, U937 is a known myeloid lineage cancer cell line that exhibits monocyte morphology and was derived in the 1970’s from malignant cells. Without wishing to be bound by any particular scientific theory, U937 cells have been described as a pro-monocytic, human myeloid leukaemia cell line. U937 cells can differentiate into macrophages or dendritic cells (antigen-presenting cells). U937 cells can differentiate along a monocyte/macrophage lineage when contacted with TPA. U937 cells can develop into macrophage-like cells when contacted with phorbol 12-myristate 13 -acetate (PMA). In various embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with one or more cells that are or include K562 cells and/or cells derived (e.g., differentiated) therefrom. In various embodiments, K562 cells are a known immortalized line of lymphoblast cells isolated from the bone marrow of a 53 -year-old chronic myelogenous leukemia patient. Without wishing to be bound by any particular scientific theory, K562 blasts are multipotential, hematopoietic malignant cells that differentiate into recognizable progenitors of the erythrocyte, granulocyte and monocytic series.

[288] In various embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with one or more cells that are or include THP-1 cells and/or cells derived (e.g., differentiated) therefrom. In certain preferred embodiments, the cells are or include THP-1 cells. In various embodiments, THP-1 is a known human monocytic leukaemia cell line established in 1980, derived from the blood of a patient with acute monocytic leukaemia. Without wishing to be bound by any particular scientific theory, THP-1 cells have been characterized as having morphology and differentiation properties characteristic of primary monocytes and macrophages. THP-1 cells show a large, round single-cell morphology and express distinct monocytic markers. THP-1 cells start to adhere to culture plates and differentiate into macrophages after exposure to phorbol-12-myristate- 13 -acetate (PMA, also known as TPA,12-O-tetradecanoylphorbol-13-acetate). THP-1 cells can also be differentiated by exposure to Phorbol 12, 13 -dibutyrate (PDBu). Monocyte-derived macrophages can be polarized into Ml, M2a, M2b, and M2c cells.

[289] Various cell types are known to those of skill in the art have characteristics similar to THP-1 cells. These include U937 cells, K562 cells, ML-2 cells, HL-60 cells, and Mono Mac 6 cells. Although U937 and THP-1 are derived from separate origins and maturation stage, at least in that U937 cells are of tissue origin while THP-1 cells originate from a blood leukaemia, the present disclosure includes that both are suitable for use in assays disclosed herein. The present disclosure further includes that THP-1 and/or U937 are representative of classes of cell types useful in the present disclosure, which classes include without limitation mammalian cells, human cells, immune cells, myeloid cells, myeloid lineage cells, cells exhibiting monocyte morphology, monocytes, immortalized cells, cancer cells, cancer-derived cells, leukemia-derived monocytes, cancer- derived monocytes, and/or leukemia-derived monocytes as well as other types and clases of cells disclosed herein, and/or cells derived (e.g., differentiated) therefrom.

[290] In some embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with differentiated cells (e.g., any of the cell types disclosed herein, and/or a cell type disclosed herein differentiated as disclosed herein). In various embodiments, diffentiation can be induced by conditions such as culture conditions, including without limitation contacting cells with an agent that induces differentiation. In various embodiments, steps to cause differentiation of cells are undertaken prior to contacting the cells with a drug product or drug substance. To provide a non-limiting example, in some embodiments, a potency assay of the present disclosure includes contacting a drug product or drug substance with cells differentiated to a monocytic phenotype (e.g., THP-1 can be treated with PMA to differentiate the cells to a macrophage phenotype).

[291] In various embodiments, cells of a potency assay disclosed herein can be engineered to encode and/or express a detectable marker. For the avoidance of doubt, a cell line, cell type, or category or class of cells disclosed herein can be referred to by the same terminology and recognized as being of the same line, type, category, class or other other grouping whether such detectable marker is present or absent.

[292] In various embodiments of the present disclosure, cells of a potency assay disclosed herein can be engineered to encode and/or express a detectable marker that produces a signal that correspondonds to expression of the transcription product. In various embodiments, cells of a potency assay disclosed herein can be engineered to encode and/or express a detectable expression product from a coding nucleic acid sequence that is operably linked with a regulatory sequence, such as a regulatory sequence that is responsive to NF-κB or is an NF-κB promoter. In various embodiments, cells of a potency assay disclosed herein can be engineered to encode and/or express a detectable expression product from a coding nucleic acid sequence that is operably linked with a regulatory sequence that is or includes at least one NF-κB response element. In various embodiments, a coding sequence that encodes a detectable expression product can be operably linked with a regulatory sequence that is or includes at least one (e.g., one, two, three, four, or five) NF-κB response elements. In various embodiments, a regulatory sequence can include a promoter such as a minimal TATA promoter. In some embodiments, a regulatory sequence can be or include at least one (e.g., one, two, three, four, or five) NF-κB response elements and a promoter such as a minimal TATA promoter, e.g., such that the regulatory sequence includes the at least one (e.g., four) NF-κB response elements positioned upstream of a promoter such as a minimal TATA promoter. In various embodiments, cells of a potency assay disclosed herein can be engineered to encode and/or express a detectable expression product from a coding nucleic acid sequence that is operably linked with an NF-κB promoter.

[293] The present disclosure includes that a promoter can be adjancent to, but is not necessarily adjacent to, a transcription start site. In various embodiments, a promoter can be positioned upstream of a transcription start site, e.g., at least or about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 500, 1,000 or more base pairs upstream of a transcription start site. The present disclosure includes that an NF-κB response element can be adjancent to, but is not necessarily adjacent to, a transcription start site and/or promoter. In various embodiments, an NF-κB response element can be positioned upstream or downstream of a transcription start site and/or promoter, e.g., at least or about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 500, 1,000 or more base pairs upstream or downstream of a transcription start site. In various embodiments including two or more NF-κB response elements (e.g., copies of an NF-κB response element), the present disclosure includes that the sequences of any two NF-κB response elements of the two or more NF-κB response elements can be adjancent to each other or non- adjacent. In various embodiments, a first NF-κB response element can be upstream or downstream of a second NF-κB response element, e.g., at least or about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 500, 1,000 or more base pairs upstream or downstream of the second NF-κB response element. In various embodiments, one or more NF-κB response elements and/or a promoter are operatively linked with a coding sequence and/or can be readily determined as being operatively linked with a coding sequence by methods familiar to those of skill in the art (e.g., by methods including measurement of expression of an associated and/or operatively linked coding sequence).

[294] In various embodiments, a detectable expression product can be a detectable expression product such as a fluorescent label, luminescent label, enzyme, or detectable tag. In various embodiments, a fluorescent label can be, e.g., yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), modified red fluorescent protein (mRFP), mCherry, red fluorescent protein tdimer2 (RFP tdimer2), and/or HcRED fluorescent protein. In various embodiments, a luminescent label can be, e.g., luciferase (e.g., firefly luciferase, Renilla luciferase, or NanoLuc Luciferase). In various embodiments, a detectable expression product that is an enzyme can be, e.g., horseradish peroxidase, alkaline phosphatase, or P-galactosidase. In various embodiments, a detectable expression product can be a tag or ligand such as avidin, streptavidin, a FLAG tag, or a His tag.

[295] In some embodiments, mammalian cells comprise a sequence encoding a fluorescent transcription product operably linked with more than one NF-κB response elements (e.g., more than one copy of an NF-κB response element, such as four copies of an NF-κB response element) positioned upstream of promoter such as a minimal TATA promoter. In some embodiments, mammalian cells comprise a sequence encoding a fluorescent transcription product operably linked with more than one NF-κB response elements (e.g., more than one copy of an NF-κB response element, such as four copies of an NF-κB response element) positioned upstream of a promoter such as a minimal TATA promoter. In some embodiments, the fluorescent transcription product is a green fluorescent polypeptide, mCherry polypeptide, yellow fluorescent polypeptide, blue fluorescent polypeptide, cyan fluorescent polypeptide, or red fluorescent polypeptide. In some embodiments, the expression of the transcription product is determined by a fluorescence assay. In some embodiments, mammalian cells comprise a luciferase coding sequence operably linked with more than one NF-κB response elements (e.g., more than one copy of an NF-κB response element, such as four copies of an NF-κB response element) positioned upstream of promoter such as a minimal TATA promoter. In some embodiments, the luciferase is a firefly luciferase and/or expression is determined by a luciferase assay.

[296] In some embodiments, mammalian cells comprise a sequence encoding a fluorescent transcription product operably linked with an NF-κB remoter. In some embodiments, the fluorescent transcription product operably linked with an NF-κB remoter is a green fluorescent polypeptide, mCherry polypeptide, yellow fluorescent polypeptide, blue fluorescent polypeptide, cyan fluorescent polypeptide, or red fluorescent polypeptide. In some embodiments, the expression of the transcription product operably linked with an NF-κB remoter is determined by a fluorescence assay. In someembodiments, mammalian cells comprise a luciferase coding sequence operably linked with an NF-κB remoter. In some embodiments, the luciferase is a firefly luciferase and/or expression is determined by a luciferase assay.

