CN117136065A

CN117136065A - Prevotella extracellular vesicle preparation

Info

Publication number: CN117136065A
Application number: CN202280023984.8A
Authority: CN
Inventors: S·阿格塔; A·卡特赖特; D·多尔曼; T·甘古利; A·伊塔诺; C·麦肯纳; D·拉古纳坦; B·王
Original assignee: Epiva Biosciences Inc
Current assignee: Epiva Biosciences Inc
Priority date: 2021-01-26
Filing date: 2022-01-25
Publication date: 2023-11-28

Abstract

Provided herein are solutions and dried forms of Prevotella denticola Extracellular Vesicles (EV), and therapeutic compositions thereof, and methods of use thereof, useful as therapeutic agents.

Description

Prevotella extracellular vesicle preparation

Cross Reference to Related Applications

The application claims the benefit of the following U.S. provisional application numbers: 63/141,693 submitted on day 26 of month 1 of 2021; 63/175,855 submitted on month 4 of 2021; 63/196,984 submitted at 4/6/2021; and 63/289,348, filed on 12/14 of 2021, the entire contents of each of these applications are incorporated herein by reference.

Disclosure of Invention

Therapeutic compositions comprising Extracellular Vesicles (EVs), such as EVs obtained from Prevotella denticola bacteria, have therapeutic effects and are useful in the treatment and/or prevention of diseases and/or health disorders. As described herein, EVs from prasuvorexant bacteria of the tissue can be prepared as biomass (e.g., isolated EVs can be resuspended in a buffer such as PBS). As described herein, EVs from the tissue prasuvorexant bacteria may be prepared as solutions, dried forms, and/or therapeutic compositions.

In some embodiments, a dry form with a moisture content of less than about 6% is more suitable for downstream processing. In some embodiments, a dry form having a moisture content of less than about 6% has improved stability. In some embodiments, the solution comprising EV from prasuvorexant bacteria of the tissue further comprises an excipient comprising a bulking agent, and optionally one or more additional ingredients, such as lyoprotectants. In some embodiments, the solution comprising EV from prasuvorexant bacteria of the tissue further comprises an excipient comprising a lyoprotectant, and optionally one or more additional ingredients, such as bulking agents. In some embodiments, the dried form comprising EV from prasugrel bacteria of the tissue also comprises an excipient comprising a bulking agent, and optionally one or more additional ingredients, such as lyoprotectants. In some embodiments, the dried form comprising EV from Prevotella denticola bacteria further comprises an excipient comprising a lyoprotectant, and optionally one or more additional ingredients, such as bulking agents.

In certain aspects, provided herein is a method of treating an immune disorder in a subject (e.g., a human subject), the method comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasugrel tissue strain and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising the extracellular vesicles. In some embodiments, the immune disorder comprises an autoimmune disease, an inflammatory disease, or an allergy. In some embodiments, the immune disorder comprises an inflammatory disease.

In certain aspects, provided herein is the use of a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles for the manufacture of a medicament for treating an immune disorder in a subject (e.g., a human subject).

In certain aspects, provided herein are doses (e.g., therapeutically effective doses) of Extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles for use in treating an immune disorder in a subject (e.g., a human subject).

In certain aspects, provided herein is a method of treating an inflammatory disease in a subject (e.g., a human subject), the method comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising the extracellular vesicles. In some embodiments, the inflammatory disease comprises a Th1 mediated inflammatory disease. In some embodiments, the inflammatory disease comprises a Th 2-mediated inflammatory disease (e.g., asthma or atopic dermatitis). In some embodiments, the inflammatory disease comprises a Th 17-mediated inflammatory disease (e.g., psoriasis).

In certain aspects, provided herein is the use of a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles for the manufacture of a medicament for treating an inflammatory disease in a subject (e.g., a human subject).

In certain aspects, provided herein are doses (e.g., therapeutically effective doses) of Extracellular Vesicles (EVs) from a prasugrel bacterial strain of the tissue of interest and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles for use in treating an inflammatory disease in a subject (e.g., a human subject).

In certain aspects, provided herein is a method of activating TLR2 in a subject (e.g., a human subject), the method comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasugrel tissue strain and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising the extracellular vesicles.

In certain aspects, provided herein is the use of a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles for the preparation of a medicament for activating TLR2 in a subject (e.g., a human subject).

In certain aspects, provided herein are doses (e.g., therapeutically effective doses) of Extracellular Vesicles (EVs) from a prasugrel bacterial strain of the tissue of interest and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles for use in activating TLR2 in a subject (e.g., a human subject).

In certain aspects, provided herein is a method of stimulating interleukin-10 receptor (IL-10R) in a subject (e.g., a human subject), the method comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexaria histolytica strain and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising the extracellular vesicles.

In certain aspects, provided herein is the use of a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles for the manufacture of a medicament for stimulating interleukin-10 receptor (IL-10R) in a subject (e.g., a human subject).

In certain aspects, provided herein are doses (e.g., therapeutically effective doses) of Extracellular Vesicles (EVs) from a prasugrel strain of tissue, and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles, for use in stimulating interleukin-10 receptor (IL-10R) in a subject (e.g., a human subject).

In certain aspects, provided herein is a method of activating an anti-inflammatory cytokine response in a subject (e.g., a human subject), the method comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexa tissue strain and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising such extracellular vesicles. In some embodiments, the anti-inflammatory cytokine response comprises interleukin-10 (IL-10) production. In some embodiments, the anti-inflammatory cytokine response comprises IL-27 production. In some embodiments, the anti-inflammatory cytokine response comprises IL-10 and IL-27 production.

In certain aspects, provided herein is the use of a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles for the manufacture of a medicament for activating an anti-inflammatory cytokine response in a subject (e.g., a human subject).

In certain aspects, provided herein are doses (e.g., therapeutically effective doses) of Extracellular Vesicles (EVs) from a prasugrel bacterial strain of the tissue, and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles, for use in activating an anti-inflammatory cytokine response in a subject (e.g., a human subject).

In certain aspects, provided herein is a method of increasing anti-inflammatory cytokine secretion in a subject (e.g., a human subject) (e.g., in PBMCs thereof), the method comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexaria histolytica strain and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising the extracellular vesicles. In some embodiments, the anti-inflammatory cytokine is IL-10. In some embodiments, the anti-inflammatory cytokine is IL-27. In some embodiments, the anti-inflammatory cytokines are IL-10 and IL-27.

In certain aspects, provided herein is the use of a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles for the preparation of a medicament for increasing anti-inflammatory cytokine secretion in a subject (e.g., a human subject).

In certain aspects, provided herein are doses (e.g., therapeutically effective doses) of Extracellular Vesicles (EVs) from a prasugrel bacterial strain of the tissue, and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles, for use in increasing anti-inflammatory cytokine secretion in a subject (e.g., a human subject).

In certain aspects, provided herein is a method of activating TLR1/2 and/or TLR2/6 heterodimers in a subject (e.g., a human subject), the method comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexaria histolytica strain and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising the extracellular vesicles.

In certain aspects, provided herein are doses (e.g., therapeutically effective doses) of Extracellular Vesicles (EVs) from a prasugrel strain of tissue, and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles, for use in activating TLR1/2 and/or TLR2/6 heterodimers in a subject (e.g., a human subject).

In certain aspects, provided herein is the use of a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles for the preparation of a medicament for activating TLR1/2 and/or TLR2/6 heterodimers in a subject (e.g., a human subject).

In some embodiments, the prasuvorexant strain of tissue (e.g., in an in vitro assay) activates TLR1/2 and/or TLR2/6 heterodimers, e.g., as described herein.

In certain aspects, provided herein is a method of directing T cells of a subject (e.g., a human subject) to reduce inflammation, the method comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexaria strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising such extracellular vesicles.

In certain aspects, provided herein are doses (e.g., therapeutically effective doses) of Extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles for use in directing T-cell reduction inflammation in a subject (e.g., a human subject).

In certain aspects, provided herein is the use of a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles for the manufacture of a medicament for directing T cell reduction inflammation in a subject (e.g., a human subject).

In some embodiments, the T cells are directed in a mesenteric lymph node.

In some embodiments, extracellular Vesicles (EVs) from a prasuvorexa strain of tissue and/or compositions comprising such extracellular vesicles (e.g., solutions, dried forms, and/or therapeutic compositions) are orally administered (e.g., and into the small intestine), dendritic cells interact with Extracellular Vesicles (EVs) from a prasuvorexa strain of tissue and/or compositions comprising such extracellular vesicles (e.g., solutions, dried forms, and/or therapeutic compositions) in the small intestine, such dendritic cells enter the mesenteric lymph nodes, and T cells transported by the mesenteric lymph nodes meet such dendritic cells.

In certain aspects, provided herein is a method of affecting T cells transported to a mesenteric lymph node of a subject (e.g., a human subject), the method comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexala strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles.

In certain aspects, provided herein are doses (e.g., therapeutically effective doses) of Extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles for use in T cells affecting a mesenteric lymph node transported to a subject (e.g., a human subject).

In certain aspects, provided herein is the use of a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles for the preparation of a medicament for affecting T cells transported to a mesenteric lymph node of a subject (e.g., a human subject).

In certain aspects, provided herein is a method of producing anti-inflammatory cd4+ T cells in a subject (e.g., a human subject), the method comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexa tissue strain and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising such extracellular vesicles. In some embodiments, extracellular vesicles produce a population of cd4+ T cells that can resolve inflammation, as demonstrated in the examples provided herein.

In certain aspects, provided herein are doses (e.g., therapeutically effective doses) of Extracellular Vesicles (EVs) from a prasugrel bacterial strain of the tissue of interest and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles for use in generating anti-inflammatory cd4+ T cells of a subject (e.g., a human subject).

In certain aspects, provided herein is the use of a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles for the manufacture of a medicament for producing anti-inflammatory cd4+ T cells in a subject (e.g., a human subject).

In certain aspects, provided herein is a method of resolving inflammation in a subject (e.g., a human subject), the method comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexa tissue strain and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising the extracellular vesicles. In some embodiments, extracellular vesicles administered in the absence of inflammation do not suppress the immune response, but rather resolve the persistent inflammatory response, as demonstrated in the examples provided herein. In some embodiments, as described in the examples herein, the antigen-independent mechanism may reduce antigen-specific inflammation.

In certain aspects, provided herein are doses (e.g., therapeutically effective doses) of Extracellular Vesicles (EVs) from a prasugrel bacterial strain of the tissue, and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles, for use in resolving inflammation in a subject (e.g., a human subject).

In certain aspects, provided herein is the use of a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles for the manufacture of a medicament for resolving inflammation in a subject (e.g., a human subject).

In some embodiments, the subject (e.g., a human subject) has Th 1-mediated inflammation.

In some embodiments, the subject (e.g., a human subject) has Th 2-mediated inflammation.

In some embodiments, the subject (e.g., a human subject) has Th 17-mediated inflammation.

In some embodiments of the methods provided herein, a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles are administered in combination with an anti-tnfα antibody. In some embodiments of the methods provided herein, a dose (e.g., a therapeutically effective dose) of prasuvorexant from the percha tissueExtracellular Vesicles (EV) of bacterial strains and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles are administered in combination with a TNFa antagonist (e.g., a TNFa antagonist or a TNFa receptor antagonist), e.g., adalimumabEtanerceptInfliximab (++>TA-650), polyethylene glycol cetuximab (>CDP 870), golimumab (>CNTO 148), anakinra- >RituximabArbazedox->Tozumazumab (Roactmura-)。

In some embodiments, the extracellular vesicles are from a prasuvorexa strain of tissue that has 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) with the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of prasuvorexa strain B of tissue (NRRL accession No. B50329). In some embodiments, the prasuvorexant strain of tissue is prasuvorexant strain B of tissue (NRRL accession No. B50329).

In some embodiments, the subject (e.g., a human subject) has an immune disorder. In some embodiments of the present invention, in some embodiments, the immune disorder is joint sclerosis, arthritis, phlebitis, vasculitis and lymphangitis, cholangitis, cholecystitis, enteritis, enterocolitis, gastritis, gastroenteritis, inflammatory bowel disease, ileitis, proctitis, crohn's disease, ulcerative colitis, irritable bowel syndrome, microscopic colitis, lymphocytic-plasmacytoid enteritis, celiac disease, collagenous colitis, lymphocytic colitis, eosinophilic enterocolitis, non-established colitis, pseudomembranous colitis (necrotizing colitis), ischemic inflammatory bowel disease, behcet's disease, sarcoidosis, scleroderma, IBD-related dysplasia, dysplasia-related mass or lesions, primary sclerosing cholangitis, cervicitis, chorioamnitis, endometritis, epididymitis, navel inflammation, oogonitis, orchitis, salpingitis, ovaginal ovarian abscess urethritis, vaginitis, vulvitis, vulvodynia, acute disseminated alopecia, behcet's disease, QIAGASHEN's disease, chronic fatigue syndrome, autonomic imbalance, encephalomyelitis, ankylosing spondylitis, aplastic anemia, suppurative sweat gland, autoimmune hepatitis, autoimmune ovaritis, celiac disease, type 1 diabetes, giant cell arteritis, goldpasm's syndrome, graves ' disease, grin-Barlich syndrome, hashimoto's disease, hun-Schenen's purple spot, kawasaki disease, lupus erythematosus, microscopic colitis, polyarteritis under microscope, mixed connective tissue disease, murray's syndrome, multiple sclerosis, myasthenia gravis, strabismus myoclonus syndrome, optic neuritis, orde thyroiditis, pemphigus, polyarteritis nodosa, oncomeningitis, polymyalgia, rheumatoid arthritis, lyter's syndrome, sjogren's syndrome, temporal arteritis, wegener's granulomatosis, warm autoimmune hemolytic anemia, interstitial cystitis, lyme disease, scleroderma, psoriasis, sarcoidosis, scleroderma, contact hypersensitivity, contact dermatitis (including contact dermatitis due to poison ivy), urticaria, skin allergy, airway allergy (hay fever, allergic rhinitis, house dust mite allergy), appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, suppurative sweat gland, iritis, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis, pleurisy, pneumonia, prostatitis, pyelonephritis, stomatitis, transplant rejection, acute pancreatitis chronic pancreatitis, acute respiratory distress syndrome, cerclage syndrome, congenital adrenal hyperplasia, non-suppurative thyroiditis, cancer-related hypercalcemia, pemphigus, bullous dermatitis herpetiformis, severe erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, seasonal or perennial allergic rhinitis, bronchial asthma, contact dermatitis, atopic dermatitis, drug hypersensitivity, allergic conjunctivitis, keratitis, ocular shingles, iritis and iridocyclitis, chorioretinitis, optic neuritis, symptomatic sarcoidosis, fulminant or disseminated tuberculosis chemotherapy, adult idiopathic thrombocytopenic purpura, adult secondary thrombocytopenia, acquired (autoimmune) hemolytic anemia, adult leukemia, lymphoma, pediatric acute leukemia, localized enteritis, autoimmune vasculitis, chronic obstructive pulmonary disease, or sepsis.

In some embodiments, the subject (e.g., a human subject) has psoriasis.

In some embodiments, the subject (e.g., a human subject) has atopic dermatitis.

In some embodiments, the subject (e.g., a human subject) has a Th 1-mediated inflammatory disease.

In some embodiments, the subject (e.g., a human subject) has a Th 2-mediated inflammatory disease.

In some embodiments, the subject (e.g., a human subject) has a Th 17-mediated inflammatory disease.

In some embodiments, EV is administered orally.

In certain aspects, extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles reduce inflammation in a model of DTH inflammation.

In certain aspects, extracellular Vesicles (EVs) from a prasugrel strain of tissue and/or compositions comprising these extracellular vesicles (e.g., solutions, dried forms, and/or therapeutic compositions) stimulate TLR2 in HEK293 reporter cell line assays cultured in vitro.

In certain aspects, extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles stimulate IL-10 secretion from U937 cells cultured in vitro (e.g., cells that have been differentiated with PMA).

In certain aspects, extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles stimulate IL-10 secretion from in vitro cultured human PBMCs.

In certain aspects, extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles stimulate IL-27 secretion from in vitro cultured human PBMCs.

In certain aspects, extracellular Vesicles (EV) from Prevotella denticola strains and/or compositions comprising such extracellular vesicles (e.g., solutions, dried forms, and/or therapeutic compositions) induce IL-10, IL-27, IL-6, IP-10, and/or TNFa secretion from in vitro cultured human PBMC.

In certain aspects, extracellular Vesicles (EV) from Prevotella denticola strains and/or compositions comprising such extracellular vesicles (e.g., solutions, dried forms, and/or therapeutic compositions) induce IL-10, IL-27, IL-6, IP-10, and/or TNFa secretion by human macrophages cultured in vitro.

In certain aspects, extracellular Vesicles (EV) from Prevotella denticola strains and/or compositions comprising such extracellular vesicles (e.g., solutions, dried forms, and/or therapeutic compositions) induce IL-10, IL-27, IL-6, IP-10, and/or TNFa secretion by human dendritic cells cultured in vitro. In certain aspects, extracellular Vesicles (EVs) from a prasuvorexant strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising these extracellular vesicles induce the secretion of IL-10, IL-6, and/or TNFa from human dendritic cells cultured in vitro.

In certain aspects, extracellular Vesicles (EV) from Prevotella denticola strains and/or compositions comprising these extracellular vesicles (e.g., solutions, dried forms, and/or therapeutic compositions) induce IL-10, IL-27, IL-6, IP-10, and TNFa secretion from U937 cells cultured in vitro.

In some embodiments, the dose is in the form of one or more capsules, which optionally comprise an enteric coating (e.g., an enteric coated capsule). In some embodiments, the dose is in the form of one or more tablets, which optionally comprise an enteric coating (e.g., an enteric coated tablet). In some embodiments, the dose is in the form of one or more miniature tablets. In some embodiments, these miniature tablets are enteric coated miniature tablets. In some embodiments, the dose is in the form of a non-enteric coated capsule comprising one or more enteric coated minitablets.

As disclosed herein, the tissue prasuvorexant Extracellular Vesicles (EVs) have therapeutic effects and are useful in the treatment and/or prevention of diseases and/or health disorders. Biomass, solution and dry forms of therapeutic compositions containing Prevotella EV, a tissue of interest, can be prepared.

Bulking agents and/or lyoprotectants are used in the preparation of Extracellular Vesicles (EVs) for drying, e.g., freeze drying and spray drying. In some embodiments, bulking agents, including but not limited to sucrose, mannitol, polyethylene glycol (PEG, such as PEG 6000), cyclodextrin, maltodextrin, and dextran (such as dextran 40 k), make the dried form (such as a powder and/or lyophilisate) easier to handle after drying. In some embodiments, the filler improves the characteristics of the dry form. In some embodiments, lyoprotectants (including, but not limited to, trehalose, sucrose, and lactose) protect the EV during drying (e.g., freeze drying or spray drying). In some embodiments, excipients are used to reduce drying cycle time. In some embodiments, the excipient is used to maintain therapeutic efficacy of the EV.

In some aspects, the disclosure provides a dry form comprising Extracellular Vesicles (EVs) from prasugrel bacteria of a tissue of interest, wherein the dry form has a moisture content of less than about 6% (e.g., as determined by the karl fischer method).

In some embodiments, the dried forms provided herein have a moisture content of less than about 5% (e.g., as determined by the karl fischer method).

In some embodiments, the dried forms provided herein have a moisture content of less than about 4% (e.g., as determined by the karl fischer method).

In some embodiments, the dry forms provided herein have a moisture content of about 1% to about 4% (e.g., as determined by the karl fischer method).

In some embodiments, the dry forms provided herein have a moisture content of about 2% to about 4% (e.g., as determined by the karl fischer method).

In some embodiments, the dry forms provided herein have a moisture content of about 2% to about 3% (e.g., as determined by the karl fischer method).

In some aspects, the disclosure provides a lyophilizate comprising Extracellular Vesicles (EVs) from prasugrel bacteria of a tissue, wherein the lyophilizate has a moisture content of less than about 6% (e.g., as determined by the karl fischer method).

In some embodiments, the lyophilizate has a moisture content of less than about 5% (e.g., as determined by the karl fischer method).

In some embodiments, the lyophilizate has a moisture content of less than about 4% (e.g., as determined by the karl fischer method).

In some embodiments, the lyophilizate has a moisture content of about 1% to about 4% (e.g., as determined by the karl fischer method).

In some embodiments, the lyophilizate has a moisture content of about 2% to about 4% (e.g., as determined by the karl fischer method).

In some embodiments, the lyophilizate has a moisture content of about 2% to about 3% (e.g., as determined by the karl fischer method).

In some aspects, the disclosure provides a powder comprising Extracellular Vesicles (EVs) from prasugrel bacteria of the tissue, wherein the powder has a moisture content of less than about 6% (e.g., as determined by the karl fischer method).

In some embodiments, the powder has a moisture content of less than about 5% (e.g., as determined by the karl fischer method).

In some embodiments, the powder has a moisture content of less than about 4% (e.g., as determined by the karl fischer method).

In some embodiments, the powder has a moisture content of about 1% to about 4% (e.g., as determined by the karl fischer method).

In some embodiments, the powder has a moisture content of about 2% to about 4% (e.g., as determined by the karl fischer method).

In some embodiments, the powder has a moisture content of about 2% to about 3% (e.g., as determined by the karl fischer method).

In some aspects, the present disclosure provides a dry form comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of a tissue, and an excipient, wherein the excipient comprises about 95% to about 99% of the total mass of the dry form.

In some aspects, the present disclosure provides a dry form comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of a tissue, and an excipient, wherein EVs may comprise about 2% to about 6% of the total mass of the dry form.

In some embodiments of the dry forms provided herein, the dry form comprises a powder. In some embodiments, the powder comprises a lyophilized powder. In some embodiments, the powder comprises a spray-dried powder.

In some embodiments of the dry forms provided herein, the dry form comprises a lyophilisate. In some embodiments, the lyophilisate comprises a lyophilized powder. In some embodiments, the lyophilisate comprises a lyophilized cake.

In some aspects, the disclosure provides Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue.

In some aspects, the present disclosure provides a therapeutic composition comprising prasuvorexant EV, a tissue, wherein the composition further comprises a pharmaceutically acceptable excipient.

In some aspects, the present disclosure provides a solution comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent.

In some aspects, the disclosure provides a solution consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent.

In some aspects, the present disclosure provides a solution comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent and a lyoprotectant.

In some aspects, the disclosure provides a solution consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent and a lyoprotectant.

In some aspects, the disclosure provides a solution comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a lyoprotectant.

In some aspects, the disclosure provides a solution consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a lyoprotectant.

In some aspects, the present disclosure provides a therapeutic composition comprising a solution, wherein the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a glidant, a lubricant, and/or a diluent.

In some aspects, the present disclosure provides a dry form comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of a tissue, and an excipient comprising a bulking agent.

In some aspects, the present disclosure provides a dry form consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent.

In some aspects, the present disclosure provides a dry form comprising Extracellular Vesicles (EVs) from prasugrel bacteria of the tissue, and an excipient comprising a bulking agent and a lyoprotectant.

In some aspects, the present disclosure provides a dry form consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent and a lyoprotectant.

In some aspects, the disclosure provides a dry form comprising Extracellular Vesicles (EVs) from prasugrel bacteria of the tissue, and an excipient comprising a lyoprotectant.

In some aspects, the disclosure provides a dry form consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a lyoprotectant.

In some aspects, the present disclosure provides a therapeutic composition comprising a dry form, wherein the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a glidant, a lubricant, and/or a diluent.

In some aspects, the present disclosure provides a powder comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent.

In some aspects, the present disclosure provides a powder consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent.

In some aspects, the present disclosure provides a powder comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent and a lyoprotectant.

In some aspects, the present disclosure provides a powder consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent and a lyoprotectant.

In some aspects, the present disclosure provides a powder comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a lyoprotectant.

In some aspects, the disclosure provides a powder consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a lyoprotectant.

In some aspects, the present disclosure provides a therapeutic composition comprising a powder, wherein the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a glidant, a lubricant, and/or a diluent.

In some aspects, the present disclosure provides a spray-dried powder comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent.

In some aspects, the present disclosure provides a spray-dried powder consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent.

In some aspects, the present disclosure provides a spray-dried powder comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent and a lyoprotectant.

In some aspects, the present disclosure provides a spray-dried powder consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent and a lyoprotectant.

In some aspects, the present disclosure provides a spray-dried powder comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a lyoprotectant.

In some aspects, the present disclosure provides a spray-dried powder consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a lyoprotectant.

In some aspects, the present disclosure provides a therapeutic composition comprising a spray-dried powder, wherein the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a glidant, a lubricant, and/or a diluent.

In some aspects, the disclosure provides a lyophilizate comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent.

In some aspects, the disclosure provides a lyophilizate consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent.

In some aspects, the disclosure provides a lyophilizate comprising Extracellular Vesicles (EVs) from prasugrel bacteria of the tissue, and an excipient comprising a bulking agent and a lyoprotectant.

In some aspects, the disclosure provides a lyophilizate consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue and an excipient comprising a bulking agent and a lyoprotectant.

In some aspects, the disclosure provides a lyophilizate comprising Extracellular Vesicles (EVs) from prasugrel bacteria of the tissue, and an excipient comprising a lyoprotectant.

In some aspects, the disclosure provides a lyophilizate consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue and an excipient comprising a lyoprotectant.

In some aspects, the present disclosure provides therapeutic compositions comprising a lyophilizate, wherein the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a glidant, a lubricant, and/or a diluent.

In some aspects, the present disclosure provides a lyophilized powder comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of a tissue, and an excipient comprising a bulking agent.

In some aspects, the present disclosure provides a lyophilized powder consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent.

In some aspects, the present disclosure provides a lyophilized powder comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of a tissue, and an excipient comprising a bulking agent and a lyoprotectant.

In some aspects, the present disclosure provides a lyophilized powder consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent and a lyoprotectant.

In some aspects, the present disclosure provides a lyophilized powder comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of a tissue, and an excipient comprising a lyoprotectant.

In some aspects, the present disclosure provides a lyophilized powder consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a lyoprotectant.

In some aspects, the present disclosure provides therapeutic compositions comprising a lyophilized powder, wherein the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a glidant, a lubricant, and/or a diluent.

In some aspects, the disclosure provides a lyophilized cake comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent.

In some aspects, the disclosure provides a lyophilized cake consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising bulking agents.

In some aspects, the disclosure provides a lyophilized cake comprising Extracellular Vesicles (EVs) from prasugrel bacteria of the tissue, and an excipient comprising a bulking agent and a lyoprotectant.

In some aspects, the present disclosure provides a lyophilized cake consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent and a lyoprotectant.

In some aspects, the disclosure provides a lyophilized cake comprising Extracellular Vesicles (EVs) from prasugrel bacteria of the tissue, and an excipient comprising a lyoprotectant.

In some aspects, the disclosure provides a lyophilized cake consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a lyoprotectant.

In some aspects, the present disclosure provides therapeutic compositions comprising Extracellular Vesicles (EVs) from prasuvorexa bacteria of the tissue, and an excipient comprising a bulking agent.

In some aspects, the present disclosure provides therapeutic compositions consisting essentially of Extracellular Vesicles (EVs) from prasuvorexa bacteria of the tissue, and an excipient comprising a bulking agent.

In some aspects, the present disclosure provides therapeutic compositions comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a bulking agent and a lyoprotectant.

In some aspects, the present disclosure provides therapeutic compositions consisting essentially of Extracellular Vesicles (EVs) from Prevotella tissue and excipients comprising bulking agents and lyoprotectants.

In some aspects, the present disclosure provides therapeutic compositions comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a lyoprotectant.

In some aspects, the present disclosure provides therapeutic compositions consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and excipients comprising lyoprotectants.

In some aspects, the disclosure provides a solution comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of a tissue, and an excipient comprising a stock solution of one or more excipients, wherein the stock solution comprises the formulation provided in table A, B, C, D, K or P.

In some aspects, the disclosure provides a solution consisting essentially of Extracellular Vesicles (EVs) from prasugrel bacteria of tissue, and an excipient comprising a stock solution of one or more excipients, wherein the stock solution comprises the formulation provided in table A, B, C, D, K or P.

In some aspects, the present disclosure provides therapeutic compositions comprising such solutions, wherein the compositions further comprise a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a glidant, a lubricant, and/or a diluent.

In some aspects, the disclosure provides a dry form comprising Extracellular Vesicles (EVs) from prasugrel bacteria of a tissue, and an excipient comprising a stock solution of one or more excipients, wherein the stock solution comprises the formulation provided in table A, B, C, D, K or P.

In some aspects, the disclosure provides a dry form consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of a tissue, and an excipient comprising a stock solution of one or more excipients, wherein the stock solution comprises the formulation provided in table A, B, C, D, K or P.

In some aspects, the present disclosure provides therapeutic compositions comprising such dry forms, wherein the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a glidant, a lubricant, and/or a diluent.

In some aspects, the present disclosure provides a powder comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of a tissue, and an excipient comprising a stock solution of one or more excipients, wherein the stock solution comprises the formulation provided in table A, B, C, D, K or P.

In some aspects, the disclosure provides a powder consisting essentially of Extracellular Vesicles (EVs) from prasugrel bacteria of tissue, and an excipient comprising a stock solution of one or more excipients, wherein the stock solution comprises the formulation provided in table A, B, C, D, K or P.

In some aspects, the present disclosure provides therapeutic compositions comprising such powders, wherein the compositions further comprise a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a glidant, a lubricant, and/or a diluent.

In some aspects, the present disclosure provides a spray-dried powder comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of a tissue, and an excipient comprising a stock solution of one or more excipients, wherein the stock solution comprises the formulation provided in table A, B, C, D, K or P.

In some aspects, the present disclosure provides a spray-dried powder consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue, and an excipient comprising a stock solution of one or more excipients, wherein the stock solution comprises the formulation provided in table A, B, C, D, K or P.

In some aspects, the present disclosure provides therapeutic compositions comprising such spray-dried powders, wherein the compositions further comprise a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a glidant, a lubricant, and/or a diluent.

In some aspects, the disclosure provides a lyophilizate comprising Extracellular Vesicles (EVs) from prasugrel bacteria of tissue and an excipient comprising a stock solution of one or more excipients, wherein the stock solution comprises the formulation provided in table A, B, C, D, K or P.

In some aspects, the disclosure provides a lyophilizate consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of tissue and an excipient comprising a stock solution of one or more excipients, wherein the stock solution comprises the formulation provided in table A, B, C, D, K or P.

In some aspects, the present disclosure provides therapeutic compositions comprising such lyophilisates, wherein the compositions further comprise a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a glidant, a lubricant, and/or a diluent.

In some aspects, the disclosure provides a lyophilized powder comprising Extracellular Vesicles (EVs) from prasugrel bacteria of a tissue, and an excipient comprising a stock solution of one or more excipients, wherein the stock solution comprises the formulation provided in table A, B, C, D, K or P.

In some aspects, the disclosure provides a lyophilized powder consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of a tissue, and an excipient comprising a stock solution of one or more excipients, wherein the stock solution comprises the formulation provided in table A, B, C, D, K or P.

In some aspects, the present disclosure provides therapeutic compositions comprising such lyophilized powders, wherein the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a glidant, a lubricant, and/or a diluent.

In some aspects, the disclosure provides a lyophilized cake comprising Extracellular Vesicles (EVs) from prasugrel bacteria of tissue and an excipient comprising a stock solution of one or more excipients, wherein the stock solution comprises the formulation provided in table A, B, C, D, K or P.

In some aspects, the disclosure provides a lyophilized cake consisting essentially of Extracellular Vesicles (EVs) from prasuvorexant bacteria of the tissue and an excipient comprising a stock solution of one or more excipients, wherein the stock solution comprises the formulation provided in table A, B, C, D, K or P.

In some aspects, the present disclosure provides therapeutic compositions comprising such lyophilized cakes, wherein the compositions further comprise a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a glidant, a lubricant, and/or a diluent.

In some aspects, the disclosure provides methods of treating a subject (e.g., a human) (e.g., a subject in need of treatment), the method comprising:

the subject is administered a prasuvorexant EV or solution, dried form, or therapeutic composition of the percha tissue described herein.

In some embodiments, a prasuvorexant EV as a tissue provided herein or a solution, dried form, or therapeutic composition is for use in treating a subject (e.g., a human) (e.g., a subject in need of treatment).

In some aspects, the disclosure provides the use of a prasuvorexant EV or solution, dried form, or therapeutic composition provided herein for the manufacture of a medicament for treating a subject (e.g., a human) (e.g., a subject in need of treatment).

In some embodiments of the methods, solutions, dried forms, therapeutic compositions, or uses provided herein, the tissue prasuvorexant EV or solution, dried form, or therapeutic composition is administered orally (e.g., for oral administration).

In some embodiments of the methods, solutions, dry forms, therapeutic compositions, or uses provided herein, a subject is in need of treatment (and/or prevention) of an immune disorder.

In some embodiments of the methods, solutions, dry forms, therapeutic compositions, or uses provided herein, a subject is in need of treatment (and/or prevention) of an autoimmune disease.

In some embodiments of the methods, solutions, dry forms, therapeutic compositions, or uses provided herein, a subject is in need of treatment (and/or prevention) of an inflammatory disease.

In some embodiments of the methods, solutions, dry forms, therapeutic compositions, or uses provided herein, a subject is in need of treatment (and/or prevention) of a metabolic disorder.

In some embodiments of the methods, solutions, dry forms, therapeutic compositions, or uses provided herein, a subject is in need of treatment (and/or prevention) of a dysbacteriosis.

In some embodiments of the methods, solutions, dry forms, therapeutic compositions, or uses provided herein, the solution, dry form, or therapeutic composition is administered in combination with an additional therapeutic agent.

In some embodiments of the methods, solutions, dry forms, therapeutic compositions, or uses provided herein, the dry form is a powder. In some embodiments, the powder is a lyophilized powder. In some embodiments, the powder is a spray-dried powder.

In some embodiments of the methods, solutions, dry forms, therapeutic compositions, or uses provided herein, the dry form is a lyophilizate. In some embodiments, the lyophilisate is a lyophilized powder. In some embodiments, the lyophilisate is a lyophilized cake.

In some aspects, the present disclosure provides a method of preparing a solution comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of a tissue, the method comprising:

a liquid formulation comprising EV from prasuvorexant bacteria of the tissue is combined with an excipient comprising (or consisting essentially of) a bulking agent to prepare a solution.

a liquid formulation comprising an EV from a prasugrel bacterium of a tissue of interest is combined with an excipient comprising (or consisting essentially of) a bulking agent and a lyoprotectant to prepare a solution.

a liquid formulation comprising an EV from a prasugrel bacterium of a tissue of interest is combined with an excipient comprising (or consisting essentially of) a lyoprotectant to prepare a solution.

In some embodiments, the present disclosure provides solutions prepared by the methods described herein.

In some aspects, the present disclosure provides a method of preparing a dried form comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of a tissue, the method comprising:

Combining a liquid formulation comprising an EV from a prasugrel bacteria of the tissue with an excipient comprising (or consisting essentially of) a bulking agent to prepare a solution; and is also provided with

The solution is dried, thereby preparing the dried form.

combining a liquid formulation comprising an EV from a prasugrel bacteria of the tissue with an excipient comprising (or consisting essentially of) a bulking agent to prepare a solution;

drying the solution to prepare a cake, and

the cake is milled (e.g., ground) to produce the dry form.

combining a liquid formulation comprising an EV from a prasugrel bacteria of the tissue with an excipient comprising (or consisting essentially of) a bulking agent and a lyoprotectant to prepare a solution; and is also provided with

The solution is dried, thereby preparing the dried form.

Combining a liquid formulation comprising an EV from a prasugrel bacteria of the tissue with an excipient comprising (or consisting essentially of) a bulking agent and a lyoprotectant to prepare a solution;

drying the solution to prepare a cake, and

the cake is milled (e.g., ground) to produce the dry form.

combining a liquid formulation comprising an EV from a prasugrel bacterium of a tissue, with an excipient comprising (or consisting essentially of) a lyoprotectant to prepare a solution; and is also provided with

The solution is dried, thereby preparing the dried form.

combining a liquid formulation comprising an EV from a prasugrel bacterium of a tissue, with an excipient comprising (or consisting essentially of) a lyoprotectant to prepare a solution;

drying the solution to prepare a cake, and

the cake is milled (e.g., ground) to produce the dry form.

In some embodiments of the methods of preparing a dried form provided herein, drying comprises lyophilization.

In some embodiments of the methods of preparing a dried form provided herein, drying comprises spray drying.

In some embodiments of the methods of preparing a dry form provided herein, the methods further comprise combining the dry form with additional ingredients. In some embodiments, the additional ingredients comprise excipients, such as glidants, lubricants, and/or diluents.

In some embodiments, the present disclosure provides a dried form prepared by the methods described herein.

In some aspects, the present disclosure provides a method of preparing a powder comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of a tissue, the method comprising:

The solution was dried to prepare the powder.

Drying the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the powder.

The solution was dried to prepare the powder.

drying the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the powder.

The solution was dried to prepare the powder.

drying the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the powder.

In some embodiments of the methods of preparing a powder provided herein, drying comprises lyophilization.

In some embodiments of the methods of preparing a powder provided herein, drying comprises spray drying.

In some embodiments of the methods of preparing a powder provided herein, the method further comprises combining the powder with additional ingredients. In some embodiments, the additional ingredients comprise excipients, such as glidants, lubricants, and/or diluents.

In some embodiments, the present disclosure provides powders prepared by the methods described herein.

In some aspects, the present disclosure provides a method of preparing a spray-dried powder comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of a tissue, the method comprising:

Spray drying the solution to produce the spray dried powder.

In some embodiments of the methods of preparing a spray-dried powder provided herein, the method further comprises combining the spray-dried powder with additional ingredients. In some embodiments, the additional ingredients comprise excipients, such as glidants, lubricants, and/or diluents.

In some embodiments, the present disclosure provides spray-dried powders prepared by the methods described herein.

In some aspects, the present disclosure provides a method of preparing a lyophilizate comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of a tissue, the method comprising:

The solution was freeze-dried (lyophilized), thereby preparing the lyophilized product.

freeze drying (lyophilization) the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the lyophilizate.

freeze drying (lyophilization) the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the lyophilizate.

Freeze drying (lyophilization) the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the lyophilizate.

In some embodiments of the methods of preparing a lyophilizate provided herein, the method further comprises combining the lyophilizate with additional ingredients. In some embodiments, the additional ingredients comprise excipients, such as glidants, lubricants, and/or diluents.

In some embodiments, the present disclosure provides a lyophilizate prepared by the methods described herein.