[297] In various embodiments, a potency assay of the present disclosure includes contacting (e.g., incubating) a drug product or drug substance with cells (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) for a time period that is sufficient for the drug product or drug substance, and/or for a reference drug product or drug substance, to induce detectable expression of the transcription product by the cells. In various embodiments, a drug product or drug substance is incubated with cells for a period of at least or about 1, at least or about 2, at least or about 3, at least or about 4, at least or about 5, at least or about 6, at least or about 8, at least or about 10, at least or about 12, at least or about 14, at least or about 16, at least or about 18, at least or about 20, at least or about 22, at least or about 24, at least or about 36, at least or about 48 hours, or at least or about 72 hours. In various embodiments, a drug product or drug substance is incubated with cells for a period having a lower boundary of at least or about 1 , at least or about 2, at least or about 3, at least or about 4, at least or about 5, at least or about 6, at least or about 8, at least or about 10, at least or about 12, at least or about 14, at least or about 16, at least or about 18, at least or about 20, at least or about 22, or at least or about 24 hours, and an upper boundary of at least or about 12, at least or about 14, at least or about 16, at least or about 18, at least or about 20, at least or about 22, at least or about 24, at least or about 36, at least or about 48 hours, or at least or about 72 hours. In various embodiments, a drug product or drug substance is incubated with cells for a period of 1 to 12 hours, 6 to 12 hours, 12 to 24 hours, 24 to 48 hours, or 48 to 72 hours.

[298] In various embodiments, a potency assay of the present disclosure includes, subsequent to contacting (e.g., incubating) a drug product or drug substance with cells, detecting and/or measuring expression of the transcription product by the cells. A variety of approaches to directly or indirectly measuring expression of a protein are known to those of skill in the art. Examples include, without limitation, measuring, detecting, and/or sequencing mRNA molecules from the cells (e.g., mRNA molecules encoding NF-κB or encoding a detectable protein) or protein from the cells. The transcription product mRNA can be measured or detected, e.g., by Northern blot, RT-PCR (reverse transcription-polymerase chain reaction), RNAseq, or microarray. The transcription product protein can be measured or detected, e.g., by a Western Blot, immunoassay, enzyme-linked immunosorbent assay (ELISA), meso scale discovery (MSD) assay, radioimmunoassay, or immunocytostaining. In some embodiments, expression of the transcription product is detected by immunohistochemistry. In some embodiments, expression of the transcription product is detected by immunostaining with a labeled antibody that binds to the expression of the transcription product.

[299] In some embodiments, antibodies specific for the transcription product or NF-κB are immobilized on a surface (e.g., are reactive elements on an array, such as a microarray, or are on another surface, such as used for surface plasmon resonance (SPR)-based technology, such as Biacore), and the transcription product or NF-κB in a sample are detected by virtue of their ability to bind specifically to the antibodies. Alternatively, the transcription product or NF-κB in the sample can be immobilized on a surface, and detected by virtue of their ability to bind specifically to the antibodies. Methods of preparing the surfaces and performing the analyses, including conditions effective for specific binding, are conventional and well-known in the art.

[300] Any of a variety of antibodies can be used in methods of the detection. Such antibodies include, for example, polyclonal, monoclonal (mAbs), recombinant, humanized or partially humanized, single chain, Fab, and fragments thereof. The antibodies can be of any isotype, e.g., IgM, various IgG isotypes such as IgGl, IgG2a, etc., and they can be from any animal species that produces antibodies, including goat, rabbit, mouse, chicken or the like.

[301] In various embodiments in which the cells are engineered to include or encode a detectable expression product operably linked to a regulatory sequence, wherein the regulatory sequence is responsive to NF-κB or is an NF-κB promoter, detection of the detectable expression product detects and/or measures expression of the transcription product by the cells. Thus, in various embodiments in which a detectable expression product is fluorescent or luminescent, transcription product expression by the cells can be detected and/or measured by the fluorescence or luminescence. In some embodiments, the expression of the transcription product is detected using fluorescence activated cell sorting (FACS). In some embodiments, expression of the transcription product is detected using microscopy (e.g., fluorescence microscopy). In various embodiments in which a detectable expression product is an enzyme, transcription product expression by the cells can be detected and/or measured by the activity of the enzyme. In various embodiments in which a detectable expression product is a tag or ligand, expression of the transcription product by the cells can be detected and/or measured by interaction of binding agents (such as anti-tag antibodies, e.g., labeled anti-tag antibodies) with the tag or ligand. [302] In various embodiments, potency is determined by quantitative, semi-quantitative, or qualitative comparison of measured expression of the transcription product of a drug product or drug substance to a reference. In various embodiments, the reference is a sample known to have, determined to have, or designated as having a target level of potency. In various embodiments, the reference can be a standard value or threshold value.

[303] In various embodiments, a drug product or drug substance and/or a reference (e.g., a reference standard) is assayed for potency according to the present disclosure using serial dilution prior to contacting the drug product(s) and/or drug substance(s) with cells. In some embodiments, the ability of the cell type to express the transcription product is confirmed, e.g., by use of positive controls, e.g., contacting an agent that is known to induce the expression of the transcription product with cells of the cell type, and/or assaying the cells for the transcription product mRNA or protein expression.

[304] In certain aspects, provided herein are methods of determining the potency of a drug product or drug substance. The method may comprise i) determining a threshold level of expression of a transcription product encoded by a nucleic acid sequence by contacting a cell of a mammalian immune cell line (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) with a reference standard, wherein the cell comprises the nucleic acid sequence operatively linked to a regulatory sequence, wherein the regulatory sequence is responsive to NF-κB or is an NF-κB promoter, and the reference standard is brought into contact with the cell (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells) for a time period sufficient to allow the reference standard to induce expression of the transcription product by the cells (e.g., THP-1 cells, such as PMA- differentiated THP-1 cells). The method may further comprise ii) determining a test level of expression the transcription product of by contacting cells (e.g., THP-1 cells, such as PMA- differentiated THP-1 cells) with the drug product or drug substance, wherein the drug product or drug substance is brought into contact with the cells (e.g., THP-1 cells, such as PMA- differentiated THP-1 cells) for a time period sufficient to allow the drug product or drug substance to induce expression of the transcription product by the cells (e.g., THP-1 cells, such as PMA-differentiated THP-1 cells). The method my further comprise iii) comparing the test level of expression of the transcription product to the level of threshold level of expression of the transcription product to determine the potency of the drug product or drug substance; wherein the drug product or drug substance comprises bacteria or mEVs. In some embodiments, the drug product or drug substance comprises a strain of bacteria (e.g., Prevotella histicola, Veillonella parvula, Harryflintia acetispora, Foumierella massiliensis, Megasphaera Sp., Selenomonas felix, Acidaminococcus intestine, and/or Propionospora sp. strain bacteria) and/or microbial extracellular vesicles (mEVs) from a strain of bacteria (e.g., Prevotella histicola, Veillonella parvula, Harryflintia acetispora, Foumierella massiliensis, Megasphaera Sp., Selenomonas felix, Acidaminococcus intestine, and/ or Propionospora sp. strain microbial extracellular vesicles (mEVs)).

[305] In some embodiments, the threshold above which a drug product or drug substance is identified as potent, and/or below which a drug product or drug substance is identified as not being potent, is expression of the transcription product that is equal to or greater than about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% the level of expression of the transcription product by cells (e.g., THP-1 cells) that have been contacted by a reference standard. In some embodiments, the threshold above which a drug product or drug substance is identified as potent, and/or below which a drug product or drug substance is identified as not being potent, is expression of the transcription product that is equal to or greater than 70% the level of expression of the transcription product by cells (e.g., THP-1 cells) that have been contacted by a reference standard. In some embodiments, the threshold above which a drug product or drug substance is identified as potent, and/or below which a drug product or drug substance is identified as not being potent, is expression of the transcription product that is at most 135% the level of expression of the transcription product by cells (e.g., THP-1 cells) that have been contacted by a reference standard.

[306] In some embodiments, the threshold above which a drug product or drug substance is identified as potent, and/or below which a drug product or drug substance is identified as not being potent, is expression of the transcription product that is equal to or greater than about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% of a determined threshold level of expression of the transcription product (e.g., from step (i) of certain exemplary methods provided herein). In some embodiments, the threshold above which a drug product or drug substance is identified as potent, and/or below which a drug product or drug substance is identified as not being potent, is expression of the transcription product that is equal to or greater than about 70% of a determined threshold level of expression of the transcription product (e.g., from step (i) of certain exemplary methods provided herein). In some embodiments, the threshold above which a drug product or drug substance is identified as potent, and/or below which a drug product or drug substance is identified as not being potent, is expression of the transcription product that is at most 135% of a determined threshold level of expression of the transcription product (e.g., from step (i) of certain exemplary methods provided herein).

[307] In various embodiments, a drug product or drug substance is identified as a potent drug product or drug substance if the level of expression of the transcription product by the cells (e.g., THP-1 cells) is at or above a reference level. In various embodiments, the reference level is determined by comparison to a reference sample known to have, determined to have, or designated as having a target level of potency. In various embodiments, the reference level is a threshold level or value.

[308] In some embodiments, the present disclosure includes formulating the drug product or drug substance into a solid dosage form if it has been identified as a potent drug product or drug substance (e.g., a tablet, a minitablet, a capsule, or a powder). In some embodiments, the solid dosage form is a tablet or capsule. In some embodiments, the present disclosure includes packaging a drug product or drug substance (e.g., a formulated drug product or drug substance) if it has been identified as a potent drug product or drug substance. In some embodiments, the present disclosure includes administering a drug product or drug substance (e.g., a formulated drug product or drug substance) to a subject in need thereof if it has been identified as a potent drug product or drug substance.

[309] In some embodiments, the present disclosure includes discarding a drug product or drug substance if it has been identified as a drug product or drug substance that is not potent. In some embodiments, the present disclosure includes discarding a drug product or drug substance if a measured sample’s level of expression of the transcription product is below a selected threshold level of expression of the transcription product. In some embodiments, the present disclosure includes not discarding a drug product or drug substance if it has been identified as a potent drug product or drug substance. In some embodiments, the present disclosure includes not discarding a drug product or drug substance if a measured sample’s level of expression of the transcription product is equal to or greater than a selected threshold level of expression of the transcription product. [310] In some embodiments, a potency assay of the present disclosure is applied to a drug product or drug substance for batch release. In some embodiments, a potency assay of the present disclosure is applied to a drug product or drug substance in order to measure, determine, and/or monitor storage stability. In some embodiments, a potency assay of the present disclosure is applied to bacteria and/or microbial extracellular vesicles (mEVs) prior to preparation of a drug substance or drug product, for example, during the manufacturing process, such as a process control, in order to measure, determine, and/or monitor the effects of a process (e.g., a step thereof) on the potency of bacteria and/or microbial extracellular vesicles (mEVs). For example, the potency assay can be used to assess the effects of changing a process parameter on the potency of bacteria and/or microbial extracellular vesicles (mEVs). Examples of process parameters include change in amount or presence of a growth media component, change in a growth condition (such as time, temperature, or optical density), change in a downstream processing step, change in excipient, and change in formulating parameter (such as compression strength).

Solid Dosage Form Compositions

[311] In certain aspects, provided herein are solid dosage forms (e.g., pharmaceutical products having a solid dosage form) comprising a pharmaceutical agent (e.g., drug substance) that contains bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs). In some embodiments, the pharmaceutical agent can optionally contain one or more additional components, such as a cryoprotectant. The pharmaceutical agent can be lyophilized (e.g., resulting in a powder). The pharmaceutical agent can be combined with one or more excipients (e.g., pharmaceutically acceptable excipients) in the solid dose form.

[312] In certain aspects, provided herein are solid dosage forms of pharmaceutical compositions. In some embodiments, the solid dosage form comprises a pharmaceutical agent, wherein the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs (e.g., bacteria and/or mEVs, and/or a powder comprising bacteria and/or mEVs)) and a diluent.

[313] In some embodiments, the solid dosage form provided herein comprises a lubricant.

[314] In some embodiments, a solid dosage form provided herein comprises a glidant. [315] Thus, in some embodiments, provided herein are solid dosage forms comprising a pharmaceutical agent that contains bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs). The bacteria can be live bacteria (e.g., powder or biomass thereof); non-live (dead) bacteria (e.g., powder or biomass thereof); non replicating bacteria (e.g., powder or biomass thereof); gamma-irradiated bacteria (e.g., powder or biomass thereof); and/or lyophilized bacteria (e.g., powder or biomass thereof).

[316] In some embodiments, the pharmaceutical agents comprise lyophilized bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs).

[317] In some embodiments, total cell count (TCC) can be used to quantify the numbers of bacteria (such as Prevotella histicola bacteria) present in a sample. In some embodiments, to quantify the numbers of bacteria (such as Prevotella histicola bacteria) present in a sample, electron microscopy (e.g., EM of ultrathin frozen sections) can be used to visualize the bacteria and count their relative numbers. Alternatively, nanoparticle tracking analysis (NTA), Coulter counting, or dynamic light scattering (DLS) or a combination of these techniques can be used. NTA and the Coulter counter count particles and show their sizes. DLS gives the size distribution of particles, but not the concentration. Bacteria frequently have diameters of 1-2 um (microns). The full range is 0.2-20 um. Combined results from Coulter counting and NTA can reveal the numbers of bacteria in a given sample. Coulter counting reveals the numbers of particles with diameters of 0.7-10 um. For most bacterial samples, the Coulter counter alone can reveal the number of bacteria in a sample. For NTA, a Nanosight instrument can be obtained from Malvern Pananlytical. For example, the NS300 can visualize and measure particles in suspension in the size range 10-2000nm. NTA allows for counting of the numbers of particles that are, for example, 50-1000 nm in diameter. DLS reveals the distribution of particles of different diameters within an approximate range of 1 nm - 3 um.

[318] In some embodiments, the bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs) may be quantified based on particle count. For example, total particle count of a bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs) preparation can be measured using NTA.

[319] In some embodiments, the bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs) may be quantified based on the amount of protein, lipid, or carbohydrate. For example, total protein content of bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs), and/or preparations thereof, can be measured using the Bradford assay or BCA. In some embodiments, bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs) may be quantified based on a lipid assay. See, e.g., WO2023114293.

[320] In certain aspects, provided herein are solid dosage forms comprising pharmaceutical agents that comprise bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs) useful for the treatment and/or prevention of disease (e.g., an autoimmune disease, an inflammatory disease, a metabolic disease, or a dysbiosis), as well as methods of making and/or identifying such bacteria, and methods of using pharmaceutical agents and solid dosage forms thereof (e.g., for the treatment of an autoimmune disease, an inflammatory disease, or a metabolic disease, either alone or in combination with other therapeutics). In some embodiments, the pharmaceutical agents comprise bacteria (such as Prevotella histicola bacteria) (e.g., whole bacteria) (e.g., live bacteria, dead (e.g., killed) bacteria, non-replicating bacteria, gamma-irradiated bacteria; attenuated bacteria). In some embodiments, the pharmaceutical agents comprise bacteria (microbial extracellular vesicles (mEVs) (such as Prevotella histicola mEVs).

[321] In certain aspects, provided are pharmaceutical agents for administration to a subject (e.g., human subject). In some embodiments, the pharmaceutical agents are combined with additional active and/or inactive materials in order to produce a final product, which may be in single dosage unit or in a multi-dose format. In some embodiments, the pharmaceutical agent is combined with an adjuvant such as an immuno-adjuvant (e.g., a STING agonist, a TLR agonist, or a NOD agonist).

[322] In some embodiments, the solid dosage form comprises at least one carbohydrate.

[323] In some embodiments, the solid dosage form comprises at least one lipid. In some embodiments, the lipid comprises at least one fatty acid selected from lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EP A), docosanoic acid (22:0), docosenoic acid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6) (DHA), and tetracosanoic acid (24:0).

[324] In some embodiments, the solid dosage form comprises at least one mineral or mineral source. Examples of minerals include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium. Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.

[325] In some embodiments, the solid dosage form comprises at least one vitamin. The at least one vitamin can be fat-soluble or water-soluble vitamins. Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin Bl 2, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin. Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.

[326] In some embodiments, the solid dosage form comprises an excipient. Non-limiting examples of suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.

[327] Suitable excipients that can be included in the solid dosage form can be one or more pharmaceutically acceptable excipients known in the art. For example, see Rowe, Sheskey, and Quinn, eAs.,_Handbook of Pharmaceutical Excipients, sixth ed.; 2009; Pharmaceutical Press and American Pharmacists Association.

Solid Dosage Forms

[328] The solid dosage form described herein can be a capsule.

[329] The solid dosage forms of a pharmaceutical agent as described herein can comprise capsules. In some embodiments, the capsule is a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule. In some embodiments, the capsule comprises HPMC (hydroxyl propyl methyl cellulose) or gelatin. In some embodiments, the capsule comprises HPMC (hydroxyl propyl methyl cellulose). In some embodiments, the capsule is banded. [330] In some embodiments, the solid dosage form is enterically coated (e.g., comprises an enteric coating; e.g., is coated with an enteric coating).

[331] In some embodiments, the solid dosage form comprises a tablet (> 4mm) (e.g., 5 mm- 17mm). For example, the tablet is a 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 11mm, 12mm, 13mm, 14mm, 15mm, 16mm, 17mm, or 18mm tablet. The size refers to the diameter of the tablet, as is known in the art. As used herein, the size of the tablet refers to the size of the tablet prior to application of an enteric coating.

[332] In some embodiments, the solid dosage form comprises a minitablet. The minitablet can be in the size range of lmm-4 mm range. E.g., the minitablet can be a 1mm minitablet, 1.5 mm minitablet, 2mm minitablet, 3mm minitablet, or 4mm minitablet. The size refers to the diameter of the minitablet, as is known in the art. As used herein, the size of the minitablet refers to the size of the minitablet prior to application of an enteric coating.

[333] The minitablets can be in a capsule. The capsule can be a size 00, size 0, size 1 , size 2, size 3, size 4, or size 5 capsule. The capsule that contains the minitablets can comprise HPMC (hydroxyl propyl methyl cellulose) or gelatin. The minitablets can be inside a capsule: the number of minitablets inside a capsule will depend on the size of the capsule and the size of the minitablets. As an example, a size 0 capsule can contain 31-35 (an average of 33) minitablets that are 3mm minitablets. In some embodiments, the capsule is banded after loading. In some embodiments, the capsule is banded with an HPMC-based banding solution.

Coating

[334] The solid dosage form (e.g., capsule or tablet or minitablet) described herein can be enterically coated. The enteric coating allows for release of the pharmaceutical agent, e.g., in the small intestine, e.g., upper small intestine, e.g., duodenum and/or jejunum. In some embodiments, the solid dosage form is enteric coated to dissolve at pH 5.5.

[335] Release of the pharmaceutical agent in the small intestine, e.g., in the upper small intestine, e.g., in the duodenum, or in the jejunum, allows the pharmaceutical agent to target and affect cells (e.g., epithelial cells and/or immune cells) located at these specific locations, e.g., which can cause a local effect in the small intestine and/or cause a systemic effect (e.g., an effect outside of the gastrointestinal tract). [336] EUDRAGIT is the brand name for a diverse range of polymethacrylate-based copolymers. It includes anionic, cationic, and neutral copolymers based on methacrylic acid and methacrylic/acrylic esters or their derivatives.