In some aspects, the present disclosure provides a method of preparing a lyophilized powder comprising Extracellular Vesicles (EVs) from prasuvorexant bacteria of a tissue, the method comprising:

The solution was freeze-dried (lyophilized), thereby preparing the lyophilized powder.

Freeze drying (lyophilization) the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the lyophilized powder.

freeze drying (lyophilization) the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the lyophilized powder.

freeze drying (lyophilization) the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the lyophilized powder.

In some embodiments of the methods of preparing a lyophilized powder provided herein, the method further comprises combining the lyophilized powder with additional ingredients. In some embodiments, the additional ingredients comprise excipients, such as glidants, lubricants, and/or diluents.

In some embodiments, the present disclosure provides a lyophilized powder prepared by the methods described herein.

In some aspects, the present disclosure provides a method of preparing a lyophilized cake comprising Extracellular Vesicles (EVs) from Prevotella denticola bacteria, the method comprising:

The solution was freeze-dried (lyophilized), thereby preparing the lyophilized cake.

In some embodiments, the present disclosure provides a lyophilized cake prepared by the methods described herein.

a liquid formulation comprising an EV from a prasugrel bacteria of the tissue, is combined with a stock solution comprising one or more excipients, wherein the stock solution comprises the formulation provided in table A, B, C, D, K or P, thereby preparing a solution.

combining a liquid formulation comprising an EV from a prasuvorexant bacterium of the tissue with a stock solution comprising one or more excipients, wherein the stock solution comprises the formulation provided in table A, B, C, D, K or P, thereby preparing a solution; and is also provided with

The solution is dried, thereby preparing the dried form.

combining a liquid formulation comprising an EV from a prasuvorexant bacterium of the tissue with a stock solution comprising one or more excipients, wherein the stock solution comprises the formulation provided in table A, B, C, D, K or P, thereby preparing a solution;

Drying the solution to prepare a cake, and

the cake is milled (e.g., ground) to produce the dry form.

The solution was dried to prepare the powder.

drying the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the powder.

Spray drying the solution to produce the spray dried powder.

Freeze drying (lyophilization) the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the lyophilizate.

Freeze drying (lyophilization) the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the lyophilized powder.

The solution was freeze-dried (lyophilized), thereby preparing a lyophilized cake.

In some embodiments of the method comprising a freeze drying step, freeze drying comprises primary drying and secondary drying. In some embodiments, the primary drying is performed at a temperature between about-35 ℃ and about-20 ℃. For example, the primary drying is performed at a temperature of about-20deg.C, about-25deg.C, about-30deg.C or about-35deg.C. In some embodiments, the secondary drying is performed at a temperature between about +20 ℃ and about +30 ℃. For example, the secondary drying is performed at a temperature of about +25℃.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, the filler comprises mannitol, sucrose, maltodextrin, dextran, ficoll, polyethylene glycol (PEG, e.g., PEG 6000), cyclodextrin, or PVP-K30.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, the filler comprises mannitol.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, the excipient comprises additional ingredients.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, additional ingredients include trehalose, mannitol, sucrose, sorbitol, dextran, poloxamer 188, maltodextrin, PVP-K30, ficoll, citrate, arginine, and/or hydroxypropyl-B-cyclodextrin.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, the excipient comprises mannitol and trehalose.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, the excipient consists essentially of mannitol and trehalose.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, the excipient comprises mannitol, trehalose, and sorbitol.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, the excipient consists essentially of mannitol, trehalose, and sorbitol.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, the excipient comprises trehalose.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, the excipient consists essentially of trehalose.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, the excipient is from a stock solution comprising one or more excipients, wherein the stock solution comprises the formulation provided in table A, B, C, D, K or P.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, the dry form is a powder. In some embodiments, the powder is a lyophilized powder. In some embodiments, the powder is a spray-dried powder.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, the dry form is a lyophilizate. In some embodiments, the lyophilisate is a lyophilized powder. In some embodiments, the lyophilisate is a lyophilized cake.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, the excipient solution comprises mannitol and trehalose, wherein the mannitol and trehalose are not present in equal amounts (e.g., mannitol and trehalose are present in unequal amounts; e.g., on a weight basis or on a weight percent basis). In some embodiments, the excipient solution comprises more mannitol than trehalose, e.g., on a weight basis or on a weight percent basis. In some embodiments, the excipient solution comprises at least twice as much mannitol as trehalose, e.g., on a weight basis or on a weight percent basis. In some embodiments, the excipient solution comprises at least three times more mannitol than trehalose, e.g., on a weight basis or on a weight percent basis. In some embodiments, the excipient in solution or dry form comprises mannitol and trehalose, wherein mannitol and trehalose are not present in equal amounts (e.g., mannitol and trehalose are present in unequal amounts; e.g., on a weight basis or a weight percent basis). In some embodiments, the excipient in solution or dry form comprises more mannitol than trehalose, e.g., on a weight basis or on a weight percent basis. In some embodiments, the excipient in solution or dry form comprises at least twice as much mannitol as trehalose, e.g., on a weight basis or on a weight percent basis. In some embodiments, the excipient in solution or dry form comprises at least three times more mannitol than trehalose, e.g., on a weight basis or on a weight percent basis.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, the excipient solution consists essentially of mannitol and trehalose. In some embodiments, the excipient solution consists essentially of mannitol and trehalose, wherein the mannitol and trehalose are not present in equal amounts (e.g., mannitol and trehalose are present in unequal amounts; e.g., on a weight basis or a weight percent basis). In some embodiments, the excipient solution consists essentially of mannitol and trehalose, wherein the excipient contains more mannitol than trehalose, e.g., on a weight basis or on a weight percent basis. In some embodiments, the excipient solution consists essentially of mannitol and trehalose, wherein the excipient solution contains at least twice as much mannitol as trehalose, e.g., on a weight basis or on a weight percent basis. In some embodiments, the excipient solution consists essentially of mannitol and trehalose, wherein the excipient solution contains at least three times more mannitol than trehalose, e.g., on a weight basis or on a weight percent basis. In some embodiments, the excipient in solution or dry form consists essentially of mannitol and trehalose, wherein the excipient in solution or dry form contains more mannitol than trehalose, e.g., on a weight basis or a weight percent basis. In some embodiments, the excipient in solution or dry form consists essentially of mannitol and trehalose, wherein the excipient in solution or dry form contains at least twice as much mannitol as trehalose, e.g., on a weight basis or on a weight percent basis. In some embodiments, the excipient in solution or dry form consists essentially of mannitol and trehalose, wherein the excipient in solution or dry form contains at least three times more mannitol than trehalose, e.g., on a weight basis or a weight percent basis.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, the excipient solution comprises or consists essentially of mannitol and trehalose, wherein neither mannitol nor trehalose is present in an amount of 5mg/ml to 15 mg/ml. In some embodiments, the excipient solution comprises or consists essentially of mannitol and trehalose, wherein mannitol is not present in an amount of 5mg/ml to 15 mg/ml. In some embodiments, the excipient solution comprises or consists essentially of mannitol and trehalose, wherein trehalose is not present in an amount of 5mg/ml to 15 mg/ml.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, the excipient solution comprises or consists essentially of mannitol and trehalose, wherein neither mannitol nor trehalose is present in an amount of 9 mg/ml. In some embodiments, the excipient solution comprises or consists essentially of mannitol and trehalose, wherein mannitol is not present in an amount of 9 mg/ml. In some embodiments, the excipient solution comprises or consists essentially of mannitol and trehalose, wherein trehalose is not present in an amount of 9 mg/ml.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, the excipient comprises or consists essentially of mannitol and trehalose, and does not comprise methionine.

In some embodiments of the dry forms or therapeutic compositions provided herein, the dry forms or therapeutic compositions comprise or consist essentially of mannitol and trehalose, and the mannitol and trehalose are not present in the dry forms or therapeutic compositions in equal amounts (e.g., mannitol and trehalose are present in unequal amounts, e.g., on a weight or weight percent basis).

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, at least about 10% (by weight) of the solution or dry form is an excipient stock.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, about 10% to about 80% (by weight) of the solution or dry form is an excipient stock.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, wherein about 20% to about 70% (by weight) of the solution or dry form is an excipient stock.

In some embodiments of the solutions, dry forms, or therapeutic compositions provided herein, about 30% to about 60% (by weight) of the solution or dry form is an excipient stock.

In some embodiments of the dry form or therapeutic compositions provided herein, EV from prasuvorexa bacteria of the tissue is at least about 1% of total solids by weight of the dry form.

In some embodiments of the dry form or therapeutic compositions provided herein, EV from the prasuvorexant bacteria of the tissue is from about 1% to about 99% of the total solids by weight of the dry form.

In some embodiments of the dry form or therapeutic compositions provided herein, EV from prasuvorexa bacteria of the tissue is from about 5% to about 90% of the total solids by weight of the dry form. In some embodiments of the dry form or therapeutic compositions provided herein, EV from the prasuvorexant bacteria of the tissue is from about 1% to about 60% of the total solids by weight of the dry form. In some embodiments of the dry form or therapeutic compositions provided herein, EV from prasuvorexa bacteria of the tissue is from about 1% to about 20% of the total solids by weight of the powder or cake. In some embodiments of the dry form or therapeutic compositions provided herein, EV from prasuvorexa bacteria of the tissue is from about 2% to about 10% of the total solids by weight of the dry form. In some embodiments of the dry form or therapeutic compositions provided herein, EV from prasuvorexa bacteria of the tissue is from about 2% to about 6% of the total solids by weight of the dry form. In some embodiments of the dry forms or therapeutic compositions provided herein, the dry form comprises a moisture content of less than about 6% (e.g., as determined by karl fischer titration).

In some embodiments of the dry forms or therapeutic compositions provided herein, the dry form comprises a moisture content of less than about 5% (e.g., as determined by karl fischer titration).

In some embodiments of the dry forms or therapeutic compositions provided herein, the dry form comprises a moisture content of about 0.5% to about 5% (e.g., as determined by karl fischer titration).

In some embodiments of the dry forms or therapeutic compositions provided herein, the dry form comprises a moisture content of about 1% to about 5% (e.g., as determined by karl fischer titration).

In some embodiments of the dry forms or therapeutic compositions provided herein, the dry form comprises a moisture content of about 1% to about 4% (e.g., as determined by karl fischer titration).

In some embodiments of the dry forms or therapeutic compositions provided herein, the dry form comprises a moisture content of about 2% to about 5% (e.g., as determined by karl fischer titration).

In some embodiments of the dry forms or therapeutic compositions provided herein, the dry form comprises a moisture content of about 2% to about 4% (e.g., as determined by karl fischer titration).

In some embodiments of the dry forms or therapeutic compositions provided herein, the dry form comprises at least 1e10 particles per mg of dry form (e.g., as determined by particles per mg, e.g., by NTA).

In some embodiments of the dry forms or therapeutic compositions provided herein, the dry form comprises from about 3e10 to about 6.5e10 particles per mg of dry form (e.g., as determined by particles per mg, e.g., by NTA).

In some embodiments of the dry forms or therapeutic compositions provided herein, the dry form comprises from about 3e10 to about 8e10 particles per mg of dry form (e.g., as determined by particles per mg, e.g., by NTA).

In some embodiments of the dry forms or therapeutic compositions provided herein, the dry form comprises from about 6e10 to about 8e10 particles per mg of dry form (e.g., as determined by particles per mg, e.g., by NTA).

In some embodiments of the dry forms or therapeutic compositions provided herein, the dry form comprises from about 6.7e8 to about 2.55e10 particles per mg of dry form (e.g., as determined by particles per mg, e.g., by NTA).

In some embodiments of the dry forms or therapeutic compositions provided herein, the dry form comprises from about 6.7e8 to about 2.89e10 particles per mg of dry form.

In some embodiments, the particle count determination is performed on the dry form by NTA. In some embodiments, particle count determinations are made on dry forms by NTA using a Zetaview camera.

In some embodiments, particle count determinations are made by NTA and using a Zetaview camera on dry forms resuspended in water.

In some embodiments of the dry forms or therapeutic compositions provided herein, the particles have a hydrodynamic diameter (zeon average, zeon) of about 100nm to about 300nm after being resuspended from the dry form (e.g., resuspended in deionized water) _ave ) (e.g.,by dynamic light scattering).

In some embodiments of the dry forms or therapeutic compositions provided herein, the particles have a hydrodynamic diameter (zeon average, zeon) of about 130nm to about 250nm after being resuspended from the dry form (e.g., resuspended in deionized water) _ave ) (e.g., as determined by dynamic light scattering).

In some embodiments of the dry forms or therapeutic compositions provided herein, the particles have a hydrodynamic diameter (zeon average, zeon) of about 200nm after being resuspended from the dry form (e.g., resuspended in deionized water) _ave ) (e.g., as determined by dynamic light scattering).

In some embodiments, the solution, dry form, or therapeutic composition provided herein may contain an EV from one or more bacterial strains in addition to an EV from Prevotella denticola. In some embodiments, the solution, dry form, or therapeutic composition provided herein may contain an EV from one bacterial strain in addition to an EV from Prevotella denticola. Bacterial strains used as sources of EV may be selected based on the characteristics of the bacteria (e.g., growth characteristics, yield, ability to modulate immune responses in an assay or subject).

In some embodiments, a prasuvorexant EV, or a solution, dried form, or therapeutic composition provided herein comprising an EV from a prasuvorexant, can be used to treat or prevent a disease and/or health disorder, for example, in a subject (e.g., a human).

In some embodiments, the dry form comprising EV from prasuvorexant bacteria of the tissue (or a therapeutic composition thereof) provided herein may be prepared as a solid dosage form, such as a tablet, mini-tablet, capsule, or powder; or a combination of these forms (e.g., miniature tablets contained in a capsule). The solid dosage form may comprise a coating (e.g., an enteric coating).

In certain embodiments, the therapeutic composition comprises a solid dosage form. In some embodiments, the therapeutic composition comprises a blend of a lyophilized powder from an EV of prasuvorexa bacteria of the tissue and an excipient (e.g., an encapsulated lyophilized powder and excipient provided herein from an EV of prasuvorexa bacteria of the tissue). In some embodiments, the therapeutic composition comprises a lyophilized (e.g., freeze-dried) powder of EV from prasuvorexant bacteria of the tissue of interest in a capsule. In some embodiments, the capsule comprises gelatin or hydroxypropyl methylcellulose HPMC. In some embodiments, the capsule is enteric coated. In some embodiments, the excipient comprises one or more of mannitol, magnesium stearate, and colloidal silica. In some embodiments, the excipients include mannitol, magnesium stearate, and colloidal silicon dioxide. In some embodiments, the therapeutic composition comprises a lyophilized (e.g., freeze-dried) powder of EV from prasuvorexant bacteria of the tissue in a tablet or minitablet. In some embodiments, the tablet or minitablet is enteric coated. In some embodiments, the excipient comprises one or more of silicified microcrystalline cellulose, crospovidone, magnesium stearate, and colloidal silicon dioxide. In some embodiments, excipients include silicified microcrystalline cellulose, crospovidone, magnesium stearate, and colloidal silicon dioxide.

In some embodiments, the dried form (or therapeutic composition thereof) provided herein comprising EV from prasuvorexant bacteria of the tissue may be reconstituted. In some embodiments, the solutions (or therapeutic compositions thereof) provided herein comprising EVs from prasuvorexant bacteria of the tissue may be used as suspensions, e.g., diluted into suspension or used in undiluted form.

In some embodiments, a therapeutic composition comprising a prasuvorexant EV of tissue or a solution and/or dried form comprising an EV from a prasuvorexant bacteria of tissue may be prepared as provided herein. Therapeutic compositions comprising a dry form may be formulated into solid dosage forms, such as tablets, minitablets, capsules or powders; or may be reconstituted in suspension.

In some embodiments, a prasuvorexant EV of the tissue, or a solution, dried form, or therapeutic composition provided herein, may comprise a gamma-irradiated EV from a prasuvorexant bacterium of the tissue. Gamma-irradiated EVs from prasuvorexant bacteria can be formulated into therapeutic compositions. The gamma-irradiated EV from the prasuvorexant bacteria of the tissue may be formulated into a solid dosage form, such as a tablet, mini-tablet, capsule or powder; or may be reconstituted in suspension.

In some embodiments, a prasuvorexant EV of the tissue or a solution, dried form, or therapeutic composition provided herein comprising an EV from a prasuvorexant bacteria of the tissue may be administered orally.

In some embodiments, the tissue Prevotella EV or the solution, dried form, or therapeutic composition provided herein comprising an EV from a tissue Prevotella bacteria may be administered intranasally.

In some embodiments, the prasuvorexant EV of the tissue or the solution, dried form, or therapeutic composition provided herein comprising an EV from a prasuvorexant bacteria of the tissue may be administered by inhalation.

In some embodiments, the tissue prasuvorexant EV or the solution, dried form, or therapeutic composition provided herein comprising an EV from a tissue prasuvorexant bacterium may be administered intravenously.

In some embodiments, the prasuvorexant EV of the tissue or the solution, dried form, or therapeutic composition provided herein comprising an EV from a prasuvorexant bacteria of the tissue may be administered by injection.

In certain aspects, provided herein are therapeutic compositions comprising a prasuvorexant EV and/or a solution and/or dried form comprising an EV from a prasuvorexant bacterium, useful for treating and/or preventing a disease or health disorder (e.g., an adverse health disorder) (e.g., an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbacteriosis, or a metabolic disease), and methods of making and/or identifying such a prasuvorexant EV and/or a solution and/or a dried form and therapeutic compositions, and methods of using such a prasuvorexant EV and/or a solution and/or a dried form and therapeutic compositions thereof (e.g., alone or in combination with other therapeutic agents for treating an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbacteriosis, or a metabolic disease).

In some embodiments, the therapeutic composition may comprise EV from, e.g., the tissue of the Prevotella denticola, and intact bacteria, e.g., the Prevotella denticola from which the EV was obtained, e.g., live bacteria, killed bacteria, attenuated bacteria. In some embodiments, in the absence of bacteria from which the therapeutic composition is obtained, the therapeutic composition comprises an EV from a prasuvorexant bacterium of the tissue such that more than about 85%, more than about 90%, or more than about 95% (or more than about 99%) of the bacterial source content in solution and/or dry form comprises a prasuvorexant EV of the tissue. The tissue Prevotella EV can be an isolated EV, for example, isolated by the methods described herein.

In some embodiments, the tissue prasuvorexant EV or the solution, dried form, or therapeutic composition comprises an isolated tissue prasuvorexant EV (e.g., from one or more bacterial strains (e.g., a therapeutically effective amount thereof)). For example, 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 prasugrel bacteria EV and/or solution and/or dry form of the tissue (e.g., content of excipients is not excluded) is an EV isolated from prasugrel bacteria of tissue (e.g., bacteria of interest).

In some embodiments, the tissue prasuvorexant EV or solution, dried form, or therapeutic composition comprises an isolated tissue prasuvorexant EV (e.g., from one bacterial strain (e.g., a bacterium of interest) (e.g., a therapeutically effective amount thereof)). For example, an isolated EV in which 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 prasuvorexa bacteria (e.g., bacteria of interest, such as those disclosed herein) of the tissue is prasuvorexa bacteria (e.g., bacteria of interest) and/or the content of the solution and/or dry form (e.g., without excluding excipients).

In some embodiments, the tissue prasuvorexant EV or the solution, dry form, or therapeutic composition comprises an EV from a tissue prasuvorexant bacterium.

In some embodiments, the solution, dry form, or therapeutic composition comprises EVs from more than one bacterial strain (e.g., EVs from strains other than prasuvorexa EV, a tissue of interest).