[337] Examples of other materials that can be used in the enteric coating (e.g., the one enteric coating or the inner enteric coating and/or the outer enteric coating) include cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), poly(vinyl acetate phthalate) (PVAP), hydroxypropyl methylcellulose phthalate (HPMCP), fatty acids, waxes, shellac (esters of aleurtic acid), plastics, plant fibers, zein, AQUA-ZEIN® (an aqueous zein formulation containing no alcohol), amylose starch, starch derivatives, dextrins, methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetate succinate), methyl methacrylate-methacrylic acid copolymers, and/or sodium alginate.

[338] The enteric coating can include a polymethacrylate-based copolymer.

[339] The enteric coating can include poly(methacrylic acid-co-ethyl acrylate).

[340] The enteric coating can include a methacrylic acid ethyl acrylate (MAE) copolymer

(1:1).

[341] The enteric coating can include methacrylic acid ethyl acrylate (MAE) copolymer (1 : 1) (such as Kollicoat MAE 100P).

[342] The enteric coating can include a Eudragit copolymer, e.g., a Eudragit L (e.g., Eudragit L 100-55; Eudragit L 30 D-55), a Eudragit S, a Eudragit RL, a Eudragit RS, a Eudragit E, or a Eudragit FS (e.g., Eudragit FS 30 D).

[343] Other examples of materials that can be used in the enteric coating (e.g., the one enteric coating or the inner enteric coating and/or the outer enteric coating) include those described in, e.g., U.S. 6312728; U.S. 6623759; U.S. 4775536; U.S. 5047258; U.S. 5292522; U.S. 6555124; U.S. 6638534; U.S. 2006/0210631; U.S. 2008/200482; U.S. 2005/0271778; U.S. 2004/0028737; WO 2005/044240.

[344] See also, e.g., U.S. 9233074, which provides pH dependent, enteric polymers that can be used with the solid dosage forms provided herein, including methacrylic acid copolymers, polyvinylacetate phthalate, hydroxypropylmethyl cellulose acetate succinate, hydroxypropylmethyl cellulose phthalate and cellulose acetate phthalate; suitable methacrylic acid copolymers include: poly(methacrylic acid, methyl methacrylate) 1 : 1 sold, for example, under the Eudragit L100 trade name; poly(methacrylic acid, ethyl acrylate) 1 : 1 sold, for example, under the Eudragit L100-55 trade name; partially-neutralized poly(methacrylic acid, ethyl acrylate) 1 : 1 sold, for example, under the Kollicoat MAE- 100P trade name; and poly(methacrylic acid, methyl methacrylate) 1:2 sold, for example, under the Eudragit S100 trade name.

Methods of Use

[345] The solid dosage forms, pharmaceutical agents (e.g., drug substances) and pharmaceutical compositions (e.g., drug products) described herein allow, e.g., for oral administration of a pharmaceutical agent contained therein.

[346] The solid dosage forms, pharmaceutical agents and pharmaceutical compositions described herein can be used in the treatment and/or prevention of inflammation, autoimmunity, a metabolic condition, or a dysbiosis.

[347] The solid dosage forms, pharmaceutical agents and pharmaceutical compositions described herein can be used in the treatment and/or prevention of bacterial septic shock, cytokine storm and/or viral infection (such as a coronavirus infection, an influenza infection, and/or a respiratory syncytial virus infection).

[348] The solid dosage forms, pharmaceutical agents and pharmaceutical compositions described herein can be used to decrease inflammatory cytokine expression (e.g., decreased IL-8, IL-6, IL-1β, and/or TNFα expression levels).

[349] Methods of using a solid dosage form and/or pharmaceutical composition (e.g., for oral administration) (e.g., for pharmaceutical use) comprising a pharmaceutical agent (e.g., a therapeutically effective amount thereof), wherein the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs), and wherein the solid dosage form and/or pharmaceutical composition further comprises the disclosed components are described herein.

[350] The methods and administered solid dosage forms and/or pharmaceutical compositions described herein allow, e.g., for oral administration of a pharmaceutical agent contained therein. The solid dosage form and/or pharmaceutical composition can be administered to a subject is a fed or fasting state. The solid dosage form and/or pharmaceutical composition can be administered, e.g., on an empty stomach (e.g., one hour before eating or two hours after eating). The solid dosage form and/or pharmaceutical composition can be administered one hour before eating. The solid dosage form and/or pharmaceutical composition can be administered two hours after eating.

[351] A solid dosage form, pharmaceutical agent, and/or pharmaceutical composition for use in the treatment and/or prevention of inflammation, autoimmunity, a metabolic condition, a cancer, or a dysbiosis is provided herein.

[352] A solid dosage form, pharmaceutical agent, and/or pharmaceutical composition for use in the treatment and/or prevention of bacterial septic shock, cytokine storm and/or viral infection (such as a coronavirus infection, an influenza infection, and/or a respiratory syncytial virus infection) is provided herein.

[353] A solid dosage form, pharmaceutical agent, and/or pharmaceutical composition for use in decrease inflammatory cytokine expression (e.g., decreased IL-8, IL-6, IL-1β, and/or TNFα expression levels) is provided herein.

[354] Use of a solid dosage form, pharmaceutical agent, and/or pharmaceutical composition for the preparation of a medicament for the treatment and/or prevention of inflammation, autoimmunity, a metabolic condition, a cancer, or a dysbiosis is provided herein.

[355] Use of a solid dosage form, pharmaceutical agent, and/or pharmaceutical composition for the preparation of a medicament for the treatment and/or prevention of bacterial septic shock, cytokine storm and/or viral infection (such as a coronavirus infection, an influenza infection, and/or a respiratory syncytial virus infection) is provided herein.

[356] Use of a solid dosage form, pharmaceutical agent, and/or pharmaceutical composition for the preparation of a medicament for decreasing inflammatory cytokine expression (e.g., decreased IL-8, IL-6, IL-1β, and/or TNFα expression levels) is provided herein.

Method of Making Solid Dosage Forms

[357] Methods of preparing a solid dosage form of a drug substance or drug product can comprise, among other things, prepration of a tablet, a minitablet, a capsule, or a powder, and steps thereof. Methods of preparing a solid dosage form of a drug substance or drug product can comprise blending, encapsulation, banding, and/or coating of a dosage form such as a tablet, a minitablet, or a capsule. [358] In certain aspects, provided herein are methods of preparing a solid dosage form of a pharmaceutical composition, the method comprising combining (e.g., blending) into a pharmaceutical composition a pharmaceutical agent (e.g., bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs) or a powder comprising bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs)) and one or more additional components described herein. In certain aspects, provided herein are methods of preparing a solid dosage form of a pharmaceutical composition, the method comprising combining into a pharmaceutical composition a pharmaceutical agent (e.g., bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs) or a powder comprising bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs)) and a diluent.

[359] In some embodiments, the method further comprises loading the pharmaceutical composition into a capsule (e.g., encapsulation).

[360] In some embodiments, the method further comprises banding the capsule after loading.

[361] In some embodiments, the method further comprises enterically coating the capsule.

[362] In some embodiments, the method further comprises compressing the pharmaceutical composition into a tablet.

[363] In some embodiments, the method further comprises enterically coating the tablet.

[364] In some embodiments, the method further comprises compressing the pharmaceutical composition into a minitablet.

[365] In some embodiments, the method further comprises enterically coating the minitablet.

Additional Aspects of the Solid Dosage Forms

[366] The solid dosage forms and/or pharmaceutical compositions, e.g., as described herein, comprising a pharmaceutical agent (e.g., a therapeutically effective amount thereof), wherein the pharmaceutical agent comprises Prevotella histicola bacteria, and wherein the solid dosage form and/or pharmaceutical composition further comprises the described components, can provide a therapeutically effective amount of the pharmaceutical agent to a subject, e.g., a human. [367] The solid dosage forms and/or pharmaceutical compositions, e.g., as described herein, comprising a pharmaceutical agent (e.g., a therapeutically effective amount thereof), wherein the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs), and wherein the solid dosage form further comprises the described components, can provide a non-natural amount of the therapeutically effective components (e.g., present in the pharmaceutical agent) to a subject, e.g., a human.

[368] The solid dosage forms and/or pharmaceutical compositions, e.g., as described herein, comprising a pharmaceutical agent (e.g., a therapeutically effective amount thereof), wherein the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs), and wherein the solid dosage form and/or pharmaceutical composition further comprises the described components, can provide an unnatural quantity of the therapeutically effective components (e.g., present in the pharmaceutical agent) to a subject, e.g., a human.

[369] The solid dosage forms and/or pharmaceutical compositions, e.g., as described herein, comprising a pharmaceutical agent (e.g., a therapeutically effective amount thereof), wherein the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs), and wherein the solid dosage form and/or pharmaceutical composition further comprises the described components, can bring about one or more changes to a subject, e.g., human, e.g., to treat or prevent a disease or a health disorder.

The solid dosage forms and/or pharmaceutical compositions, e.g., as described herein, comprising a pharmaceutical agent (e.g., a therapeutically effective amount thereof), wherein the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) (such as Prevotella histicola bacteria and/or mEVs), and wherein the solid dosage form and/or pharmaceutical composition further comprises the described components, has potential for significant utility, e.g., to affect a subject, e.g., a human, e.g., to treat or prevent a disease or a health disorder.

Administration

[370] In certain aspects, provided herein is a method of delivering a solid dosage form and/or pharmaceutical composition described herein to a subject. In some embodiments of the methods provided herein, the solid dosage form and/or pharmaceutical composition is administered in conjunction with the administration of an additional therapeutic agent. In some embodiments, the solid dosage form and/or pharmaceutical composition comprises a pharmaceutical agent co-formulated with the additional therapeutic agent. In some embodiments, the solid dosage form and/or pharmaceutical composition is co-administered with the additional therapeutic agent. In some embodiments, the additional therapeutic agent is administered to the subject before administration of the solid dosage form (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes before, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours before, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days before). In some embodiments, the additional therapeutic agent is administered to the subject after administration of the solid dosage form and/or pharmaceutical composition (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes after, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours after, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days after). In some embodiments, the same mode of delivery is used to deliver both the solid dosage form and/or pharmaceutical composition and the additional therapeutic agent. In some embodiments, different modes of delivery are used to administer the solid dosage form and/or pharmaceutical composition and the additional therapeutic agent. For example, in some embodiments the solid dosage form and/or pharmaceutical composition is administered orally while the additional therapeutic agent is administered via injection (e.g., an intravenous and/or intramuscular).

[371] The dosage regimen can be any of a variety of methods and amounts, and can be determined by one skilled in the art according to known clinical factors. As is known in the medical arts, dosages for any one patient can depend on many factors, including the subject's species, size, body surface area, age, sex, immunocompetence, and general health, the particular microorganism to be administered, duration and route of administration, the kind and stage of the disease, and other compounds such as drugs being administered concurrently or near- concurrently. In addition to the above factors, such levels can be affected by the infectivity of the microorganism, and the nature of the microorganism, as can be determined by one skilled in the art. In the present methods, appropriate minimum dosage levels of microorganisms can be levels sufficient for the microorganism to survive, grow and replicate. The dose of a pharmaceutical agent (e.g., in a solid dosage form) described herein may be appropriately set or adjusted in accordance with the dosage form, the route of administration, the degree or stage of a target disease, and the like. For example, the general effective dose of the agents may range between 0.01 mg/kg body weight/day and 1000 mg/kg body weight/day, between 0.1 mg/kg body weight/day and 1000 mg/kg body weight/day, 0.5 mg/kg body weight/day and 500 mg/kg body weight/day, 1 mg/kg body weight/day and 100 mg/kg body weight/day, or between 5 mg/kg body weight/day and 50 mg/kg body weight/day. The effective dose may be 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, or 1000 mg/kg body weight/day or more, but the dose is not limited thereto.

[372] In some embodiments, the dose administered to a subject is sufficient to prevent disease (e.g., autoimmune disease, inflammatory disease, or metabolic disease), delay its onset, or slow or stop its progression, or relieve one or more symptoms of the disease. One skilled in the art will recognize that dosage will depend upon a variety of factors including the strength of the particular agent (e.g., pharmaceutical agent) employed, as well as the age, species, condition, and body weight of the subject. The size of the dose will also be determined by the route, timing, and frequency of administration as well as the existence, nature, and extent of any adverse sideeffects that might accompany the administration of a particular pharmaceutical agent and the desired physiological effect.

[373] Suitable doses and dosage regimens can be determined by conventional range-finding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages, which are no more than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. An effective dosage and treatment protocol can be determined by routine and conventional means, starting e.g., with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Animal studies are commonly used to determine the maximal tolerable dose ("MTD") of bioactive agent per kilogram weight. Those skilled in the art regularly extrapolate doses for efficacy, while avoiding toxicity, in other species, including humans.

[374] In accordance with the above, in therapeutic applications, the dosages of the pharmaceutical agents used as provided herein vary depending on the active agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage. As another example, the dose should be sufficient to result in slowing of progression of the disease for which the subject is being treated, and preferably amelioration of one or more symptoms of the disease for which the subject is being treated.

[375] Separate administrations can include any number of two or more administrations, including two, three, four, five or six administrations. One skilled in the art can readily determine the number of administrations to perform or the desirability of performing one or more additional administrations according to methods known in the art for monitoring therapeutic methods and other monitoring methods provided herein. Accordingly, the methods provided herein include methods of providing to the subject one or more administrations of a solid dosage form and/or pharmaceutical composition, where the number of administrations can be determined by monitoring the subject, and, based on the results of the monitoring, determining whether or not to provide one or more additional administrations. Deciding on whether or not to provide one or more additional administrations can be based on a variety of monitoring results.

[376] The time period between administrations can be any of a variety of time periods. The time period between administrations can be a function of any of a variety of factors, including monitoring steps, as described in relation to the number of administrations, the time period for a subject to mount an immune response. In one example, the time period can be a function of the time period for a subject to mount an immune response; for example, the time period can be more than the time period for a subject to mount an immune response, such as more than about one week, more than about ten days, more than about two weeks, or more than about a month; in another example, the time period can be no more than the time period for a subject to mount an immune response, such as no more than about one week, no more than about ten days, no more than about two weeks, or no more than about a month.

[377] In some embodiments, the delivery of an additional therapeutic agent in combination with the solid dosage form and/or pharmaceutical composition described herein reduces the adverse effects and/or improves the efficacy of the additional therapeutic agent.

[378] The effective dose of an additional therapeutic agent described herein is the amount of the additional therapeutic agent that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, with the least toxicity to the subject. The effective dosage level can be identified using the methods described herein and will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions or agents administered, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts. In general, an effective dose of an additional therapeutic agent will be the amount of the additional therapeutic agent which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[379] The toxicity of an additional therapeutic agent is the level of adverse effects experienced by the subject during and following treatment. Adverse events associated with additional therapy toxicity can include, but are not limited to, abdominal pain, acid indigestion, acid reflux, allergic reactions, alopecia, anaphylasix, anemia, anxiety, lack of appetite, arthralgias, asthenia, ataxia, azotemia, loss of balance, bone pain, bleeding, blood clots, low blood pressure, elevated blood pressure, difficulty breathing, bronchitis, bruising, low white blood cell count, low red blood cell count, low platelet count, cardiotoxicity, cystitis, hemorrhagic cystitis, arrhythmias, heart valve disease, cardiomyopathy, coronary artery disease, cataracts, central neurotoxicity, cognitive impairment, confusion, conjunctivitis, constipation, coughing, cramping, cystitis, deep vein thrombosis, dehydration, depression, diarrhea, dizziness, dry mouth, dry skin, dyspepsia, dyspnea, edema, electrolyte imbalance, esophagitis, fatigue, loss of fertility, fever, flatulence, flushing, gastric reflux, gastroesophageal reflux disease, genital pain, granulocytopenia, gynecomastia, glaucoma, hair loss, hand-foot syndrome, headache, hearing loss, heart failure, heart palpitations, heartburn, hematoma, hemorrhagic cystitis, hepatotoxicity, hyperamylasemia, hypercalcemia, hyperchloremia, hyperglycemia, hyperkalemia, hyperlipasemia, hypermagnesemia, hypernatremia, hyperphosphatemia, hyperpigmentation, hypertriglyceridemia, hyperuricemia, hypoalbuminemia, hypocalcemia, hypochloremia, hypoglycemia, hypokalemia, hypomagnesemia, hyponatremia, hypophosphatemia, impotence, infection, injection site reactions, insomnia, iron deficiency, itching, joint pain, kidney failure, leukopenia, liver dysfunction, memory loss, menopause, mouth sores, mucositis, muscle pain, myalgias, myelosuppression, myocarditis, neutropenic fever, nausea, nephrotoxicity, neutropenia, nosebleeds, numbness, ototoxicity, pain, palmar- plantar erythrodysesthesia, pancytopenia, pericarditis, peripheral neuropathy, pharyngitis, photophobia, photosensitivity, pneumonia, pneumonitis, proteinuria, pulmonary embolus, pulmonary fibrosis, pulmonary toxicity, rash, rapid heartbeat, rectal bleeding, restlessness, rhinitis, seizures, shortness of breath, sinusitis, thrombocytopenia, tinnitus, urinary tract infection, vaginal bleeding, vaginal dryness, vertigo, water retention, weakness, weight loss, weight gain, and xerostomia. In general, toxicity is acceptable if the benefits to the subject achieved through the therapy outweigh the adverse events experienced by the subject due to the therapy.

Exemplary Embodiments

1. A method for assaying a drug product or drug substance, the method comprising:

(a) contacting a cell of a mammalian immune cell line with the drug product or drug substance, wherein:

(i) the cell comprises a nucleic acid sequence operatively linked to a NF-κB regulatory sequence, wherein the NF-κB regulatory sequence is a sequence that is responsive to NF-κB or is an NF-κB promoter; and

(ii) the drug product or drug substance comprises bacteria (e.g., Prevotella histicola, Veillonella parvula, Harryflintia acetispora, Foumierella massiliensis, Megasphaera Sp., Selenomonas felix, Acidaminococcus intestine, and/or Propionospora sp. bacteria) and/or microbial extracellular vesicles (mEVs) (e.g, from Prevotella histicola, Veillonella parvula, Harryflintia acetispora, Foumierella massiliensis, Megasphaera Sp., Selenomonas felix, Acidaminococcus intestine, and/ or Propionospora sp. bacteria); and

(b) detecting the level of expression of a transcription product encoded by the nucleic acid sequence by the cell.

2. The method of embodiment 1, wherein the bacteria and/or mEVs are from a strain of Prevotella histicola.

3. The method of embodiment 2, wherein the Prevotella histicola strain comprises 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).

4. The method of embodiment 3, wherein the Prevotella histicola strain is the Prevotella Strain B (NRRL accession number B 50329). 5. The method of any one of embodiments 1-4, wherein the transcription product is a protein.

6. The method of embodiment 5, wherein the expression of the protein is determined by an enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay, or immunocytostaining.