In some embodiments, the tissue Prevotella EV is lyophilized.

In some embodiments, the Prevotella EV is gamma irradiated to the tissue.

In some embodiments, the tissue Prevotella EV is subjected to UV irradiation.

In some embodiments, the prasuvorexant EV is heat-inactivated (e.g., at 50 ℃ for two hours or at 90 ℃ for two hours).

In some embodiments, the tissue Prevotella EV is subjected to an acid treatment.

In some embodiments, the oxygen injection is performed on the tissue Prevotella EV (e.g., at 0.1vvm for two hours).

In some embodiments, the tissue prasuvorexant EV is from a strain comprising at least 90% (or at least 97%) genomic, 16S, and/or CRISPR sequence identity to the nucleotide sequence of prasuvorexant strain B50329 (NRRL accession No. B50329). In some embodiments, the tissue prasuvorexant EV is from a strain comprising at least 99% genomic, 16S, and/or CRISPR sequence identity to the nucleotide sequence of prasuvorexant strain B50329 (NRRL accession No. B50329). In some embodiments, the tissue-dwelling prasuvorexant bacteria are from prasuvorexant strain B50329 (NRRL accession No. B50329).

In certain aspects, the tissue prasuvorexant EV is obtained from bacteria that have been selected based on certain desired characteristics, such as reduced toxicity and adverse effects (e.g., by removal or deletion of Lipopolysaccharide (LPS)), enhanced oral delivery (e.g., by improved acid resistance, mucoadhesion and/or permeability and/or resistance to bile acids, resistance to antibacterial peptides and/or antibody neutralization), targeting of desired cell types (e.g., M cells, goblet cells, intestinal epithelial cells, dendritic cells, macrophages), improved bioavailability (e.g., mesenteric lymph nodes, peyer's patches, lamina propria, lymph nodes and/or blood) systemic or in a suitable niche, enhanced immunomodulation and/or therapeutic effects (e.g., alone or in combination with another therapeutic agent), enhanced immune activation and/or manufacturing attributes (e.g., growth characteristics, yield, higher stability, improved freeze-thaw tolerance, shorter production time).

In certain aspects, the tissue prasuvorexant EV is derived from engineered bacteria that are modified to enhance certain desired properties. In some embodiments, the engineered bacteria are modified such that EVs produced therefrom will have reduced toxicity and adverse effects (e.g., by removal or deletion of Lipopolysaccharide (LPS)), enhanced oral delivery (e.g., by improved acid resistance, mucoadhesion and/or permeability and/or resistance to bile acids, resistance to antimicrobial peptides and/or antibody neutralization), targeting desired cell types (e.g., M cells, goblet cells, intestinal epithelial cells, dendritic cells, macrophages), improved bioavailability (e.g., mesenteric lymph nodes, peyer's patches, lamina propria, lymph nodes and/or blood), enhanced immunomodulation and/or therapeutic effects (e.g., alone or in combination with another therapeutic agent), enhanced immune activation and/or manufacturing attributes (e.g., growth characteristics, yield, higher stability, improved freeze-thaw tolerance, shorter production time). In some embodiments, provided herein are methods of manufacturing such EVs.

In certain aspects, provided herein are prasugrel bacteria EV, which are useful for treating and/or preventing a disease or health disorder (e.g., an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbacteriosis, or a metabolic disease) and/or a solution and/or dried form (or therapeutic composition thereof) comprising an EV from prasugrel bacteria, which are useful for treating and/or preventing a disease or health disorder (e.g., an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbacteriosis, or a metabolic disease), and methods of making and/or identifying such a solution and/or dried form (or therapeutic composition thereof), as well as methods of using such a solution and/or dried form (e.g., alone or in combination with one or more other therapeutic agents for treating an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbacteriosis, or a metabolic disease).

Therapeutic compositions containing Prevotella denticola EV and/or in solution and/or dried form may provide efficacy comparable to or greater than therapeutic compositions containing Prevotella denticola bacteria from which EV was obtained. For example, at the same dose of EV (e.g., based on particle count or protein content), a therapeutic composition containing a solution and/or dry form may provide comparable or higher efficacy as compared to a comparative therapeutic composition containing intact bacteria of the same tissue prasuvorexant bacterial strain from which the EV was obtained. Such therapeutic compositions containing EVs and/or solutions and/or dried forms may allow for higher doses to be administered and elicit comparable or greater (e.g., more effective) responses observed with comparative therapeutic compositions containing intact bacteria of the same tissue prasuvorexant bacterial strain from which EVs were obtained.

As another example, at the same dose (e.g., based on particle count or protein content), a therapeutic composition containing prasuvorexant EV and/or a solution and/or dried form of prasuvorexant bacteria may contain less microorganism-derived material (based on particle count or protein content) than a therapeutic composition containing whole prasuvorexant bacteria of the same bacterial strain from which EV was obtained, while providing comparable or greater therapeutic benefit to a subject receiving such a therapeutic composition.

As another example, EV from Prevotella denticola bacteria may be found, for example, at about 1X10 ⁷ Up to about 1x10 ¹⁵ The dosage of individual particles is administered, for example, as measured by NTA. In some embodiments, the dose of EV is about 1x10 ⁵ Up to about 7x10 ¹³ Individual particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)). In some embodiments, the dose of EV from Prevotella denticola bacteria is about 1x10 ¹⁰ Up to about 7x10 ¹³ Individual particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)). NTA may be performed using Zetaview.

As another example, EV from a prasuvorexant bacterium of the tissue may be administered at a dose of, for example, about 5mg to about 900mg total protein, e.g., as measured by the brabender assay. As another example, EV from a prasuvorexant bacterium of the percha tissue may be administered at a dose of, for example, about 5mg to about 900mg total protein, e.g., as measured by BCA assay.

In certain embodiments, provided herein are methods of treating a subject having an immune disorder (e.g., an autoimmune disease, an inflammatory disease, or an allergy) comprising administering to the subject a therapeutic composition or a tissue prasuvorexa EV and/or a solution and/or a dry form described herein. In certain embodiments, provided herein are methods of treating a subject having a metabolic disorder comprising administering to the subject a therapeutic composition or a prasuvorexant EV and/or solution and/or dry form described herein. In certain embodiments, provided herein are methods of treating a subject having a dysbacteriosis comprising administering to the subject a therapeutic composition or a prasuvorexant EV and/or solution and/or dried form described herein. In certain embodiments, provided herein are methods of treating a subject having a neurological disorder comprising administering to the subject a therapeutic composition described herein or a prasuvorexant EV and/or solution and/or dry form of the tissue.

In some embodiments, the method further comprises administering an antibiotic to the subject. In some embodiments, the method further comprises administering an immunosuppressant and/or an anti-inflammatory agent. In some embodiments, the therapeutic composition or the prasuvorexant EV and/or solution, dried form, and/or lyophilisate may be used in combination with one or more other immune effect modulators. In some embodiments, the method further comprises administering a metabolic disease therapeutic agent.

In certain aspects, provided herein are therapeutic compositions or tissue prasuvorexant EV and/or solution and/or dry forms for use in treating and/or preventing a disease (e.g., an immune disorder (e.g., autoimmune disease, inflammatory disease, allergy), a dysbacteriosis, or a metabolic disease) or a health disorder, alone or in combination with one or more other (e.g., additional) therapeutic agents.

In certain embodiments, provided herein are therapeutic compositions or tissue-dwelling prasuvorexant EVs and/or solutions and/or dried forms for use in treating and/or preventing an immune disorder (e.g., autoimmune disease, inflammatory disease, allergy) in a subject (e.g., human). The therapeutic composition or the tissue-dwelling Prevotella EV and/or the solution and/or dried form may be used alone or in combination with one or more other therapeutic agents for the treatment of immune disorders. In certain embodiments, provided herein are therapeutic compositions or tissue-dwelling prasuvorexant EVs and/or solutions and/or dried forms for use in treating and/or preventing dysbacteriosis in a subject (e.g., a human). The therapeutic composition or the tissue-dwelling Prevotella EV and/or the solution and/or dried form may be used alone or in combination with a therapeutic agent for the treatment of dysbacteriosis. In certain embodiments, provided herein are therapeutic compositions or tissue-dwelling prasuvorexant EVs and/or solutions and/or dried forms for use in treating and/or preventing a metabolic disease in a subject (e.g., a human). The therapeutic composition or the prasugrel tissue EV and/or the solution and/or dried form may be used alone or in combination with a therapeutic agent for the treatment of metabolic diseases. In certain embodiments, provided herein are therapeutic compositions or tissue-dwelling prasuvorexant EVs and/or solutions and/or dried forms for use in treating and/or preventing dysbacteriosis in a subject (e.g., a human). The therapeutic composition or the tissue-dwelling Prevotella EV and/or the solution and/or dried form may be used alone or in combination with a therapeutic agent for the treatment of dysbacteriosis. In certain embodiments, provided herein are therapeutic compositions or tissue-dwelling prasuvorexant EVs and/or solutions and/or dried forms for use in treating and/or preventing a neurological disorder in a subject (e.g., a human). The therapeutic composition or tissue-dwelling Prevotella EV and/or the solution and/or dried form may be used alone or in combination with one or more other therapeutic agents for the treatment of neurological disorders.

In some embodiments, the therapeutic composition or the prasuvorexant EV and/or the solution and/or dried form of tissue may be used in combination with an antibiotic. In some embodiments, the therapeutic composition or the prasuvorexant EV of the percha tissue and/or the solution and/or dried form may be used in combination with another therapeutic bacterium and/or an EV obtained from one or more other bacterial strains (e.g., therapeutic bacteria). In some embodiments, the therapeutic composition or the prasuvorexant EV for the tissue and/or the solution and/or dried form may be used in combination with one or more immunosuppressants and/or one or more anti-inflammatory agents. In some embodiments, the therapeutic composition or the prasuvorexant EV and/or the solution and/or dried form of tissue may be used in combination with one or more other metabolic disease therapeutic agents.

In certain aspects, provided herein is the use of a therapeutic composition or a prasugrel bacteria EV and/or a solution and/or dried form of a tissue for the preparation of a medicament for the treatment and/or prevention of a disease (e.g., an immune disorder (e.g., autoimmune disease, inflammatory disease, allergy), a dysbacteriosis, or a metabolic disease), alone or in combination with another therapeutic agent. In some embodiments, the use is used in combination with another therapeutic bacterium and/or an EV obtained from one or more other bacterial strains (e.g., therapeutic bacteria).

In certain embodiments, provided herein is a use of a therapeutic composition or a tissue prasuvorexant EV and/or a solution and/or dried form (for the manufacture of a medicament for the treatment and/or prevention of an immune disorder (e.g., autoimmune disease, inflammatory disease, allergy) in a subject (e.g., human). The therapeutic composition or the prasugrel bacteria EV and/or the solution and/or dried form of tissue may be used alone or in combination with another therapeutic agent for immune disorders. In certain embodiments, provided herein is the use of a therapeutic composition or a tissue-dwelling prasuvorexa EV and/or a solution and/or dried form for the manufacture of a medicament for the treatment and/or prevention of dysbacteriosis in a subject (e.g., a human). The therapeutic composition or the tissue-dwelling Prevotella EV and/or the solution and/or dried form may be used alone or in combination with another therapeutic agent for dysbacteriosis. In certain embodiments, provided herein is the use of a therapeutic composition or a tissue-dwelling prasuvorexa EV and/or a solution and/or dried form for the manufacture of a medicament for the treatment and/or prevention of a metabolic disease in a subject (e.g., a human). The therapeutic composition or the prasugrel tissue EV and/or the solution and/or dried form may be used alone or in combination with another therapeutic agent for metabolic diseases. In certain embodiments, provided herein is the use of a therapeutic composition or a tissue-dwelling prasuvorexa EV and/or a solution and/or dried form for the manufacture of a medicament for the treatment and/or prevention of dysbacteriosis in a subject (e.g., a human). The therapeutic composition or the tissue-dwelling Prevotella EV and/or the solution and/or dried form may be used alone or in combination with another therapeutic agent for dysbacteriosis. In certain embodiments, provided herein is the use of a therapeutic composition or a tissue-dwelling prasuvorexant EV and/or a solution and/or dried form for the manufacture of a medicament for the treatment and/or prevention of a neurological disease in a subject (e.g., a human). The therapeutic composition or the tissue-dwelling Prevotella EV and/or the solution and/or dried form may be used alone or in combination with another therapeutic agent for neurological disorders.

In some embodiments, the therapeutic composition or the prasuvorexant EV and/or the solution and/or dried form of tissue may be used in combination with an antibiotic. In some embodiments, the therapeutic composition or the prasuvorexant EV of the percha tissue and/or the solution and/or dried form may be used in combination with another therapeutic bacterium and/or an EV obtained from one or more other bacterial strains (e.g., therapeutic bacteria). In some embodiments, the therapeutic composition or the prasuvorexant EV for the tissue and/or the solution and/or dried form may be used in combination with one or more other immunosuppressants and/or one or more anti-inflammatory agents. In some embodiments, the therapeutic composition or the prasuvorexant EV and/or the solution and/or dried form of tissue may be used in combination with one or more other metabolic disease therapeutic agents.

For example, a therapeutic composition comprising an EV from a prasuvorexa tissue bacterium or a prasuvorexa tissue EV and/or a solution and/or dried form as described herein may provide a therapeutically effective amount of a prasuvorexa tissue EV to a subject (e.g., a human).

For example, a therapeutic composition comprising an EV from a prasuvorexa tissue bacterium or a prasuvorexa tissue EV and/or a solution and/or dried form as described herein may provide a subject (e.g., a human) with an unnatural amount of a therapeutically effective component (e.g., present in a prasuvorexa tissue EV).

For example, a therapeutic composition comprising an EV from a prasuvorexa tissue bacterium as described herein or a prasuvorexa tissue EV and/or a solution and/or dried form may provide a subject (e.g., a human) with an unnatural amount of a therapeutically effective component (e.g., present in an EV).

For example, a therapeutic composition comprising an EV from a prasuvorexa tissue bacterium as described herein or a prasuvorexa tissue EV and/or a solution and/or dried form may bring about one or more changes to a subject (e.g., a human), e.g., to treat or prevent a disease or health disorder.

For example, a therapeutic composition comprising an EV from a prasuvorexa tissue bacterium as described herein or a prasuvorexa tissue EV and/or a solution and/or dried form thereof has potentially significant utility, e.g., affects a subject (e.g., a human), e.g., for treating or preventing a disease or health disorder.

In certain aspects, provided herein are stock solutions comprising one or more excipients, wherein the stock solutions comprise a bulking agent, wherein the stock solutions are for use in combination with Extracellular Vesicles (EVs) from prasuvorexa bacteria of the tissue (e.g., liquid formulations thereof).

In certain aspects, provided herein are stock solutions comprising one or more excipients, wherein the stock solutions comprise a bulking agent and a lyoprotectant, wherein the stock solutions are for use in combination with Extracellular Vesicles (EVs) from prasuvorexa bacteria of the tissue (e.g., liquid formulations thereof).

In certain aspects, provided herein are stock solutions comprising one or more excipients, wherein the stock solutions comprise a lyoprotectant, wherein the stock solutions are for use in combination with Extracellular Vesicles (EVs) (e.g., liquid formulations thereof) from prasuvorexa bacteria of the tissue.

In some embodiments, the bulking agent comprises mannitol, sucrose, maltodextrin, dextran, ficoll, or PVP-K30.

In some embodiments, the filler comprises mannitol.

In some embodiments, the excipient solution comprises additional ingredients.

In some embodiments, the additional ingredients comprise trehalose, mannitol, sucrose, sorbitol, dextran, poloxamer 188, maltodextrin, PVP-K30, ficoll, citrate, arginine, and/or hydroxypropyl-B-cyclodextrin.

In some embodiments, the excipient solution comprises mannitol and trehalose.

In some embodiments, the excipient solution consists essentially of mannitol and trehalose.

In some embodiments, the excipient solution comprises mannitol, trehalose, and sorbitol.

In some embodiments, the excipient solution consists essentially of mannitol, trehalose, and sorbitol.

In some embodiments, the excipient solution comprises trehalose.

In some embodiments, the excipient solution consists essentially of trehalose.

In some embodiments, the excipient solution comprises mannitol and trehalose, wherein the mannitol and trehalose are not present in equal amounts (e.g., mannitol and trehalose are present in unequal amounts; e.g., on a weight basis or a weight percent basis). In some embodiments, the excipient solution comprises more mannitol than trehalose, e.g., on a weight basis or on a weight percent basis. In some embodiments, the excipient solution comprises at least twice as much mannitol as trehalose, e.g., on a weight basis or on a weight percent basis. In some embodiments, the excipient solution comprises at least three times more mannitol than trehalose, e.g., on a weight basis or on a weight percent basis. In some embodiments, the excipient in solution or dry form comprises mannitol and trehalose, wherein mannitol and trehalose are not present in equal amounts (e.g., mannitol and trehalose are present in unequal amounts; e.g., on a weight basis or a weight percent basis). In some embodiments, the excipient in solution or dry form comprises more mannitol than trehalose, e.g., on a weight basis or on a weight percent basis. In some embodiments, the excipient in solution or dry form comprises at least twice as much mannitol as trehalose, e.g., on a weight basis or on a weight percent basis. In some embodiments, the excipient in solution or dry form comprises at least three times more mannitol than trehalose, e.g., on a weight basis or on a weight percent basis.

In some embodiments, the excipient solution consists essentially of mannitol and trehalose. In some embodiments, the excipient solution consists essentially of mannitol and trehalose, wherein the mannitol and trehalose are not present in equal amounts (e.g., mannitol and trehalose are present in unequal amounts; e.g., on a weight basis or a weight percent basis). In some embodiments, the excipient solution consists essentially of mannitol and trehalose, wherein the excipient solution contains more mannitol than trehalose, e.g., on a weight basis or on a weight percent basis. In some embodiments, the excipient solution consists essentially of mannitol and trehalose, wherein the excipient solution contains at least twice as much mannitol as trehalose, e.g., on a weight basis or on a weight percent basis. In some embodiments, the excipient solution consists essentially of mannitol and trehalose, wherein the excipient solution contains at least three times more mannitol than trehalose, e.g., on a weight basis or on a weight percent basis. In some embodiments, the excipient in solution or dry form consists essentially of mannitol and trehalose, wherein the excipient in solution or dry form contains more mannitol than trehalose, e.g., on a weight basis or a weight percent basis. In some embodiments, the excipient in solution or dry form consists essentially of mannitol and trehalose, wherein the excipient in solution or dry form contains at least twice as much mannitol as trehalose, e.g., on a weight basis or on a weight percent basis. In some embodiments, the excipient in solution or dry form consists essentially of mannitol and trehalose, wherein the excipient in solution or dry form contains at least three times more mannitol than trehalose, e.g., on a weight basis or a weight percent basis.

In some embodiments, the excipient solution comprises or consists essentially of mannitol and trehalose, wherein neither mannitol nor trehalose is present in an amount of 5mg/ml to 15 mg/ml. In some embodiments, the excipient solution comprises or consists essentially of mannitol and trehalose, wherein mannitol is not present in an amount of 5mg/ml to 15 mg/ml. In some embodiments, the excipient solution comprises or consists essentially of mannitol and trehalose, wherein trehalose is not present in an amount of 5mg/ml to 15 mg/ml.

In some embodiments, the excipient solution comprises or consists essentially of mannitol and trehalose, wherein neither mannitol nor trehalose is present in an amount of 9 mg/ml. In some embodiments, the excipient solution comprises or consists essentially of mannitol and trehalose, wherein mannitol is not present in an amount of 9 mg/ml. In some embodiments, the excipient solution comprises or consists essentially of mannitol and trehalose, wherein trehalose is not present in an amount of 9 mg/ml.

In some embodiments, the excipient solution comprises or consists essentially of mannitol and trehalose, and does not comprise methionine.

In certain aspects, provided herein are stock solutions comprising one or more excipients, wherein the stock solutions comprise the formulations provided in table A, B, C, D, K or P.

In certain aspects, provided herein are stock solutions comprising one or more excipients, wherein the stock solutions comprise the formulations provided in table A, B, C, D, K or P, wherein the stock solutions are for use in combination with Extracellular Vesicles (EVs) (e.g., liquid formulations thereof) from prasuvorexant bacteria of the tissue.

In some embodiments of the solutions and dry forms and methods described herein, the liquid formulation comprises a cell culture supernatant, e.g., a bacterial cell culture supernatant as described herein. In some embodiments of the solutions and dry forms and methods described herein, the liquid formulation comprises a retentate, such as a concentrated retentate as described herein.

In some embodiments of the methods provided herein, the excipient is present in (e.g., provided in) an excipient solution. Examples of excipient solutions include stock solutions comprising one or more of the excipients provided in table A, B, C, D, K or P. For example, once the moisture has been removed, such as by drying, the dried forms provided herein contain excipients from an excipient solution (e.g., stock solution). For example, a liquid formulation comprising EV from prasugrel bacteria of the tissue is combined with the stock solution of formula 7a of table a (which comprises the excipients mannitol and trehalose) to prepare a solution. The solution was dried to prepare a dried form. The dry form contains EV, mannitol and trehalose from a prasugrel bacterium of the tissue. As used herein, "stock solution" refers to a solution comprising one or more excipients but no active ingredient (e.g., extracellular vesicles). In some embodiments, the stock solution is used to introduce one or more excipients into a formulation (e.g., a liquid formulation) comprising the EV. In some embodiments, the stock solution is a concentrated solution comprising a known amount of one or more excipients. In some embodiments, the stock solution is combined with a formulation (e.g., a liquid formulation) comprising the EV to prepare a solution or dried form provided herein.

Drawings

FIGS. 1A and 1B are graphs showing that oral administration of Prevotella EV requires multiple pathways for anti-inflammatory action in a delayed hypersensitivity (DTH) inflammation model. Inflammation was assessed as a change in ear thickness (mm). The effect of antibodies against TLR2 (anti-TLR 2) or antibodies against IL-10 receptor (IL-10R) (fig. 1A) and antibodies against CD62 and LPAM-1 (anti-CD 62/LPAM-1) (fig. 1B) on the ability of prasuvorexant EV to reduce inflammation was evaluated. Dexamethasone was used as a positive control for reducing inflammation.

Figure 2 is a graph showing that Prevotella EV has potent human TLR2 agonist activity in HEK293 reporter cell assays. Activation of TLR1/2/6, TLR1/2 or TLR2/6 at different concentrations of Prevotella EV was determined as a normalized response (OD 630 nm).

FIG. 3 is a graph showing that TLR1/2 signaling is required to induce U937 cells to release IL-10 in response to Prevotella EV. The effect of antibodies to TLR2 (anti-TLR 2), to TLR1 (anti-TLR-1) and to TLR6 (anti-TLR 6) on the ability of prasuvorexa EV to cause U937 cells to release IL-10 cytokines (pg/ml) was measured.

FIGS. 4A and 4B are graphs showing that Prevotella EV induces concentration-dependent production of IL-10 (FIG. 4A) and IL-27 (FIG. 4B) in human PBMC. IL-10 and IL-27 levels were measured as pg/ml at various concentrations of Prevotella EV.

Fig. 5 is a graph showing the effect of orally administered prasuvorexa EV powder prepared in formulation 7a in a Delayed Type Hypersensitivity (DTH) inflammation model. Inflammation was assessed as a change in ear thickness (mm).

Fig. 6 is a graph showing the effect of orally administered prasuvorexa smEV (EV) and intraperitoneally administered anti-tnfα antibodies in a Delayed Type Hypersensitivity (DTH) inflammation model. Inflammation was assessed as a change in ear thickness (mm).

FIG. 7 is a graph showing ear thickness variation 24 hours after KLH ear challenge following (pre) or post) dosing of vehicle (PBS), dexamethasone, or Prevotella smeV (EV) or a smeV (EV) from another bacterial strain (other strain EV).

FIG. 8 is a graph showing ear thickness variation 24 hours after KLH ear challenge following immunization with IFA-PBS or CFA-PBS, administration of vehicle (PBS), dexamethasone or Prevotella smeV (EV), followed by KLH-CFA.

FIG. 9 is a graph showing the change in ear thickness 24 hours after KLH ear challenge in recipient mice after transfer of CD4+ T cells from donor mice immunized with vehicle or Prevotella smeV (EV) or a smeV (EV) from another bacterial strain (other strain EV).

Fig. 10 is a graph showing ear thickness changes 24 hours after KLH ear challenge in recipient mice after transfer of cd4+ T cells from vehicle-administered or prevotella smEV (EV) isotype control or anti-TLR 2 treated KLH-CFA immunized mice to KLH-CFA immunized recipient mice.

FIG. 11 is a graph showing the change in ear thickness 24 hours after KLH ear challenge in recipient mice after transfer of CD8+ T cells from donor mice immunized with a vehicle or Prevotella smeV (EV) or a smeV (EV) from another bacterial strain (other strain EV).

Detailed Description

The present disclosure provides for Prevotella denticola EV, as well as solutions, dried forms, and therapeutic compositions containing Extracellular Vesicles (EV) from Prevotella denticola bacteria, and methods of making and using the same.

The small intestine axis is the anatomic and functional connection network that connects the small intestine with the rest of the body. It senses external signals in the intestinal lumen and converts them into systemic immune effects. We have previously shown that oral microbial drug candidates induce anti-inflammatory activity in preclinical models of inflammation by acting directly on host cells without colonizing the gut or modulating microbiomes. We now extend these observations to Prevotella EV, a tissue of Prevotella which has potent anti-inflammatory activity in a preclinical model. EV is a non-replicating bacterial membrane vesicle with a parent cell volume of about 1/1000. Prevotella EV, a perchloric tissue, is orally delivered and limits intestinal distribution, which reduces systemic inflammatory response by modulating innate and adaptive immunity in the small intestine.

The present disclosure provides solutions and dried forms containing Extracellular Vesicles (EVs) from Prevotella denticola bacteria, as well as methods of making and using the same. The present disclosure also provides therapeutic compositions comprising the solutions and/or in dry form. In some embodiments, the EV is secreted (e.g., produced) by the bacterial cell in culture. Such secreted extracellular vesicles may be referred to as secreted microbial extracellular vesicles (smevs). In some embodiments, the EV is prepared (e.g., artificially prepared) by treating the bacterial cells, e.g., by a method that disrupts the bacterial membrane, e.g., sonication. Such artificially prepared microbial extracellular vesicles (pmevs) may be referred to as processing.

As used herein, a "dried form" containing Extracellular Vesicles (EVs) (e.g., from a prasuvorexa bacterium of the tissue of interest) refers to the product resulting from drying a solution containing EVs. In some embodiments, drying is performed, for example, by freeze-drying (lyophilization) or spray drying. In some embodiments, the dry form is a powder. As used herein, powder refers to a dry form and includes lyophilized powder and spray dried powder obtained by a method such as spray drying.

When freeze-drying (lyophilization) is performed, the resulting dry form is a lyophilisate. In some embodiments, the dry form is a lyophilisate. For example, in some embodiments, the lyophilisate is a lyophilized powder or a lyophilized cake. In some embodiments, the lyophilized cake is milled to produce a lyophilized powder.

In some embodiments, the solution and dry form containing EV from Prevotella denticola bacteria also comprise one or more excipients, such as bulking agents and/or lyoprotectants.

In some embodiments, bulking agents and lyoprotectants are used when preparing Extracellular Vesicles (EVs) for lyophilization. In some embodiments, bulking agents, including but not limited to sucrose, mannitol, polyethylene glycol (PEG, e.g., PEG 6000), cyclodextrin, maltodextrin, and dextran (e.g., dextran 40 k), are added (e.g., as a stock solution containing the same) to a liquid formulation of an EV (e.g., obtained by isolating the EV from a bacterial culture) to prepare a dry form, e.g., a lyophilizate, that is easier to handle after drying (and optionally, further formulated into, e.g., a therapeutic composition). In some embodiments, lyoprotectants (including, but not limited to, trehalose, sucrose, and lactose) are added (e.g., as stock solutions containing them) to a liquid formulation of an EV (e.g., obtained by isolating the EV from a bacterial culture) to protect the EV upon lyophilization or spray drying. In some embodiments, bulking agents and/or lyoprotectants are included in an excipient stock that is added to an EV (e.g., purified and/or concentrated EV) to produce a solution, and/or after subsequent drying to produce a dried form of the solution, for example. In some embodiments, the dry form, such as a lyophilisate, contains from about 5% to about 100% EV solids by weight. In some embodiments, the total solids including EV and excipients is between about 2% to about 20% (by weight) prior to drying (e.g., by lyophilization).

In some embodiments, the excipient comprises about 95% to about 99% of the total mass of the powder or cake in a lyophilizate comprising Prevotella denticola EV.

As described herein, in some embodiments, in a lyophilizate containing a prasuvorexant EV, the EV comprises about 2% to about 6% (e.g., about 2% to about 5%, about 2% to about 3%, or about 3% to about 5%) of the total mass of the lyophilizate.

In some embodiments, excipients are used to maintain EV efficacy and/or reduce drying (e.g., lyophilization) cycle time. In some embodiments, the lyoprotectant protects the EV (e.g., its protein component) during lyophilization. In some embodiments, bulking agents improve lyophilizate properties, for example, for further downstream processing (e.g., milling, blending, and/or preparing a therapeutic composition).

The length of the lyophilization cycle is important for cost considerations. Critical temperature modifiers such as bulking agents and/or lyoprotectants can significantly shorten drying times. In some embodiments, an excipient stock including one or more excipients (e.g., including bulking agents and/or lyoprotectants) is added to a concentrated EV (e.g., a liquid formulation thereof) to bring the total solids between about 2% and about 20%. In some embodiments, the EV is concentrated to 5 to 100 times or Volume Concentration Factor (VCF). The examples provided herein target about 10% total solids, with actual dissolved solids ranging from about 6% to about 8%. In some embodiments, an excipient stock (e.g., stock of excipients comprising the formulation provided in one of tables A, B, C, D, K or P) containing one or more excipients (e.g., containing bulking agents and/or lyoprotectants) is prepared as a stock solution in deionized water and sterile filtered with a 0.2mm filter prior to use. In some embodiments, the stock solution is added to the concentrated EV, for example up to 80% by weight. In some embodiments, the percentage to be added is based on the estimated solids contribution of the EV plus the dissolved solids of the excipient stock to achieve the desired total solids content prior to lyophilization.

After freeze-drying the tissue prasugrel bacteria EV (e.g., with an excipient comprising a bulking agent, e.g., as described herein), in some embodiments, the resulting lyophilizate (e.g., a lyophilized cake) has a uniform appearance and is white to off-white. In some embodiments, the resulting lyophilizate (e.g., a lyophilized cake) obtained after lyophilization is a white to off-white, fine, and smooth particulate powder (e.g., after grinding (e.g., milling) the lyophilized cake). In some embodiments, dynamic Light Scattering (DLS) is used to obtain the hydrodynamic diameter (zaverage, Z) of particles present after resuspension of a lyophilizate (e.g., a lyophilized powder) in deionized water or a buffer such as PBS (e.g., 0.1X PBS) _ave ). In some embodiments, Z _ave For quantifying the effectiveness of the stabilizer. For example, if it is managedImagined Z _ave The grain diameter is 200nm; thus, having the lowest Z closest to the particle size _ave Is considered to be sufficiently stable. In some embodiments, the particle size ranges from, for example, 130nm to 300nm. In some embodiments, dynamic Light Scattering (DLS) is used to obtain the mean size of the optimal potential DLS integral peak of particles present after resuspension of a lyophilizate (e.g., a lyophilized powder) in deionized water or a buffer such as PBS (e.g., 0.1X PBS). Notably, the mean size of the particles, whether measured by Z-average or by the mean size of the most dominant DLS integral peak, is not necessarily the same as the mean size of EV before lyophilization. For example, in some embodiments, the mean size of the particles after lyophilization (e.g., after resuspension of the lyophilizate in deionized water or a buffer such as PBS (e.g., 0.1X PBS)) is greater than or less than the mean EV size prior to lyophilization, or the mean size after separation or preparation of the EV from the bacterial culture (e.g., the mean size after gradient purification of the EV from the bacterial culture). The particles in the lyophilisate (after lyophilization of the EV-containing solution) contain the prasuvorexant EV, a tissue of interest, and may also include other components from the culture medium, such as cell debris, LPS, and/or proteins.

The lyophilisates obtained after lyophilization with the excipients and/or conditions provided herein do not have a porous sponge shape. In some embodiments, after milling, the lyophilizate obtained after lyophilization with the excipients and/or conditions provided herein is a white to off-white, fine and smooth particulate lyophilized powder.

Also as described herein, the use of the excipients provided herein allows the solution comprising Prevotella denticola EV to be freeze-dried at higher temperatures and shorter drying times. For example, the excipients and methods provided herein allow for EV freeze-drying in less than 4000 minutes, such as from about 2800 to about 3200 minutes. As another example, in some embodiments, the freezing step is performed in less than 225 minutes, rather than 10 to 15 hours (600 to 900 minutes). As another example, in some embodiments, primary drying is performed at a temperature between about-35 ℃ and about-20 ℃, such as about-20 ℃, about-25 ℃, about-30 ℃, or about-35 ℃, rather than, for example, -50 ℃, using the excipients and methods provided herein. As another example, in some embodiments, primary drying is performed for about 42 hours or less (e.g., 2500 minutes or less), rather than, for example, 50-60 hours (3000 to 3600 minutes) using the excipients and methods provided herein. In some embodiments, using the excipients and methods provided herein, the total drying time is, for example, about 72 hours or less, such as about 48 to about 72 hours, such as less than about 48 hours. In some embodiments, primary drying is performed for about 65 hours or less (e.g., about 60 hours or less) using the excipients and methods provided herein. In some embodiments, using the excipients and methods provided herein, the secondary drying is performed for about 12 hours or less (e.g., about 10 to about 12 hours, about 5 to about 10 hours, about 10 hours or less, or about 5 hours or less). As another example, in some embodiments, using the excipients and methods provided herein, the secondary drying is performed at a temperature between about +20 ℃ and about +30 ℃, such as room temperature, e.g., about +25 ℃, instead of, e.g., -20 ℃. In some embodiments, the use of shorter drying times and/or higher drying temperatures makes the EV lyophilization process commercially more viable.

In some embodiments, a lyophilizate comprising a prasuvorexant EV of tissue described herein (e.g., prepared using the excipients and/or methods described herein) is prepared to have a moisture content (e.g., as determined by the karl fischer method) of less than about 10% (e.g., less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, or less than about 4%, e.g., from about 1% to about 4%, from about 1.5% to about 4%, from about 2% to about 3%) after lyophilization is complete. In some embodiments, the lyophilisate is more suitable for downstream processing, e.g., for use in a therapeutic composition, by preparing the lyophilisate to have a moisture content of less than about 6%. In some embodiments, the lyophilizate has improved stability, e.g., after storage, by preparing the lyophilizate with a moisture content of less than about 6%.

As described in the examples provided herein, the moisture content of the lyophilizate containing the prasugrel bacteria EV as a tissue of interest (as determined by the karl fischer method) has a moisture content of about 1.8% to about 3.8%. The components of the excipients may be selected to achieve the desired moisture content. The drying conditions may be selected to achieve the desired moisture content.

In some embodiments, a lyophilizate comprising a prasuvorexant EV of the tissue described herein (e.g., prepared using the excipients and/or methods described herein) is prepared to have a particle count of about 3.25e10 to about 7.77e10 particles/mg lyophilizate. In some embodiments, the particle count is determined on a lyophilisate resuspended in water, for example by NTA and using a Zetaview camera. In some embodiments, a lyophilizate containing the prasuvorexant EV tissue described herein (e.g., prepared using the excipients and/or methods described herein) is prepared to have a particle count of about 3.25e10 to about 6.45e10 particles/mg lyophilizate. In some embodiments, the particle count is determined on a lyophilisate resuspended in water, for example by NTA and using a Zetaview camera. The components of the excipients may be selected to achieve the desired particle count. The drying conditions may be selected to obtain the desired particle count.

In some embodiments, the particles (e.g., prepared using the excipients and/or methods described herein) in a lyophilizate (e.g., a lyophilized powder) described herein are prepared to have a hydrodynamic diameter (zeaverage, Z) of about 137.4nm to about 226.1nm _ave ). In some embodiments, the particles (e.g., prepared using the excipients and/or methods described herein) in a lyophilizate (e.g., a lyophilized powder) described herein are prepared to have a hydrodynamic diameter (zeaverage, Z) of about 137.4nm to about 212.8nm _ave ). In some embodiments, dynamic Light Scattering (DLS) is used to obtain the hydrodynamic diameter (zeaverage, Z) of particles present after resuspension of the lyophilizate in deionized water or a buffer such as PBS (e.g., 0.1X PBS) _ave ). The components of the excipients can be selected to achieve the desired Z _ave . The drying conditions can be selected to obtain the desired Z _ave 。

In some embodiments, a spray-dried powder (e.g., prepared using the excipients and/or methods described herein) containing an EV described herein is prepared to have a moisture content (e.g., as determined by the karl fischer method) of less than about 10% (e.g., less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, or less than about 4%, e.g., from about 1% to about 4%, from about 1.5% to about 4%, from about 2% to about 3%) after spray-drying is complete. In some embodiments, the spray-dried powder is more suitable for downstream processing, such as for use in therapeutic compositions, by preparing the spray-dried powder to have a moisture content of less than about 6%. In some embodiments, the spray-dried powder has improved stability, e.g., after storage, by preparing the spray-dried powder to have a moisture content of less than about 6%.

As described in the examples provided herein, the moisture content of the spray-dried powder containing prasuvorexant EV as a tissue (as determined by the karl fischer method) has a moisture content of about 2.54% to about 8.38%. The components of the excipients may be selected to achieve the desired moisture content. The drying conditions may be selected to achieve the desired moisture content.

In some embodiments, a spray-dried powder (e.g., prepared using the excipients and/or methods described herein) containing an EV described herein is prepared to have a particle count of about 6.7e8 to about 2.55e10 particles/mg spray-dried powder. In some embodiments, the particle count is determined, for example, by NTA using a Zetaview camera.

As described in the examples provided herein, the spray-dried powder containing prasuvorexant EV, a tissue of interest, had a particle count of about 8.05e9 to about 2.e10 particles/mg spray-dried powder. The components of the excipients may be selected to achieve the desired particle count. The drying conditions may be selected to obtain the desired particle count.

Definition of the definition

As used herein, the term "or" is to be interpreted as inclusive unless otherwise indicated or apparent from the context. The terms "a" and "an" and "the" as used herein are to be understood as singular or plural unless otherwise indicated herein or apparent from the context.