7. The method of embodiment 5, wherein the protein is a luciferase protein.

8. The method of embodiment 7, wherein the luciferase protein is a firefly luciferase protein.

9. The method of embodiment 7 or embodiment 8, wherein expression of the luciferase is determined by a luciferase assay.

10. The method of embodiment 5, wherein the protein is a fluorescent protein.

11. The method of embodiment 10, wherein the fluorescent protein is a green fluorescent protein, mCherry protein, a yellow fluorescent protein, a blue fluorescent protein, a cyan fluorescent protein, or a red fluorescent protein.

12. The method of embodiment 10 or embodiment 11, wherein expression of the fluorescent protein is determined by detecting fluorescence.

13. The method of any one of embodiments 1-4, wherein the transcription product is an RNA.

14. The method of embodiment 13, wherein the expression of the RNA is determined by a northern hybridization method or using reverse transcription-polymerase chain reaction (RT- PCR).

15. The method of any one of embodiment 1-14, wherein the NF-κB regulatory sequence is a sequence that is responsive to NF-κB.

16. The method of embodiment 15, wherein the sequence that is responsive to NF-κB is an NF-κB response element.

17. The method of embodiment 16, wherein the NF-κB response element comprises a sequence according to SEQ ID NO: 1, 2, or 3.

18. The method of any one of embodiments 15-17, wherein the sequence that is responsive to NF-κB is positioned upstream of a promoter.

19. The method of embodiment 18, wherein the promoter is a minimal TATA promoter. 20. The method of any one of embodiments 15-19, wherein the nucleic acid sequence is operably linked to at least two NF-κB response elements.

21. The method of embodiment 20, wherein each of the at least two NF-κB response elements comprises a sequence according to SEQ ID NO: 1, 2, or 3.

22. The method of embodiment 20 or 21, wherein each of the at least two NF-κB response elements has the same sequence.

23. The method of embodiment 20 or 21, wherein each of the at least two NF-κB response elements has a distinct sequence.

24. The method of any one of embodiments 20-23, wherein the at least two NF-κB response elements are positioned upstream of a promoter.

25. The method of embodiment 24, wherein the promoter is a minimal TATA promoter.

26. The method of any one of embodiment 1-14, wherein the NF-κB regulatory sequence is an NF-κB promoter.

27. The method of any one of embodiments 1-26, wherein the mammalian immune cell line is a monocyte cell line.

28. The method of embodiment 27, wherein the cell is a THP-1 cell.

29. The method of embodiment 27 or 28, further comprising the step of contacting the cells withPhorbol 12-myristate 13-acetate (PMA).

30. The method of any one of embodiments 1-29, further comprising serially diluting the drug product or drug substance prior to contacting the cell with the drug product or drug substance.

31. The method of any one of embodiments 30, wherein the drug product or drug substance is contacted with the cell for a time period sufficient for the drug product or drug substance to induce expression of the transcription product by the cell.

32. The method of any one of embodiment 1-31, further comprising comparing the level of expression of the transcription product to a threshold level of expression of the transcription product.

33. The method of embodiment 32, further comprising determining the threshold level by contacting a cell of the mammalian immune cell line with a reference standard of the drug product or drug substance and detecting the level of expression of the transcription product by the cell contacted with the reference standard, wherein the level of expression by the cell contacted with the reference standard is the threshold level.

34. The method of embodiment 32, wherein the threshold level is about 50%-200% the level of expression of the transcription product by a cell of the mammalian immune cell line that has been contacted by a reference standard.

35. The method of embodiment 32, wherein the threshold level is about 70%-135% the level of expression of the transcription product by a cell of the mammalian immune cell line that has been contacted by a reference standard.

36. The method any one of embodiments 32-35, wherein the method further comprises discarding the drug product or drug substance if level of expression of the transcription product is below the threshold level.

37. The method of any one of embodiments 32-36, further comprising identifying the drug product or drug substance as a potent drug product or drug substance if the level of expression of the transcription product is at or above the threshold level.

38. The method of embodiment 37, further comprising formulating the drug product or drug substance into a solid dosage form if it has been identified as a potent drug product or drug substance.

39. The method of embodiment 38, wherein the solid dosage form is a tablet or a capsule.

40. The method of any one of embodiments 37-39, further comprising packaging the drug product or drug substance if it has been identified as a potent drug product or drug substance.

41. The method of any one of embodiments 37-40, further comprising administering the drug product or drug substance to a subject in need thereof if it has been identified as a potent drug product or drug substance.

42. The method of any one of embodiments 37-41, further comprising not discarding the drug product or drug substance if it has been identified as a potent drug product or drug substance.

43. A method of determining the potency of a drug product or drug substance, comprising the steps of: i) determining a threshold level of expression of a transcription product encoded by a nucleic acid sequence by contacting a cell of a mammalian immune cell line with a reference standard, wherein the cell comprises the nucleic acid sequence operatively linked to a NF-κB regulatory sequence, wherein the NF-κB regulatory sequence is a sequence that is responsive to NF-κB or is an NF-κB promoter, and the reference standard is brought into contact with the cell for a time period sufficient to allow the reference standard to induce expression of the transcription product by the cell; ii) determining a test level of expression of the transcription product by contacting the cell with the drug product or drug substance, wherein the drug product or drug substance is brought into contact with the cell for a time period sufficient to allow the drug product or drug substance to induce expression of the transcription product by the cell; and iii) comparing the test level of expression of the transcription product to the level of threshold level of expression of the transcription product to determine the potency of the drug product or drug substance; wherein the drug product or drug substance comprises bacteria (e.g., Prevotella histicola, Veillonella parvula, Harryflintia acetispora, Foumierella massiliensis, Megasphaera Sp., Selenomonas felix, Acidaminococcus intestine, and/or Propionospora sp. bacteria) and/or microbial extracellular vesicles (mEVs) (e.g, from Prevotella histicola, Veillonella parvula, Harryflintia acetispora, Foumierella massiliensis, Megasphaera Sp., Selenomonas felix, Acidaminococcus intestine, and/ or Propionospora sp. bacteria).

44. The method of embodiment 43, wherein the bacteria and/or mEVs are from a strain of Prevotella histicola.

45. The method of embodiment 44, wherein the Prevotella histicola strain comprises 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).

46. The method of embodiment 44, wherein the Prevotella histicola strain is the Prevotella Strain B (NRRL accession number B 50329).

47. The method of any one of embodiments 43-46, wherein the transcription product is a protein.

48. The method of embodiment 47, wherein the expression of the protein is determined by an enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay, or immunocy tostaining .

49. The method of embodiment 47, wherein the protein is a luciferase protein. 50. The method of embodiment 49, wherein the luciferase protein is a firefly luciferase protein.

51. The method of embodiment 49 or embodiment 50, wherein expression of the luciferase is determined by a luciferase assay.

52. The method of embodiment 47, wherein the protein is a fluorescent protein.

53. The method of embodiment 52, wherein the fluorescent protein is a green fluorescent protein, mCherry protein, a yellow fluorescent protein, a blue fluorescent protein, a cyan fluorescent protein, or a red fluorescent protein.

54. The method of embodiment 52 or embodiment 53, wherein expression of the fluorescent protein is determined by detecting fluorescence.

55. The method of any one of embodiments 43-46, wherein the transcription product is an RNA.

56. The method of embodiment 55, wherein the expression of the RNA is determined by a northern hybridization method or using reverse transcription-polymerase chain reaction (RT- PCR).

57. The method of any one of embodiment 43-56, wherein the NF-κB regulatory sequence is a sequence that is responsive to NF-κB.

58. The method of embodiment 57, wherein the sequence that is responsive to NF-κB is an NF-κB response element.

59. The method of embodiment 58, wherein the NF-κB response element comprises a sequence according to SEQ ID NO: 1, 2, or 3.

60. The method of any one of embodiments 57-59, wherein the sequence that is responsive to NF-κB is positioned upstream of a promoter.

61. The method of embodiment 60, wherein the promoter is a minimal TATA promoter.

62. The method of embodiment 58, wherein the nucleic acid sequence is operably linked to at least two NF-κB response elements.

63. The method of embodiment 62, wherein each of the at least two NF-κB response elements comprises a sequence according to SEQ ID NO: 1, 2, or 3.

64. The method of embodiment 62 or 63, wherein each of the at least two NF-κB response elements has the same sequence. 65. The method of embodiment 62 or 63, wherein each of the at least two NF-κB response elements has a distinct sequence.

66. The method of any one of embodiments 62-65, wherein the at least two NF-κB response elements are positioned upstream of a promoter.

67. The method of embodiment 66, wherein the promoter is a minimal TATA promoter.

68. The method of any one of embodiment 43-56, wherein the NF-κB regulatory sequence is an NF-κB promoter.

69. The method of any one of embodiments 43-68, wherein the mammalian immune cell line is a monocyte cell line.

70. The method of embodiment 69, wherein the cell is a THP-1 cell.

71. The method of embodiment 69 or 70, further comprising the step of contacting the cell withPhorbol 12-myristate 13-acetate (PMA).

72. The method of any one of embodiments 43-71, further comprising serially diluting the drug product or drug substance prior to bringing the drug product or drug substance into contact with the cell.

73. The method of one of embodiments 43-72, wherein the time period sufficient to allow the reference standard and the drug product or drug substance to induce expression of the transcription product by the cell is at least 24 hours.

74. The method of any one of embodiments 43-73, wherein the threshold level of transcription product expression is about 50-200% of the level of transcription product expression by the mammalian cells in step i).

75. The method of any one of embodiments 43-74, wherein the threshold level of transcription product expression is about 70-135% of the level of transcription product expression by the mammalian cells in step i).