"adjuvant" or "adjuvant therapy" refers broadly to an agent that affects an immunological or physiological response in a patient or subject (e.g., a human). For example, adjuvants may increase the presence of antigen over time or in a region of interest (e.g., tumor), help to take up antigen presenting cell antigens, activate macrophages and lymphocytes, and support cytokine production. By altering the immune response, adjuvants may allow for the use of smaller doses of the immune-interacting agent to increase the effectiveness or safety of a particular dose of the immune-interacting agent. For example, an adjuvant may prevent T cell depletion and thereby increase the effectiveness or safety of a particular immune interactive agent.

"administration" broadly refers to the route by which a composition (e.g., a therapeutic composition) is administered to a subject. Examples of routes of administration include oral administration, rectal administration, topical administration, inhalation (nasal) or injection. Injection administration includes Intravenous (IV), intramuscular (IM) and Subcutaneous (SC) administration. The therapeutic compositions described herein may be administered in any form by any effective route including, but not limited to: in a preferred embodiment, the therapeutic compositions described herein are administered orally, rectally, topically, intravesically, by injection into or adjacent to a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously.

As used herein, the term "antibody" may refer to both whole antibodies and antigen-binding fragments thereof. An intact antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as V _H ) A heavy chain constant region. Each light chain comprises a light chain variable region(abbreviated herein as V _L ) A light chain constant region. V (V) _H V (V) _L The regions may be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), and regions of more conservation, termed Framework Regions (FR), interspersed with each other. Each V _H V (V) _L Consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The term "antibody" includes, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies, and antigen-binding antibody fragments.

As used herein, the terms "antigen-binding fragment" and "antigen-binding portion" of an antibody refer to one or more fragments of an antibody that retain the ability to bind antigen. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include Fab, fab ', F (ab') ₂ Fv, scFv, disulfide-linked Fv, fd, diabodies, single chain antibodies,CDRH3 and other antibody fragments that retain at least a portion of the variable region of the intact antibody are isolated. These antibody fragments can be obtained using conventional recombinant and/or enzymatic techniques and can be screened for antigen binding in the same manner as whole antibodies.

"carbohydrate" refers to a sugar or sugar polymer. The terms "sugar", "polysaccharide", "carbohydrate" and "oligosaccharide" are 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 formula C _n H _2n O _n . The carbohydrate may be a monosaccharide, disaccharide, trisaccharide, oligosaccharide or polysaccharide. The most basic carbohydrates are monosaccharides such as glucose, galactose, mannose, ribose, arabinose, xylose and fructose. Disaccharides are two joined monosaccharides. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose. In general, the number of the devices used in the system,oligosaccharides include 3 and 6 monosaccharide units (e.g., raffinose, stachyose), and polysaccharides include 6 or more monosaccharide units. Exemplary polysaccharides include starch, glycogen, and cellulose. The carbohydrate may contain modified sugar units, such as 2 '-deoxyribose, wherein the hydroxyl group, 2' -fluororibose, is removed, wherein the hydroxyl group is replaced with fluorine; or N-acetylglucosamine, which is a nitrogen-containing form of glucose (e.g., 2' -fluororibose, deoxyribose, and hexose). Carbohydrates may exist in many different forms, such as conformational isomers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers and isomers.

"cell enhancement" broadly refers to the influx of cells or the expansion of cells in an environment in which they were not substantially present prior to administration of the composition and in the composition itself. The environmental enhancing cells include immune cells, stromal cells, bacterial and fungal cells.

"clades" refer to OTUs or members of a phylogenetic tree that are downstream of statistically significant nodes in the phylogenetic tree. The clade comprises a set of end leaves in the phylogenetic tree, which are distinct single-line evolutionary units and share sequence similarity to some extent.

"combination" may refer to an EV from one source strain with another agent, such as another EV (e.g., from another strain), with bacteria (e.g., a strain that is the same as or different from the strain from which the EV was obtained), or with another therapeutic agent. Such a combination may be physically co-located, either in the same material or product, or in physically linked products, and the time of EV and other agents.

As used herein, the term "consisting essentially of … … (consists essentially of or consisting essentially of)" means limited to those recited elements and/or steps and does not materially affect the basic and novel characteristics of the invention as claimed.

"dysbacteriosis" refers to a state of microbiota or microbiome of the intestinal tract or other body area, including, for example, mucosal or skin surfaces (or any other microbiome niches) in which the normal diversity and/or function of the host intestinal microbiome ecological network "microbiome" is disrupted. Dysbacteriosis may lead to a disease state, or may be unhealthy only under certain conditions or only when present for a long period of time. Dysbacteriosis may be due to a variety of factors including environmental factors, infectious agents, host genotype, host diet and/or stress. Dysbacteriosis may result in: a change (e.g., an increase or decrease) in the prevalence of one or more bacterial types (e.g., anaerobes), species, and/or strains, a change (e.g., an increase or decrease) in the diversity of the host microbiome population composition; a change (e.g., an increase or decrease) in one or more symbiotic populations that results in a decrease 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 symbiota that cause disease only in some cases, and/or overgrowth.

The term "reduce" or "deplete" means a change such that the difference (as the case may be) from the pre-treatment state after treatment is 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. The properties that can be reduced include the number of immune cells, bacterial cells, stromal cells, myeloid-derived suppressor cells, fibroblasts, metabolites; levels of cytokines; or another physical parameter such as ear thickness (e.g., in a DTH animal model) or tumor size (e.g., in an animal tumor model).

The term "effective dose" is the amount of therapeutic composition effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration with minimal toxicity to the subject.

As used herein, an "engineered bacterium" is any bacterium that has been genetically altered from a natural state by human activity and the progeny of any such bacterium. Engineered bacteria include, for example, products targeted for genetic modification, products screened by random mutagenesis, and products of directed evolution.

The term "epitope" means a protein determinant that can specifically bind to an antibody or T cell receptor. Epitopes are generally composed of chemically active surface groupings of molecules such as amino acids or sugar side chains. Certain epitopes can be defined by the specific sequence of amino acids to which an antibody can bind.

An "extracellular vesicle" (EV) may be a naturally occurring vesicle of bacterial origin, such as a smEV. EV is composed of bacterial lipids and/or bacterial proteins and/or bacterial nucleic acids and/or bacterial carbohydrate fractions and is isolated from the culture supernatant. The natural production of these vesicles can be artificially enhanced (e.g., increased) or decreased by manipulating the environment in which the bacterial cells are being cultured (e.g., by a medium or temperature change). Furthermore, EV compositions may be modified to reduce, increase, add or remove bacterial components or foreign substances to alter efficacy, immunostimulation, stability, immunostimulation ability, stability, organ targeting (e.g., lymph nodes), absorption (e.g., gastrointestinal tract), and/or yield (e.g., thereby altering efficacy). As used herein, the term "purified EV composition" or "EV composition" refers to a formulation of an EV that has been separated from at least one related substance found in the source material or any material associated with the EV in any method used to produce the formulation (e.g., separated from at least one other bacterial component). Compositions that have been significantly enriched for a particular component may also be referred to. Extracellular vesicles can also be obtained from mammalian cells, and can be obtained from microorganisms such as archaebacteria, fungi, microalgae, protozoa and parasites. Extracellular vesicles from any of these sources can be prepared in the solution and/or dried forms described herein. Extracellular vesicles can be artificially produced vesicles prepared from bacteria, such as pmEV, for example, obtained by chemical disruption (e.g., by lysozyme and/or lysostaphin) and/or physical disruption (e.g., by mechanical force) of bacterial cells and separation of bacterial membrane components from intracellular components by centrifugation and/or ultracentrifugation or other methods, and can also be prepared in solution and/or dried form as described herein.

The term "gene" is used in a broad sense to refer to any nucleic acid associated with a biological function. The term "gene" applies to a particular genomic sequence and to the cDNA or mRNA encoded by that genomic sequence.

"identity" between nucleic acid sequences of two nucleic acid molecules can be determined as a percentage of identity using known computer algorithms (e.g., the "FASTA" program) using, for example, preset parameters as in Pearson et al (1988) Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. USA ]85:2444 (other programs include GCG program package (Devereux, J. Et al, nucleic Acids Research [ nucleic acids research ]12 (I): 387 (1984)), BLASTP, BLASTN, FASTA Atschul, S.F. et al, J. Molecular Biol [ J. Mol. Biol ]215:403 (1990); guide to Huge Computers [ giant computer guide ], martin J. Bishop, academic Press, sanilemic Diego [ San Diego ],1994 and Carlo et al (1988) SIAM J Applied Math [ applied mathematics ] 48:1073). For example, the BLAST function of the national center for Biotechnology information database (National Center for Biotechnology Information database) can be used to determine identity. Other commercially or publicly available programs include the DNAStar "MegAlign" program (Madison, wis.)) and the university of wisconsin genetics computer group (University of Wisconsin Genetics Computer Group) (UWG) "Gap" program (Madison, wis.)).

As used herein, the term "immune disorder" refers to any disease, disorder or disease symptom caused by the 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 myopathies), 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 allergies (e.g., food allergy, drug allergy, and/or environmental allergies).

"immunotherapy" is a treatment that uses the immune system of a subject to treat a disease (e.g., an immune disease, an inflammatory disease, a metabolic disease, cancer) and includes, for example, checkpoint inhibitors, cancer vaccines, cytokines, cell therapies, CAR-T cells, and dendritic cell therapies.

The term "increase" means a change such that the difference (as the case may be) from the pre-treatment state after treatment is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 4-fold, 10-fold, 100-fold, 10-3-fold, 10-4-fold, 10-5-fold, 10-6-fold and/or 10-7-fold higher. The properties that can be increased include the number of immune cells, bacterial cells, stromal cells, myeloid-derived suppressor cells, fibroblasts, metabolites; levels of cytokines; or another physical parameter such as ear thickness (e.g., in a DTH animal model) or tumor size (e.g., in an animal tumor model).

An "innate immune agonist" or "immune adjuvant" is a small molecule, protein, or other agent that specifically targets an innate immune receptor (including Toll-like receptors (TLRs), NOD receptors, RLRs, C-lectin receptors, STING-cGAS pathway components, inflammatory complexes). For example, LPS is a bacterial or synthetic TLR-4 agonist and aluminum can be used as an immunostimulating adjuvant. Immunoadjuvants are a specific class of broader adjuvants or adjuvant therapies. Examples of STING agonists include, but are not limited to, rp, sp isomers of 2'3' -cGAMP, 3'-cGAMP, c-di-AMP, c-di-GMP, 2' -cGAMP, and 2'3' -cGAM (PS) 2 (Rp/Sp) (dithiophosphate analogs of 2'3' -cGAMP). Examples of TLR agonists include, but are not limited to, TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLRl0 and TLR11. Examples of NOD agonists include (but are not limited to): n-acetyl muramyl-L-alanyl-D-isoglutamine (muramyl dipeptide (MDP)), gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP), and desmoleyl peptide (desmuramylpeptide (DMP)).

An "internal transcribed spacer" or "ITS" is a segment of nonfunctional RNA located between structural ribosomal RNAs (rrnas) on common precursor transcripts that are typically used to identify eukaryotic species (particularly fungi). rRNA of the ribosomal nucleus forming fungus is transcribed into a signal gene and consists of 8S, 5.8S and 28S regions and ITS4 and 5 between 8S and 5.8S and between 5.8S and 28S regions, respectively. As previously described, such two double-translated gene blocks (intercistronic segment) between 18S and 5.8S and between 5.8S and 28S regions are removed by splicing and contain significant variation between species for the purpose of barcodes (Schoch et al Nuclear ribosomal Internal Transcribed Spacer (ITS) region as a universal DNA barcode marker for Fungi [ the in-ribosome transcriptional spacer (ITS) is a universal DNA barcode tag for fungi ]. PNAS [ Proc. Natl. Acad. Sci. USA ] 109:6241-6246.2012). 18S rDNA is traditionally used for phylogenetic reconstruction, however ITS can do this because it is usually highly conserved but contains hypervariable regions with sufficient nucleotide diversity to distinguish the genus and species of most fungi.

The term "isolated" or "enriched" encompasses microorganisms, EVs (e.g., bacterial EVs), or other entities or substances that have been (1) separated from at least some components associated therewith when initially produced (whether in nature or in an experimental environment), and/or (2) produced, prepared, purified, and/or manufactured by man. The isolated bacteria or EV 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 it was originally associated. In some embodiments, the isolated bacteria or EV is greater 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 greater than about 99% pure, e.g., substantially free of other components.

As used herein, "lipid" includes fats, oils, triglycerides, cholesterol, phospholipids, any form of fatty acid (including free fatty acids). Fats, oils and fatty acids may be saturated, unsaturated (cis or trans) or partially unsaturated (cis or trans).

As used herein, "metabolite" refers to any and all molecular compounds, compositions, molecules, ions, cofactors, catalysts, or nutrients that are used as substrates or as product compounds, compositions, molecules, ions, cofactors, catalysts, or nutrients in any cellular or bacterial metabolic reaction resulting from any cellular or bacterial metabolic reaction.

"microbiome" broadly refers to a microorganism residing on or in a body part of a subject or patient. The microorganisms in the microbiome may include bacteria, viruses, eukaryotic microorganisms and/or viruses. The individual microorganisms in the microbiome may be metabolically active, dormant, latent or present as spores, may be present in planktonic form or in a biofilm, or may be present in the microbiome in a sustainable or transient manner. The microbiome may be a symbiotic or healthy state microbiome or a disease state or dysbacteriosis microbiome. The microbiome can be native to the subject or patient, or components of the microbiome can be adjusted, introduced, or consumed as a result of changes in health status or treatment conditions (e.g., antibiotic treatment, exposure to different microorganisms). In some aspects, the microbiome is present at a mucosal surface. In some aspects, the microbiome is an intestinal microbiome.

"microbiome profile (microbiome profile)" or "microbiome signature (microbiome signature)" of a tissue or sample refers to at least a partial characterization of the bacterial composition of a microbiome. In some embodiments, the microbiome profile indicates whether at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bacterial strains are present in the microbiome or are not present in the microbiome.

"modified" with respect to bacteria broadly refers to bacteria that have been altered from the wild-type form. Bacterial modifications may be produced from engineering bacteria. Examples of bacterial modifications include genetic modifications, genetic expression modifications, phenotypic modifications, formulation modifications, chemical modifications, and dosages or concentrations. Examples of improved properties are described throughout the specification and include, for example, attenuation, auxotrophy, homing, or antigenicity. Phenotypic modifications may include (as exemplified) growth of a bacterium in a medium that modifies the phenotype of the bacterium such that it increases or decreases virulence.

"operational taxon" and "OTU" refer to the terminal leaves in a phylogenetic tree and are defined by nucleic acid sequences (e.g., the entire genome or a specific gene sequence and all sequences sharing sequence identity at the species level with such nucleic acid sequences). In some embodiments, the specific gene sequence may be a 16S sequence or a portion of a 16S sequence. In other embodiments, the entire genomes of the two entities are sequenced and compared. In another embodiment, selected regions (e.g., multiple Locus Sequence Tags (MLST), specific genes, or gene sets) can be compared genetically. For 16S, OTUs sharing > 97% average nucleotide identity throughout 16S or some 16S variable regions can be considered the same OTU. See, e.g., claesson MJ, wang Q, O 'Sullivan O, greene-Dinitz R, cole JR, ross RP, and O' Toole PW.2010.Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable16S rRNA gene regions [ comparison of two next-generation sequencing techniques using tandem variable16S rRNA gene regions to resolve highly complex microbiota compositions ]. Nucleic Acids Res [ nucleic acids research ]38:e200.Konstantinidis KT,Ramette A and Tiedje JM.2006.the bacterial species definition in the genomic era [ definition of bacterial species in genome age ]. Philos Trans R Soc Lond B Biol Sci [ London family, proc. B.): biological science philosophy report 361:1929-1940. OTUs sharing an average nucleotide identity of > 95% can be considered as identical OTUs for the whole genome, MLST, a specific gene (except 16S) or a gene set. See, for example, achtman M and Wagner m.2008.microbal diversity and the genetic nature of microbial species [ microbial diversity and genetic nature of microbial species ]. Nat.rev. Microbiol. [ natural reviews of microorganisms ]6:431-440.Konstantinidis KT,Ramette A and Tiedje jm.2006.the bacterial species definition in the genomic era [ definition of bacterial species in genomic age ]. Philos Trans R Soc Lond B Biol Sci [ london Royal society B edition: biological science philosophy report 361:1929-1940. OTUs are typically defined by comparing sequences between organisms. Typically, sequences having less than 95% sequence identity are not considered to form part of the same OTU. OTUs may also be characterized by nucleotide markers or genes, particularly highly conserved genes (e.g. "housekeeping" genes), or any combination thereof. Provided herein are Operational Taxonomies (OTUs) that can allocate, for example, genus, species, and phylogenetic clades.

As used herein, a gene is "overexpressed" in a bacterium if the expression level of the gene in an engineered bacterium under at least some conditions is higher than the expression level of a wild-type bacterium of the same species under the same conditions. Similarly, a gene is "under-expressed" in a bacterium if the expression level of the gene in the engineered bacterium under at least some conditions is lower than the expression level of a wild-type bacterium of the same species under the same conditions.

The term "polynucleotide" and "nucleic acid" are used interchangeably. They refer to polymeric forms of nucleotides of any length (deoxyribonucleotides or ribonucleotides) or analogs thereof. The polynucleotide may have any three-dimensional structure and may perform any function. Non-limiting examples of polynucleotides are as follows: coding or non-coding regions of a gene or gene fragment, multiple loci (loci) defining a self-interlocking analysis, exons, introns, messenger RNAs (mRNA), micrornas (miRNA), silencing RNAs (siRNA), transfer RNAs, ribosomal RNAs, ribozymes, cdnas, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modification of the nucleotide structure, if present, may be imparted before or after assembly of the polymer. The polynucleotide may be further modified, such as by conjugation with a labeling component. U nucleotides may be interchanged with T nucleotides in all nucleic acid sequences provided herein.

As used herein, a substance is "pure" when it is substantially free of other components. The terms "purifying" or "purifying (ing)" and "purified" refer to an EV (e.g., an EV from bacteria) preparation or other material that has been separated from at least some components associated therewith when initially produced or formed (e.g., whether in nature or in an experimental environment) or during any time after initial production. An EV formulation or composition may be considered purified if it is separated from, for example, one or more other bacterial components at the time of or after production, and the purified microorganism or bacterial population may contain up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more than about 90% of other materials and still be considered "purified". In some embodiments, the purified EV is greater 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 greater than about 99% pure. EV compositions (or formulations) are, for example, purified from residual habitat products.

As used herein, the term "purified EV composition" or "EV composition" refers to a formulation of: it includes an EV from a bacterium that has been separated from the source material or from at least one related substance found in any material associated with the EV in any method used to produce the formulation (e.g., separated from at least one other bacterial component). It also refers to compositions that have been significantly enriched or concentrated. In some embodiments, these EVs are concentrated 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold, or more than 10,000-fold.

"residual habitat product" refers to a material derived from a microbiota habitat within or on a subject. For example, a fermentation culture of a microorganism may contain contaminants, such as other microbial strains or forms (e.g., bacteria, viruses, mycoplasma, and/or fungi). For example, microorganisms are born in the feces of the gastrointestinal tract, in the skin itself, in saliva, in the mucus of the respiratory tract, or in secretions of the genitourinary tract (i.e., biological substances associated with the microflora). By substantially free of residual habitat products is meant that the microbial composition no longer contains biological material associated with the microbial environment on or in culture or in a human or animal subject and is 100% free, 99% free, 98% free, 97% free, 96% free or 95% free of any contaminating biological material associated with the microbial community. The residual habitat product may include non-biological materials (including undigestedFood) or it may include undesirable microorganisms. Substantially free of residual habitat products may also mean that the microbial composition does not contain detectable cells from culture contaminants or humans or animals and that only microbial cells are detectable. In one embodiment, substantially free of residual habitat products may also mean that the microbial composition is free of detectable viruses, including bacteria, viruses (e.g., phage), fungi, mycoplasma contaminants. In another embodiment, this means that less than 1x10 in the microbial composition compared to microbial cells ^-2 ％、1x10 ^-3 ％、1x10 ^-4 ％、1x10 ^-5 ％、1x10 ^-6 ％、1x10 ^-7 ％、1x10 ^-8 % of the viable cells are human or animal cells. There are many ways to achieve this purity, none of which are limiting. Thus, contaminants can be reduced by performing multiple streaking steps on a single colony on solid medium until replications (such as, but not limited to, two) from a series of single colonies have shown only a single colony morphology to isolate the desired component. Alternatively, the reduction of contaminants may be accomplished by multiple rounds of serial dilution to a single desired cell (e.g., 10 ^-8 Or 10 ^-9 Dilution of (c) as done by multiple 10-fold serial dilutions. This can be further confirmed by showing that a plurality of isolated colonies have similar cell shapes and gram staining behavior. Other methods for confirming adequate purity include genetic analysis (e.g., PCR, DNA sequencing), serological and antigenic analysis, enzymatic and metabolic analysis, and methods using instrumentation, such as flow cytometry using reagents that distinguish the desired components from contaminants.

As used herein, "specific binding" refers to an antibody that is capable of binding to a predetermined antigen or polypeptide that is capable of binding to its predetermined binding partner. Typically, the antibody or polypeptide will correspond to about 10 ^-7 M or less K _D Specifically binds to its intended antigen or binding partner, and with an affinity that is at least 10-fold smaller, at least 100-fold smaller, or at least 1000-fold smaller (e.g., by K) than its affinity for binding to a non-specific and unrelated antigen/binding partner (e.g., BSA, casein) _D Represented by) Binds to a predetermined antigen/binding partner. Alternatively, specific binding is more broadly applicable to two-component systems in which one component is a protein, lipid, or carbohydrate, or combination thereof, and is conjugated in a specific manner with a second component that is a protein, lipid, carbohydrate, or combination thereof.

"Strain" refers to a member of a bacterial species having a genetic signature such that it is distinguishable from closely related members of the same bacterial species. The genetic signature may be the absence of all or a portion of at least one gene, the absence of all or a portion of at least one regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), the absence ("elimination") of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutant gene, the presence of at least one foreign gene (a gene derived from another species), the presence of at least one mutant regulatory region (e.g., promoter, terminator, riboswitch, 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. The genetic signature between different strains can be identified by PCR amplification and optionally followed by DNA sequencing of one or more genomic regions or whole genomes of interest. If one strain has acquired or lost antibiotic resistance or acquired or lost biosynthetic capacity (e.g., an auxotrophic strain) as compared to another strain of the same species, the strain can be distinguished by selection or counter-selection using an antibiotic or nutrient/metabolite, respectively.

The term "subject" or "patient" refers to any mammal. A subject or patient described as "in need" refers to a person in need of treatment (or prevention) of a disease. Mammals (i.e., mammals) include humans, laboratory animals (e.g., primates, rats, mice), domestic animals (e.g., cows, sheep, goats, pigs), and domestic 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: dogs, cats, cattle, horses, pigs, donkeys, goats, camels, mice, rats, guinea pigs, sheep, camels, monkeys, gorillas, or chimpanzees. The subject may be healthy or may have a disease or disorder at any stage of development.

As used herein, the term "therapeutic agent" refers to an agent for therapeutic use. In some embodiments, the therapeutic agent is a composition comprising an EV that is useful for treating and/or preventing a disease and/or disorder ("EV composition"). In some embodiments, the therapeutic agent is a pharmaceutical formulation. In some embodiments, the pharmaceutical product, medical food, food or dietary supplement comprises a therapeutic agent. In some embodiments, the therapeutic agent is in solution, while in other embodiments, in dry form. Embodiments in dry form may be produced, for example, by lyophilization or spray drying. In some embodiments, the therapeutic agent in dry form is a lyophilized cake or powder. In some embodiments, the therapeutic agent in dry form is a spray-dried powder.

As used herein, the term "therapeutic composition" or "pharmaceutical composition" refers to a composition (e.g., an EV composition as described herein) that comprises a therapeutically effective amount of a therapeutic agent. In some embodiments, the therapeutic composition is (or is present in) a pharmaceutical product, medical food, food or dietary supplement.

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 a drug therapy (e.g., administration of one or more agents) to the subject, thereby reducing at least one symptom of the disease or preventing its exacerbation. Thus, in one embodiment, "treatment" refers to, inter alia, delaying progression, promoting relief, inducing relief, increasing relief, accelerating recovery, increasing efficacy, or decreasing resistance to an alternative treatment, or a combination thereof. As used herein, the term "preventing" a disease in a subject refers to administering a pharmaceutical treatment to the subject, e.g., administering one or more agents such that the onset of at least one symptom of the disease is delayed or prevented.

Bacteria and method for producing same

In certain aspects, provided herein are Prevotella denticola Extracellular Vesicles (EV), as well as solutions and/or dried forms and therapeutic compositions comprising Prevotella denticola Extracellular Vesicles (EV). In certain aspects, provided herein are solution and/or dry forms and therapeutic compositions comprising an EV obtained from a prasuvorexa bacteria of the tissue of interest.

In some embodiments, the serratia bacteria of the percha tissue from which the EV was obtained are lyophilized.

In some embodiments, the Prevotella bacteria of the percha tissue from which the EV was obtained are gamma irradiated (e.g., to 17.5 or 25 kGy).

In some embodiments, the EV-derived tissue prasuvorexant bacteria are subjected to UV irradiation.

In some embodiments, the Prevotella bacteria of the percha tissue from which the EV was obtained are heat-inactivated (e.g., at 50℃for two hours or at 90℃for two hours).

In some embodiments, the serratia bacteria of the percha tissue from which the EV was obtained are subjected to an acid treatment.

In some embodiments, the serratia bacteria, the percha tissue from which the EV was obtained, are subjected to oxygen sparging (e.g., at 0.1vvm for two hours).

In some embodiments, the tissue Prevotella EV is lyophilized.

In some embodiments, the tissue Prevotella EV is spray dried.

In some embodiments, the Prevotella EV is gamma irradiated (e.g., at 17.5 or 25 kGy) to the tissue.

In some embodiments, the tissue Prevotella EV is subjected to UV irradiation.

The growth stage may affect the amount or nature of EV produced by the bacteria and/or the tissue Prevotella bacteria. For example, in the EV preparation methods provided herein, the EV may be isolated from the culture, for example, at the beginning of the logarithmic growth phase, at the middle of the logarithmic growth phase, and/or once the stationary growth phase is reached.

In some embodiments, the tissue Prevotella EV is from a bacterial strain, such as the strains provided herein.

In some embodiments, the tissue prasuvorexant EV is from one bacterial strain (e.g., the strains provided herein) or from more than one strain.

In some embodiments, the EV is from a prasuvorexant bacterium, e.g., from a strain comprising at least 90% or at least 99% genomic, 16S, and/or CRISPR sequence identity to the nucleotide sequence of a prasuvorexant strain B50329 (NRRL accession No. B50329). In some embodiments, the EV is from a prasuvorexant bacterium, e.g., from prasuvorexant strain B50329 (NRRL accession No. B50329).

In some embodiments, the EV-obtaining tissue Prevotella bacteria are modified (e.g., engineered) to reduce toxicity or other adverse effects; enhanced delivery of EV (e.g., oral delivery) (e.g., by improving acid resistance, mucoadhesion and/or permeability and/or resistance to bile acids, digestive enzymes, resistance to antimicrobial peptides, and/or antibody neutralization); targeting desired cell types (e.g., M cells, goblet cells, intestinal epithelial cells, dendritic cells, macrophages); enhancing the immunomodulatory and/or therapeutic effects of EV (e.g., alone or in combination with another therapeutic agent); and/or enhancing immune activation or inhibition by EV (e.g., via modified manufacture of polysaccharides, cilia, pili, adhesins). In some embodiments, the engineered bacteria described herein are modified to improve EV manufacturing (e.g., higher oxygen tolerance, stability, improved freeze-thaw tolerance, shorter production time). For example, in some embodiments, the engineered bacteria described herein include bacteria having one or more genetic alterations that result in over-expression and/or under-expression of one or more genes comprising one or more nucleotide insertions, deletions, translocations, or substitutions on bacterial chromosomes or endogenous plasmids and/or one or more exogenous plasmids, or any combination thereof. Engineered bacteria may be produced using any technique known in the art, including, but not limited to, site-directed mutagenesis, transposon mutagenesis, knockout, knock-in, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet mutagenesis, transformation (chemical or by electroporation), phage transduction, directed evolution, or any combination thereof.

Modified EV

In some aspects, the prasuvorexant EVs described herein are modified such that they comprise, are linked to, and/or bind to a therapeutic moiety.

In some embodiments, the tissue prasuvorexant EVs described herein are engineered such that they comprise, are linked to, and/or bind to magnetic and/or paramagnetic moieties (e.g., magnetic beads). In some embodiments, the magnetic and/or paramagnetic moiety comprises a bacterium and/or is directly attached to a bacterium. In some embodiments, the magnetic and/or paramagnetic moiety is attached to and/or is part of an EV-binding moiety that binds to an EV. In some embodiments, the EV-binding moiety is a fragment of a full-length peptidoglycan recognition protein (e.g., PGRP) or a full-length peptidoglycan recognition protein. In some embodiments, the EV-binding moiety has binding specificity for EV (e.g., by having binding specificity for a bacterial antigen). In some embodiments, the EV binding moiety comprises an antibody or antigen-binding fragment thereof. In some embodiments, the EV binding moiety comprises a T cell receptor or Chimeric Antigen Receptor (CAR).

Production of bacterial Extracellular Vesicles (EV)

Secreted EV.In certain aspects, EVs (e.g., secreted EVs (smevs) from the bacteria described herein) are prepared using any method known in the art.

In some embodiments, the smEV is prepared without a smEV purification step. For example, in some embodiments, the bacteria described herein are killed by using a method that leaves the smEV intact and the resulting bacterial composition (including the smEV) is used in the methods and compositions described herein. In some embodiments, the bacteria are killed by use of an antibiotic (e.g., using an antibiotic as described herein). In some embodiments, the bacteria are killed by using UV radiation. In some embodiments, the bacteria are heat killed.

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, smEV is prepared from bacterial cultures using the methods described in s.bin Park et al PLoS ONE [ public science library-complex ].6 (3): e17629 (2011) or g.norheim et al PLoS ONE [ public science library-complex ].10 (9): e 0134553 (2015) or jepepsen et al Cell [ Cell ]177:428 (2019), each of which is hereby incorporated by reference in its entirety. In some embodiments, these bacteria are cultured to high optical density and then centrifuged to pellet the bacteria (e.g., centrifugation at 10,000Xg for 30min at 4 ℃, centrifugation at 15,500Xg for 15min at 4 ℃). In some embodiments, the culture supernatant is then passed through a filter to exclude intact bacterial cells (e.g., a 0.22 μm filter). In some embodiments, the supernatant is then subjected to tangential flow filtration, during which the supernatant is concentrated, less than 100kDa material is removed, and the medium is partially exchanged with PBS. In some embodiments, the filtered supernatant is centrifuged to pellet the bacterial smEV (e.g., at 100,000 to 150,000x g for 1 to 3 hours at 4 ℃ and at 200,000x g for 1 to 3 hours at 4 ℃). In some embodiments, these smevs are further purified by resuspension of the resulting smEV pellet (e.g., in PBS) and application of the resuspended smEV to an Optiprep (iodixanol) gradient or gradient (e.g., 30% to 60% discontinuous gradient, 0-45% discontinuous gradient), followed by centrifugation (e.g., at 200,000x g for 4-20 hours at 4 ℃). The smEV bands can be collected, diluted with PBS and centrifuged to pellet the smEV (e.g., at 150,000x g for 3 hours at 4 ℃ and at 200,000x g for 1 hour at 4 ℃). Purified smevs can be stored (e.g., at-80 ℃ or-20 ℃) until use. In some embodiments, the smEV is further purified by treatment with dnase and/or proteinase K.

For example, in some embodiments, a culture of bacteria may be centrifuged at 11,000Xg for 20-40 minutes at 4 ℃ to pellet the bacteria. The culture supernatant may be passed through a 0.22 μm filter to exclude intact bacterial cells. The filtered supernatant may then be concentrated using methods that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration. For example, in the case of ammonium sulfate precipitation, 1.5-3M ammonium sulfate may be slowly added to the filtered supernatant while stirring at 4 ℃. The pellet may be incubated at 4℃for 8 to 48 hours and then centrifuged at 11,000Xg for 20-40 minutes at 4 ℃. The resulting precipitate contained bacterial smEV and other debris. Ultracentrifugation can be used and the filtered supernatant centrifuged at 100,000 to 200,000Xg for 1-16 hours at 4 ℃. This centrifuged sediment contains bacterial smEV and other debris (e.g. large protein complexes). In some embodiments, using filtration techniques, such as by using Amicon super spin filters or by tangential flow filtration, the supernatant may be filtered so as to retain substances with molecular weights >50 or 100 kDa.

Alternatively, the smEV may be obtained continuously from the bacterial culture at a selected point in time during growth or during growth, for example by connecting the bioreactor to an Alternating Tangential Flow (ATF) system (e.g. XCell ATF from Repligen). The ATF system retains intact cells (> 0.22 μm) in the bioreactor and allows smaller components (e.g., smEV, free protein) to pass through the filter for collection. For example, the system may be structured such that <0.22 μm filtrate is then passed through a 100kDa second filter, allowing collection of material such as smEV between 0.22 μm and 100kDa and pumping of species less than 100kDa back into the bioreactor. Alternatively, the system may be structured to allow the medium in the bioreactor to be replenished and/or modified during the growth of the culture. The smEV collected by this method can be further purified and/or concentrated by ultracentrifugation or filtration as described above for the filtered supernatant.

The smevs obtained by the methods provided herein can be further purified by size-based column chromatography, by affinity chromatography, by ion exchange chromatography, and by gradient ultracentrifugation using methods that can include, but are not limited to, using sucrose gradients or Optiprep gradients. Briefly, when using the sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation is used to concentrate the filtered supernatant, the precipitate is resuspended in 60% sucrose, 30mM pH 8.0 Tris. If filtration is used to concentrate the filtered supernatant, the concentrate buffer is exchanged into 60% sucrose, 30mM pH 8.0Tris using an Amicon Ultra column. Samples were applied to a 35% -60% discontinuous sucrose gradient and centrifuged at 200,000Xg at 4 ℃ for 3-24 hours. Briefly, when using the Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation is used to concentrate the filtered supernatant, the precipitate is resuspended in PBS and 3 volumes of 60% Optiprep are added to the sample. In some embodiments, if filtration is used to concentrate the filtered supernatant, the concentrate is diluted to a final concentration of 35% Optiprep using 60% Optiprep. Samples were applied to an Optiprep gradient of 0-45% discontinuity and centrifuged at 200,000x g for 3 to 24 hours at 4 ℃, e.g., 4 to 24 hours at 4 ℃.

In some embodiments, to confirm sterility and isolation of the smEV formulation, the smEV is serially diluted onto agar medium (which is used for routine culture of the bacteria under test) and incubated using routine conditions. The unsterilized formulation was passed through a 0.22 μm filter to remove intact cells. To further increase purity, the isolated smevs may be treated with dnase or proteinase K.

In some embodiments, to prepare a smEV for in vivo injection, the purified smEV is treated as previously described (g.norheim et al, PLoS ONE. [ public science library. Complex ]10 (9): e 0134553 (2015)). Briefly, after sucrose gradient centrifugation, the smEV-containing bands were resuspended to a final concentration of 50 μg/mL in a solution containing 3% sucrose or other solutions known to those skilled in the art to be suitable for in vivo injection. The solution may also contain an adjuvant (e.g., aluminum hydroxide) at a concentration of 0-0.5% (w/v). In some embodiments, to prepare a smEV for in vivo injection, the smEV in PBS is sterile filtered to <0.22 μm.

In certain embodiments, to make samples compatible with other tests (e.g., to remove sucrose prior to TEM imaging or in vitro analysis), sample buffers are exchanged into PBS or 30mM pH 8.0Tris, dialyzed, or ultracentrifuged (200,000Xg,. Gtoreq.3 hours, 4 ℃) using filtration (e.g., an Amicon Ultra column) and resuspended.

In some embodiments, sterility of a smEV formulation can be confirmed by inoculating a portion of the smEV onto agar medium (which is used for standard culture of bacteria used to produce the smEV) and incubating using standard conditions.

In some embodiments, the selected smevs are isolated and enriched by chromatography and binding surface moieties on the smevs. In other embodiments, the selected smevs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins, or other methods known to those of skill in the art.

In some embodiments, the smEV is analyzed, for example, as described by Jeppesen et al Cell [ Cell ]177:428 (2019).

In some embodiments, the smEV is lyophilized.

In some embodiments, the smEV is spray dried.

In some embodiments, the smEV is gamma irradiated (e.g., at 17.5 or 25 kGy).

In some embodiments, the smEV is UV irradiated.

In some embodiments, the smEV is heat-inactivated (e.g., at 50 ℃ for two hours or at 90 ℃ for two hours).

In some embodiments, the smEV is acid treated.

In some embodiments, the smEV is oxygen injected (e.g., at 0.1vvm for two hours).

The growth stage may affect the amount or nature of the bacteria and/or the smEV produced by the bacteria. For example, in the methods of making a smEV provided herein, the smEV can be isolated from the culture, for example, at the beginning of the logarithmic growth phase, at the middle of the logarithmic growth phase, and/or once the stationary growth phase is reached.

The growth environment (e.g., culture conditions) can affect the amount of the bacteria that produce the smEV. For example, a smEV-inducing factor may increase the yield of smEV, as shown in table 4.

Table 4: culture technique for increasing smEV production

In the methods of making a smEV provided herein, the methods can optionally include exposing the bacterial culture to a smEV-inducing factor prior to isolating the smEV from the bacterial culture. The bacterial culture may be exposed to the smEV-inducing factor at the beginning of the logarithmic growth phase, at the middle of the logarithmic growth phase, and/or once the stationary growth phase is reached.

Processed EV. In certain aspects, EVs (processed EVs (pmevs) as described herein) are prepared (e.g., artificially prepared) using any method known in the art.

In some embodiments, the pmEV is prepared without a pmEV purification step. For example, in some embodiments, bacteria that release the pmevs described herein are killed using a method that leaves the bacterial pmevs intact, and the resulting bacterial components (including pmevs) are used in the methods and compositions described herein. In some embodiments, the bacteria are killed by use of an antibiotic (e.g., using an antibiotic as described herein). In some embodiments, the bacteria are killed by using UV radiation.