76. The method of any one of embodiments 43-75, wherein the method further comprises discarding the drug product or drug substance if the test level of expression of the transcription product is below the threshold level of expression of the transcription product.

77. The method of any one of embodiments 43-76, further comprising identifying the drug product or drug substance as a potent drug product or drug substance if the test level of expression of the transcription product is at or above a threshold level of expression of the transcription product. 78. The method of embodiment 77, further comprising formulating the drug product or drug substance into a solid dosage form if it has been identified as a potent drug product or drug substance.

79. The method of embodiment 78, wherein the solid dosage form is a tablet or a capsule.

80. The method of any one of embodiments 77-79, further comprising packaging the drug product or drug substance it has been identified as a potent drug product or drug substance.

81. The method of any one of embodiments 77-80, further comprising administering the drug product or drug substance to a subject in need thereof if it has been identified as a potent drug product or drug substance.

82. The method of any one of embodiments 77-81, further comprising not discarding the drug product or drug substance if it has been identified as a potent drug product or drug substance.

83. The method of any one of embodiments 1 to 82, wherein the drug product or drug substance comprises bacteria.

84. The method of any one of embodiments 1 to 83, wherein the drug product or drug substance comprises bacteria and/or mEVs that have been gamma irradiated, UV irradiated, heat inactivated, acid treated, or oxygen sparged.

85. The method of any one of embodiments 83 to 84, wherein the drug product or drug substance comprises live bacteria.

86. The method of any one of embodiments 83 to 84, wherein the drug product or drug substance comprises dead bacteria.

87. The method of any one of embodiments 83 to 86, wherein the drug product or drug substance comprises non-replicating bacteria.

88. The method of any one of embodiments 1 to 87, wherein the drug product or drug substance comprises one strain of bacteria or mEVs from one strain of bacteria.

89. The method of any one of embodiments 1 to 88, wherein the bacteria and/or mEVs are lyophilized (e.g., the lyophilized product further comprises a pharmaceutically acceptable excipient) (e.g., a powder form).

90. The method of any one of embodiments 1 to 89, wherein the bacteria and/or mEVs are gamma irradiated. 91. The method of any one of embodiments 1 to 89, wherein the bacteria and/or mEVs are UV irradiated.

92. The method of any one of embodiments 1 to 91, wherein the bacteria and/or mEVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).

93. The method of any one of embodiments 1 to 92, wherein the bacteria and/or mEVs are acid treated.

94. The method of any one of embodiments 1 to 93, wherein the bacteria and/or mEVs are oxygen sparged (e.g., at 0.1 wm for two hours).

95. The method of any one of embodiments 1 to 94, wherein the drug product or drug substance comprises microbial extracellular vesicles (mEVs).

96. The method of embodiment 95, wherein the drug product or drug substance comprises isolated mEVs (e.g., from one or more strains of bacteria (e.g., bacteria of interest)) (e.g., a therapeutically effective amount thereof).

97. The method of embodiments 95 or 96, wherein the mEVs comprise secreted mEVs (smEVs).

98. The method of embodiments 95 or 96, wherein the mEVs comprise processed mEVs (pmEVs).

99. The method of embodiment 98, wherein the pmEVs are produced from bacteria that have been gamma irradiated, UV irradiated, heat inactivated, acid treated, or oxygen sparged.

100. The method of embodiment 98 or 99, wherein the pmEVs are produced from live bacteria.

101. The method of any one of embodiments 98 to 100, wherein the pmEVs are produced from dead bacteria.

102. The method of any one of embodiments 98 to 99, wherein the pmEVs are produced from non-replicating bacteria.

103. The method of any one of embodiments 1 to 102, wherein the mEVs are from one strain of bacteria.

104. The method of any one of embodiments 1 to 102, wherein the bacteria are from one strain of bacteria.

105. A method for assaying a drug product or drug substance, the method comprising:

(a) contacting a cell of THP-1 cell line with the drug product or drug substance, wherein: (i) the cell comprises a nucleic acid sequence operatively linked to a NF-κB regulatory sequence, wherein the NF-κB regulatory sequence is a sequence that is responsive to NF-κB or is an NF-κB promoter; and

(ii) the drug product or drug substance comprises bacteria and/or microbial extracellular vesicles (mEVs), wherein the bacteria and/or mEVs are from a strain of Prevotella histicola (such as Prevotella Strain B 50329 (NRRL accession number B 50329); and

(b) detecting the level of expression of a transcription product encoded by the nucleic acid sequence by the cell.

106. A method of determining the potency of a drug product or drug substance, comprising the steps of: i) determining a threshold level of expression of a transcription product encoded by a nucleic acid sequence by contacting a cell of THP-1 cell line with a reference standard, wherein the cell comprises the nucleic acid sequence operatively linked to a NF-κB regulatory sequence, wherein the NF-κB regulatory sequence is a sequence that is responsive to NF-κB or is an NF-κB promoter, and the reference standard is brought into contact with the cell for a time period sufficient to allow the reference standard to induce expression of the transcription product by the cell; ii) determining a test level of expression of the transcription product by contacting the cell with the drug product or drug substance, wherein the drug product or drug substance is brought into contact with the cell for a time period sufficient to allow the drug product or drug substance to induce expression of the transcription product by the cell; and iii) comparing the test level of expression of the transcription product to the level of threshold level of expression of the transcription product to determine the potency of the drug product or drug substance; wherein the drug product or drug substance comprises bacteria and/or microbial extracellular vesicles (mEVs), wherein the bacteria and/or mEVs are from a strain of Prevotella histicola (such as Prevotella Strain B 50329 (NRRL accession number B 50329). EXAMPLES

Example 1: Measuring the relative potency of mEVs

[380] The present Example demonstrates the determination of potency of secreted microbial extracellular vesicles (smEVs) derived from a Prevotella histicola microbial strain in a Prevotella extracellular vesicle (smEV) drug substance (DS) formulation. In this Example, the Prevotella extracellular vesicles (smEVs) were isolated from Prevotella Strain B (NRRL accession number B 50329). The smEVs were contacted with differentiated THP-1 NF-κB luciferase (luc) reporter cells.

[381] The NF-κB reporter (Luc)-THP-1 cell line includes a transgene that encodes a firefly luciferase gene driven by four copies of the NF-κB response element positioned upstream of a minimal TATA promoter. After activation, endogenous NF-κB transcription factors bind to the DNA response elements, inducing transcription of the luciferase reporter gene.

[382] THP-1 NF-κB luc reporter cells were cultured using complete RPMI-1640 cell culture media containing puromycin selection marker. THP-1 NF-κB luc reporter cells were passaged twice a week for up to 30 passages, maintaining a 0.4 - 1.6× 10 ⁶ cells/mL density. Cultured THP-1 NF-κB luc reporter cells were seeded in four white opaque tissue culture treated flat bottom 96-well plates at 7.0× 10 ⁴ cells/well. THP-1 cells were incubated in the presence of PMA 50 nM for 24 hours at 37°C, 5% CO ₂ . Differentiated THP-1 cells were rested without PMA for 24 hours at 37°C, 5% CO ₂ .

[383] Prevotella Strain B smEVs DS reference standard and test sample powders were resuspended in THP-1 cell culture media. Prevotella Strain B smEVs reference standard and test sample DS suspensions were serially diluted to create eleven multiplicity-of-infection (MOI; that is, the ratio of EVs to cells) dilution points. Serial dilutions of Prevotella Strain B smEVs reference standard and DS test samples were added to four 96-well plates containing PMA- differentiated THP-1 cells. Differentiated THP-1 cells were cultured with Prevotella Strain B smEVs reference standard and DS test sample dilutions for 4 hours at 37°C, 5% CO ₂ .

[384] Steady-Gio® reagent was added to the four plates to lyse the THP-1 cells and generate the luminescence signal. Luminescence intensity generated by NF-κB luc reporter was quantified for each dilution of Prevotella Strain B smEVs reference standard and test DS samples using a luminescence microplate reader. Luminescence values were plotted against the corresponding MOI dilution values to obtain a sigmoidal dose response curve using four- parameter logistics (4-PL) regression. The EC50 values calculated for Prevotella Strain B smEVs reference standard and test sample DS samples were used to calculate the relative potency value for Prevotella Strain B smEVs test DS samples.

[385] Experiments of this Example were carried out according to the protocol provided below.

Reagent Preparation

[386] Preparation of PMA 5 mg/mL (8.1 mM) stock solution: Reconstitute lyophilized PMA powder by adding 2 mL of sterile DMSO to a vial containing 10 mg of PMA to form a 5 mg/mL stock concentration, equivalent to 8.1 mM stock concentration. Prepare 50 μL aliquots of PMA 8.1 mM stock solution. Assign a 6-month expiration date from the date of preparation. Store at ≤ -20°C.

[387] Preparation of THP-1 cell culture media: Combine 900 mL of RPMI 1640 media, 100 mL of heat-inactivated FBS (10% final concentration) and 0.1 mL of puromycin 10 mg/mL (1 pg/mL final concentration) inside a biosafety cabinet. Sterilize the complete THP-1 cell culture media using a 0.2 μm vacuum filtration system. Assign a 1 -month expiration date from the date of preparation. Store at 2 - 8°C.

[388] Preparation of Steady-Gio® reagent: Reconstitute lyophilized Steady-Gio® reagent by adding 100 mL of included buffer to the bottle. Mix by inverting the bottle until the lyophilized Steady-Gio® reagent is fully resuspended. Aliquot the resuspended Steady-Gio® reagent into amber 50 mL conical tubes. Assign a 1 -month expiration date from the date of preparation. Keep at -20°C.

Dav 1 : Seed THP-1 Cells and Differentiate with PMA for 24 Hours

[389] Count THP-1 cells: Measure the pre-spin THP-1 cell density and viability using NucleoCounter NC-200 or a similar cell counter. Transfer the required volume of cell suspension from a T175 tissue culture flask to the 50 mL conical tube. Centrifuge the 50 mL conical tube at 300 x g for 5 minutes at room temperature in a swinging bucket rotor. Aspirate the supernatant using vacuum. Resuspend the cell pellet in THP-1 cell culture media. Measure the post-spin THP-1 cell density and viability. Adjust the final concentration of THP-1 cells to 7.0×10 ⁵ cells/mL using THP-1 cell culture media.

[390] Differentiate THP-1 cells with PMA: Thaw an aliquot of PMA 8.1 mM (equivalent to 5 mg/mL) stock solution at room temperature. Label a sterile 1.5 mL microcentrifuge tube as “PMA”. Transfer 990 μL of THP-1 cell culture media to the “PMA” labeled tube. Prepare PMA 81 μM working solution by transferring 10 μL of PMA 8.1 mM (5 mg/mL) stock solution to the “PMA” labeled tube to create a 1 : 100 dilution. Add PMA to the THP-1 cell suspension to achieve a 50 nM final PMA concentration. For example, add 22.2 μL of PMA 81 μM working solution to 36 mL of THP-1 cell suspension.

[391] Plate THP-1 cells + PMA in four white 96- well tissue culture treated plates: Homogenize the THP-1 7.0× 10 ⁵ cells/mL suspension containing PMA 50 nM inside the 50 mL conical tube by pipetting up and down with a 25 mL serological pipette. Transfer the cell suspension a 50 mL sterile reservoir. Mix the cell suspension before addition to plates. Add 100 μL of cell suspension to rows B - G of four white tissue culture treated flat-bottom 96-well plates to achieve 7.0×10 ⁴ cells per well. Transfer 25 mL of sterile PBS pH 7.4 to a 50 mL sterile reservoir. Add 200 μL of PBS to rows A and H of each 96-well plate. Incubate the four 96-well plates for 24 hours at 37°C, 5% CO ₂ .

Dav 2: Media Exchange

[392] Warm the THP-1 cell culture media inside a 37°C water bath. Transfer 70 mL of THP-1 cell culture media to 100 mL sterile reservoir. Remove PMA-containing cell culture media from each 96-well plate using a P200 multichannel pipette. Hold the 96-well plate at a 45° angle and aspirate from the corner of the wells. Be careful not to disrupt the adherent THP-1 cells. Add 200 μL of fresh THP-1 cell culture media to each well using a P200 multichannel pipette. Incubate the differentiated THP-1 cells for additional 24 hours at 37°C, 5% CO ₂ .

Dav 3 - Morning: Addition of Prevotella Strain B smEVs DS Reference Standard and Test Lot Dilutions

[393] The dilutions of Prevotella Strain B smEVs DS reference standard and test samples will be prepared using THP-1 cell culture media inside a designated biosafety cabinet using aseptic techniques. Preparation of Prevotella Strain B smEVs reference standard suspension. Resuspend 50 mg of Prevotella Strain B smEVs DS powder in 1 mL of THP-1 cell culture media. Mix by vortexing at full speed until the Prevotella Strain B smEVs DS powder is fully resuspended. Calculate the concentration (EVs per mL) of the Prevotella Strain B smEVs DS suspension, using the following equation:

[394] Calculate the initial multiplicity of infection (MOI, that is ratio of EVs to cells) value for the Prevotella Strain B smEVs DS reference standard suspension, using the following equation:

[395] Keep the Prevotella Strain B smEVs DS suspension on ice.

Preparation of Prevotella Strain B smEVs DS test sample suspension

[396] Weigh out 50- 100 mg of the Prevotella Strain B smEVs DS test sample powder inside a screw-cap vial using an analytical balance. Resuspend the weighted Prevotella Strain B smEVs DS test sample powder in 2 mL of THP-1 cell culture media. Mix by vortexing at full speed until the Prevotella Strain B smEVs DS test sample powder is fully resuspended. Calculate the concentration (EVs per mL) of the Prevotella Strain B smEVs DS test sample suspension, using the following equation:

[397] For example, resuspending 100 mg of Prevotella Strain B smEVs DS test lot powder, which has 1.6E+14 EVs/gram packing density, in 2 mL of THP-1 cell culture media will result in 8.0E+12 EVs/mL suspension.

[398] Calculate the initial multiplicity of infection (MOI) value for the Prevotella Strain B smEVs DS test sample, using the following equation:

[399] Keep the Prevotella Strain B smEVs DS test sample suspension on ice.

Preparation of preliminary Prevotella Strain B smEVs DS reference standard MOI dilutions

[400] Two independently prepared MOI dilution series of Prevotella Strain B smEVs DS reference standard are created, acting as reference standard 1 (RSI) and reference standard 2 (RS2) for the relative potency analysis of Prevotella Strain B smEVs DS test samples. 2 mL deep-well block #1 is used for preparing preliminary dilutions of Prevotella Strain B smEVs DS reference standard suspensions and test sample suspensions. Prepare three preliminary dilutions of Prevotella Strain B smEVs DS reference standard suspension in a 2 mL deep-well block #1, based on the initial MOI value calculated above.

[401] Mix each dilution by pipetting up and down 5 - 10 times using a Pl 000 multichannel pipette. Change tips after every transfer.

[402] Preparation of preliminary Prevotella Strain B smEVs DS test sample MOI dilutions. Depending on the strength of Prevotella Strain B smEVs DS test sample, the first preliminary MOI dilution may be 1.0× 10 ⁶ or 1.0× 10 ⁵ , based on the initial MOI value of Prevotella Strain B smEVs DS test sample suspension. Prepare preliminary dilutions of Prevotella Strain B smEVs DS test sample suspensions in a 2 mL deep-well block #1, based on the initial MOI value calculated previously. Mix each dilution by pipetting up and down 5 - 10 times using a P1000 multichannel pipette. Change tips after every transfer.

Preparation of Prevotella Strain B smEVs DS reference standard and test sample dilutions

[403] Prepare 4-fold serial dilutions of Prevotella Strain B smEVs DS reference standard and test sample suspensions in a 2 mL deep-well block #2 to create samples with indicated MOI values.

[404] Mix by pipetting up and down 5 - 10 times using Pl 000 multichannel pipette. Change pipette tips after each sample transfer. Position of Prevotella Strain B smEVs reference standard and test sample MOI dilutions. Addition of reference standard and test sample MOI dilutions to PMA-differentiated THP-1 cells.

[405] Handle one row of the 96-well plate at a time. Change tips after every transfer. Remove cell culture media from each 96-well plate using a P300 multichannel pipette. Hold the 96-well plate at a 45° angle and aspirate from the corner of the wells. Be careful not to disrupt the adherent THP-1 cells. Follow the plate map shown below for the addition of Prevotella Strain B smEVs DS reference standard and Prevotella Strain B smEVs DS test sample MOI dilutions to the four 96-well plates. Transfer 100 μL of Prevotella Strain B smEVs DS reference standard 1 (RSI) MOI dilutions to row C using P200 multichannel pipette. Transfer 100 μL of Prevotella Strain B smEVs DS reference standard 2 (RS 2) MOI dilutions to row F using P200 multichannel pipette. Transfer 100 μL of Prevotella Strain B smEVs DS test sample 1 (SI) MOI dilutions to row B on plate 1, row D on plate 2, row E on plate 3 and row G on plate 4 using P200 multichannel pipette.

[406] Incubate the four 96-well plates for 4 hours ± 15 minutes at 37°C and 5% CO ₂ . Transfer an aliquot of the Steady-Gio® reagent from -20°C to the bench.

Dav 3 - Afternoon: Analysis of NF-κB Luciferase Reporter Activity using Promega Steady- Glo® Luciferase Assay System

[407] Remove the four 96-well plates from the microbial cell culture 37°C incubator. Remove the lid from each plate. Incubate the four 96-well plates at room temperature for 10 - 15 minutes. Add 50 μL of Steady-Gio® reagent to rows B - G of each plate using P300 multichannel pipette. Immediately cover each plate with a black lid. Shake the four 96-well plates at 450 rpm at room temperature for 20 - 30 minutes. Launch the software for the spectrophotometer. Create a new experiment using a luminescence protocol template. Measure the luminescence of the four 96-well plates in the same order that the Steady-Gio® reagent was added. Export the results of each experiment as an Excel file.

Example 2: Measuring the relative potency of additional mEVs

[408] The assay detailed in Example 1 was used to determine the potency of smEVs from two additional bacterial strains: Veillonella bacteria deposited as ATCC designation number PTA-125691 and Fournier ella massiliensis bacteria deposited as ATCC designation number PTA- 126696. [409] The general assay steps of number of cells used for seeding plates, concentration and incubation time for PMA differentiation, media exchange and rest period incubation time, incubation time of smEVs with differentiated THP-1 NF-κB luc cells were kept the same as in Example 1.

[410] For each of the two additional smEVs, the required smEV dilution range was determined by performing an extended (24 dose point) dose finding experiment.

[411] smEVs were derived from Veillonella bacteria deposited as ATCC designation number PTA-125691. Uses for the potency assay for these smEVs include assessment of bioprocess development, such as comparison of batch culture conditions.

[412] smEVs were derived from Foumierella massiliensis bacteria deposited as ATCC designation number PTA-126696. Uses for the potency assay for these smEVs include drug substance stability assessments.

Incorporation by Reference

[413] 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

[414] 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.