In some embodiments, the pmevs described herein are purified from one or more other bacterial components. Methods for purifying pmEV from bacteria (and optionally other bacterial components) are known in the art. In some embodiments, pmevs are prepared from bacterial cultures by using the methods described in Thein, et al (j. Proteome Res. [ journal of proteomics research ]9 (12): 6135-6147 (2010)) or Sandrini, et al (Bio-protocol [ biological protocol ]4 (21): e1287 (2014)), each of which is incorporated herein by reference in its entirety. In some embodiments, these bacteria are cultured to high optical density and then centrifuged to pellet the bacteria (e.g., at 10,000-15,000Xg for 10-15 minutes at room temperature or 4 ℃). In some embodiments, the supernatant is discarded and the cell pellet is frozen at-80 ℃. In some embodiments, the cell pellet is thawed on ice and resuspended in 100mM Tris-HCl (pH 7.5) supplemented with 1mg/mL DNase I. In some embodiments, the cells are lysed using Emulsiflex C-3 (ovistin, inc.) under conditions recommended by the manufacturer. In some embodiments, debris and uncleaved cells are pelleted by centrifugation at 10,000Xg for 15 minutes at 4 ℃. In some embodiments, the supernatant is then centrifuged at 120,000Xg for 1 hour at 4 ℃. In some embodiments, the pellet is resuspended in ice-cold 100mM sodium carbonate pH 11, incubated with stirring at 4℃for 1 hour, and then centrifuged at 120,000Xg at 4℃for 1 hour. In some embodiments, the pellet is resuspended in 100mM Tris-HCl, pH 7.5, centrifuged at 120,000Xg for 20 minutes at 4℃and then resuspended in 0.1M Tris-HCl (pH 7.5) or in PBS. In some embodiments, the sample is stored at-20 ℃.

In certain aspects, pmEV is obtained by adapting the method from Santrini et al, 2014. In some embodiments, the bacterial culture is centrifuged at 10,000-15,500x g for 10-15 minutes at room temperature or 4 ℃. In some embodiments, the cell pellet is frozen at-80 ℃ and the supernatant discarded. In some embodiments, the cell pellet is thawed on ice and resuspended in 10mM Tris-HCl (pH 8.0), 1mM EDTA supplemented with 0.1mg/mL lysozyme. In some embodiments, the samples are mixed and incubated for 30 minutes at room temperature or 37 ℃. In some embodiments, the sample is re-frozen at-80 ℃ and then thawed again on ice. In some embodiments, DNase I is added to a final concentration of 1.6mg/mL and MgCl is added ₂ The final concentration was 100mM. In some embodiments, samples were sonicated using a QSonica Q500 sonicator with 7 cycles of 30 seconds on and 30 seconds off. In some embodiments, debris and uncleaved cells are pelleted by centrifugation at 10,000Xg for 15 minutes at 4 ℃. In some embodiments, the supernatant is then centrifuged at 110,000Xg for 15 minutes at 4 ℃. In some embodiments, the pellet is resuspended in 10mM Tris-HCl (pH 8.0), 2% Triton X-100, and mixed incubated at room temperature for 30-60min. In some embodiments, the sample is centrifuged at 110,000Xg for 15 minutes at 4 ℃. In some embodiments, the pellet is resuspended in PBS and stored at-20deg.C.

In certain aspects, the methods of forming (e.g., preparing) an isolated bacterial pmEV described herein comprise the steps of: (a) Centrifuging the bacterial culture, thereby forming a first precipitate and a first supernatant, wherein the first precipitate comprises cells; (b) discarding the first supernatant; (c) resuspending the first precipitate in solution; (d) lysing the cells; (e) Centrifuging the lysed cells, thereby forming a second precipitate and a second supernatant; (f) Discarding the second precipitate and centrifuging the second supernatant, thereby forming a third precipitate and a third supernatant; (g) The third supernatant was discarded and the third precipitate was resuspended in the second solution, thereby forming isolated bacterial pmEV.

In some embodiments, the method further comprises the steps of: (h) Centrifuging the solution of step (g) to form a fourth precipitate and a fourth supernatant; (i) The fourth supernatant was discarded and the fourth precipitate was resuspended in the third solution. In some embodiments, the method further comprises the steps of: (j) Centrifuging the solution of step (i) to form a fifth precipitate and a fifth supernatant; and (k) discarding the fifth supernatant and resuspending the fifth precipitate in a fourth solution.

In some embodiments, the centrifugation of step (a) is performed at 10,000×g. In some embodiments, the centrifugation of step (a) is performed for 10-15 minutes. In some embodiments, the centrifugation of step (a) is at 4 ℃ or room temperature. In some embodiments, step (b) further comprises freezing the first precipitate at-80 ℃. In some embodiments, the solution in step (c) is 100mM Tris-HCl (pH 7.5) supplemented with 1mg/ml DNase I. In some embodiments, the solution in step (c) is 10mM Tris-HCl (pH 8.0), 1mM EDTA, supplemented with 0.1mg/ml lysozyme. In some embodiments, step (c) further comprises incubating at 37 ℃ or room temperature for 30 minutes. In some embodiments, step (c) further comprises freezing the first precipitate at-80 ℃. In some embodiments, step (c) further comprises adding dnase I to a final concentration of 1.6 mg/ml. In some embodiments, step (c) further comprises adding MgCl ₂ To a final concentration of 100 mM. In some embodiments, the cells are lysed by homogenization in step (d). In some embodiments, the cells are lysed by emulisflex C3 in step (d). In some embodimentsIn an example, the cells are lysed by ultrasound in step (d). In some embodiments, the cells are sonicated for 7 cycles, wherein each cycle includes 30 seconds of sonication and 30 seconds of non-sonication. In some embodiments, the centrifugation of step (e) is performed at 10,000×g. In some embodiments, the centrifugation of step (e) is performed for 15 minutes. In some embodiments, the centrifugation of step (e) is at 4 ℃ or room temperature.

In some embodiments, the centrifugation of step (f) is performed at 120,000×g. In some embodiments, the centrifugation of step (f) is performed at 110,000×g. In some embodiments, the centrifugation of step (f) is performed for 1 hour. In some embodiments, the centrifugation of step (f) is performed for 15 minutes. In some embodiments, the centrifugation of step (f) is at 4 ℃ or room temperature. In some embodiments, the second solution in step (g) is 100mM sodium carbonate at 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 at 4 ℃ for 1 hour. In some embodiments, step (g) further comprises incubating the solution at room temperature for 30-60 minutes. In some embodiments, the centrifugation of step (h) is performed at 120,000×g. In some embodiments, the centrifugation of step (h) is performed at 110,000×g. In some embodiments, the centrifugation of step (h) is performed for 1 hour. In some embodiments, the centrifugation of step (h) is performed for 15 minutes. In some embodiments, the centrifugation of step (h) is at 4 ℃ 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 performed at 120,000×g. In some embodiments, the centrifugation of step (j) is performed for 20 minutes. In some embodiments, the centrifugation of step (j) is at 4 ℃ or room temperature. In some embodiments, the fourth solution in step (k) is 100mM Tris-HCl (pH 7.5) or PBS.

The pmevs obtained by the methods provided herein can be further purified by size-based column chromatography, by affinity chromatography, and by gradient ultracentrifugation using methods that can include, but are not limited to, the use of sucrose gradients or Optiprep gradients. Briefly, when using the sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation is used to concentrate the filtered supernatant, the precipitate is resuspended in 60% sucrose, 30mM pH 8.0 Tris. If filtration is used to concentrate the filtered supernatant, the concentrate buffer is exchanged into 60% sucrose, 30mM pH 8.0Tris using an Amicon Ultra column. Samples were applied to a 35% -60% discontinuous sucrose gradient and centrifuged at 200,000Xg at 4 ℃ for 3-24 hours. Briefly, when using the Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation is used to concentrate the filtered supernatant, the pellet is resuspended in 35% Optiprep in PBS. In some embodiments, if filtration is used to concentrate the filtered supernatant, the concentrate is diluted to a final concentration of 35% Optiprep using 60% Optiprep. Samples were applied to a 35% -60% discontinuous sucrose gradient and centrifuged at 200,000Xg at 4 ℃ for 3-24 hours.

In some embodiments, to confirm sterility and isolation of the pmEV formulation, the pmevs are serially diluted onto agar medium (which is used for routine culture of the bacteria under test) and incubated using routine conditions. The unsterilized formulation was passed through a 0.22 μm filter to remove intact cells. To further increase purity, the isolated pmEV may be treated with DNase or proteinase K.

In some embodiments, sterility of the pmEV formulation can be confirmed by inoculating a portion of the pmEV onto agar medium (which is used for standard culture of bacteria used to produce the pmEV) and incubating using standard conditions.

In some embodiments, the selected pmEV is isolated and enriched by chromatography and binding surface moieties on the pmEV. In other embodiments, the selected pmevs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins, or other methods known to those of skill in the art.

In some embodiments, pmEV is analyzed, for example, as described by Jeppesen et al Cell [ Cell ]177:428 (2019).

In some embodiments, pmEV is lyophilized.

In some embodiments, pmEV is spray dried.

In some embodiments, pmEV is gamma irradiated (e.g., at 17.5 or 25 kGy).

In some embodiments, pmEV is UV irradiated.

In some embodiments, pmEV is heat-inactivated (e.g., at 50 ℃ for two hours or at 90 ℃ for two hours).

In some embodiments, pmEV is acid treated.

In some embodiments, the pmEV is subjected to oxygen injection (e.g., at 0.1vvm for two hours).

The growth stage may affect the amount or nature of the bacteria. In the pmEV preparation methods provided herein, the pmEV can be isolated from the culture, for example, at the beginning of the logarithmic growth phase, in the middle of the logarithmic growth phase, and/or once the stationary growth phase is reached.

Solution and dry form

The present disclosure provides solutions (e.g., liquid mixtures) comprising a prasuvorexant EV (e.g., a combination of prasuvorexant EVs and/or EVs described herein) of a tissue. For example, in some embodiments, the solution includes Prevotella denticola EV and an excipient comprising a filler. As another example, in some embodiments, the solution includes prasuvorexant EV, a tissue, and an excipient comprising a bulking agent and a lyoprotectant. As another example, in some embodiments, the solution includes prasuvorexant EV, a tissue, and an excipient comprising a lyoprotectant.

For example, in some embodiments, the bulking agent comprises mannitol, sucrose, maltodextrin, dextran, ficoll, or PVP-K30. In some embodiments, the excipient optionally includes additional components, such as trehalose, mannitol, sucrose, sorbitol, maltodextrin, dextran, poloxamer 188, maltodextrin, PVP-K30, ficoll, citrate, arginine, and/or hydroxypropyl-B-cyclodextrin. For example, in some embodiments, the solution contains a liquid formulation of the EV and an excipient comprising a filler, such as an excipient from a stock of the formulation provided in one of tables A, B, C, D, K or P. For example, in some embodiments, the solution includes a liquid formulation comprising a prasuvorexant EV of tissue (e.g., obtained by isolating a prasuvorexant EV of tissue from a bacterial culture (e.g., supernatant) or retentate) and an excipient comprising a bulking agent, e.g., combining the liquid formulation comprising a prasuvorexant EV of tissue with an excipient stock comprising a bulking agent (e.g., an excipient stock of the formulation provided in one of tables A, B, C, D, K or P) to prepare the solution.

The "dried form" containing the Prevotella denticola Extracellular Vesicles (EV) refers to a product obtained by drying a solution containing Prevotella denticola EV. In some embodiments, drying is performed by freeze drying (lyophilization) or spray drying. In some embodiments, the dry form is a powder. As used herein, powder refers to a dry form and includes lyophilized powder, but includes powders obtained by a process such as spray drying, such as spray dried powders.

When freeze-drying (lyophilization) is performed, the resulting product is a lyophilisate. In some embodiments, the dry form is a lyophilisate. As used herein, lyophilisate refers to a dry form and includes lyophilized powders and lyophilized cakes. In some embodiments, the lyophilized cake is milled (e.g., ground) to produce a lyophilized powder. Milling refers to the reduction in mechanical size of the solid. Milling is, for example, of the type which can be carried out on dry form. See, e.g., seibert et al, "MILLING OPERATIONS IN THE PHARMACEUTICAL INDUSTRY [ milling operations in the pharmaceutical industry ]]”Chemical Engineering in the Pharmaceutical Industry:R&D to Manufacturing[Chemical engineering in the pharmaceutical industry: from research and development to production Manufacturing process]David j.am end edit (2011).

In some embodiments, the disclosure also provides a dry form, such as a lyophilizate, comprising a prasuvorexant EV (e.g., a prasuvorexant EV and/or combination of EVs described herein) and an excipient. For example, the dry form may include Prevotella denticola EV and an excipient comprising a filler. As another example, the dry form may include prasugrel tissue EV and an excipient comprising a bulking agent and a lyoprotectant. As another example, the dry form may include prasugrel tissue EV and an excipient comprising a lyoprotectant. For example, as described herein, in some embodiments, the prasuvorexant EV is combined with an excipient comprising a bulking agent and/or lyoprotectant, e.g., to prepare a solution. In some embodiments, the solution is dried. The resulting dry form (e.g., lyophilisate) contains the prasugrel tissue EV and one or more components of excipients, such as bulking agents and/or lyoprotectants (e.g., dry form).

The present disclosure also provides for a dry form of Prevotella EV and excipients, which are tissue-dwelling. In some embodiments, the dry form is a lyophilizate, such as a lyophilized cake or a lyophilized powder. In some embodiments, the dry form is a powder, such as a spray-dried powder or a lyophilized powder. For example, in some embodiments, the bulking agent comprises mannitol, sucrose, maltodextrin, dextran, ficoll, or PVP-K30. In some embodiments, the excipient comprises additional components, such as trehalose, mannitol, sucrose, sorbitol, dextran, poloxamer 188, maltodextrin, PVP-K30, ficoll, citrate, arginine, and/or hydroxypropyl-B-cyclodextrin. For example, in some embodiments, the dry form contains prasugrel tissue EV and an excipient, e.g., the excipient comprises a filler, e.g., an excipient from a stock of the formulation provided in one of tables A, B, C, D, K or P. In some embodiments, the dry form has a moisture content of less than about 6% (or less than about 5%) (e.g., as determined by karl fischer titration). In some embodiments, the dry form has about 10% to about 80% (by weight) of an excipient, such as an excipient comprising a filler. In some embodiments, the dry form has about 10% to about 80% (by weight) of excipients, for example, excipients from stock solutions of the formulations provided in one of tables A, B, C, D, K or P. In some embodiments, the prasuvorexant EV tissue comprises from about 1% to about 99% of the total solids by weight of the dry form. In some embodiments, the dry form has at least about 1e10 particles of prasugrel tissue per mg of dry form (e.g., as determined by particles/mg, e.g., by NTA). In some cases In embodiments, the particles in dry form have a hydrodynamic diameter (zeaverage, Z) of about 130nm to about 300nm after being resuspended from dry form (e.g., resuspended in deionized water) _ave ) (e.g., as determined by dynamic light scattering).

In some embodiments, the solution and/or dried form comprises a prasuvorexant EV that is substantially or completely free of intact prasuvorexant bacteria (e.g., live bacteria, killed bacteria, and/or attenuated bacteria). In some embodiments, the solution and/or dried form comprises Prevotella denticola EV and Prevotella denticola whole bacteria (e.g., live bacteria, killed bacteria, and/or attenuated bacteria). In some embodiments, the solution and/or dry form comprises gamma-irradiated Prevotella EV as a tissue of interest. In some embodiments, after isolating (e.g., preparing) the EV, the prasuvorexant EV is gamma irradiated to the tissue.

In some embodiments, to quantify the number of bacteria present in a bacterial sample and/or Prevotella EV, a tissue from the bacteria, electron microscopy (e.g., ultra-thin frozen section, EM) is used to observe the EV and/or bacteria and count their relative numbers. Alternatively, nanoparticle Tracking Analysis (NTA), coulter counting or Dynamic Light Scattering (DLS) or a combination of these techniques is used. NTA and coulter counters count particles and display their size. DLS gives the particle size distribution of the particles, not the concentration. Bacteria typically have a diameter of 1-2 μm (micrometers). The complete range is 0.2-20 μm. The combined results from the coulter counts and NTA may reveal the number of bacteria and/or EVs from the bacteria in a given sample. The coulter count reveals the number of particles having a diameter of 0.7-10 μm. For most bacterial and/or EV samples, the coulter counter alone may reveal the number of bacteria and/or EVs in the sample. For NTA, nanosight instruments are available from malvern general analysis company (Malvern Pananlytical). For example, NS300 can observe and measure suspended particles in the size range of 10-2000 nm. NTA allows counting the number of particles, for example, 50-1000nm in diameter. DLS reveals the distribution of particles having different diameters in the approximate range of 1nm-3 μm.

In some embodiments, prevotella denticola EV is characterized by analytical methods known in the art, such as Jeppesen et al Cell [ Cell ]177:428 (2019).

In some embodiments, the prasuvorexant EV is quantified based on particle count. For example, the particle count of EV formulations can be measured using NTA. For example, particle counts of EV formulations can be measured using NTA and Zetaview.

In some embodiments, the prasuvorexant EV is quantified based on the amount of protein, lipid, or carbohydrate. For example, in some embodiments, the total protein content of the EV formulation is measured using a braytod assay or BCA.

In some embodiments, the tissue Prevotella EV is separated from one or more other bacterial components of the source bacterium. In some embodiments, the solution and/or dry form further comprises other bacterial components.

In certain embodiments, a prasuvorexant EV liquid formulation of the tissue obtained from the source bacteria may be fractionated into subpopulations based on the physical characteristics of the subpopulations (e.g., size, density, protein content, and/or binding affinity). One or more EV sub-populations (e.g., as a liquid formulation) can then be incorporated into the solutions, powders and/or lyophilisates of the present invention.

In certain aspects, provided herein are solutions and/or dry forms (and therapeutic compositions thereof) comprising a bacterial-derived, tissue-dwelling prasuvorexant EV that are useful for treating and/or preventing diseases (e.g., immune disorders (e.g., autoimmune diseases, inflammatory diseases, allergies), dysbacterioses, or metabolic diseases), as well as methods of making and/or identifying such EVs, and methods of using such solutions and/or dry forms (and therapeutic compositions thereof) (e.g., alone or in combination with other therapeutic agents for treating immune disorders (e.g., autoimmune diseases, inflammatory diseases, allergies), dysbacterioses, or metabolic diseases). In some embodiments, the therapeutic composition comprises Prevotella denticola EV and Prevotella denticola whole bacteria (e.g., live bacteria, killed bacteria, and/or attenuated bacteria). In some embodiments, the solution and/or dry form comprises an EV of bacteria from one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) taxonomic groups (e.g., class, order, family, genus, species, or strain). In some embodiments, the solution and/or dry form comprises an EV from one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) bacterial strains or species of bacteria. In some embodiments, the therapeutic composition comprises a prasuvorexant EV that is a percha tissue that is free of bacteria (e.g., at least about 85%, at least about 90%, at least about 95%, or at least about 99% free of bacteria). In some embodiments, the therapeutic composition comprises an EV from one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) bacteria of the taxonomic group (e.g., class, order, family, genus, species, or strain). In some embodiments, the therapeutic composition comprises an EV from bacteria of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) bacterial strains or species.

In some embodiments, the solution and/or dry form is added or incorporated into a food (e.g., a food or beverage), such as a healthy food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, elderly people, or other specific populations, a functional food, beverage, a food or beverage for specified health applications, a dietary supplement, a probiotic, a food or beverage for patients, or an animal feed. Specific examples of foods and beverages include various beverages such as fruit juice, soft drink, tea drink, beverage preparation, jelly drink, and functional drink; alcoholic beverages, such as beer; carbohydrate-containing foods such as rice foods, noodles, bread and dough; paste products, such as fish ham, sausage, seafood paste products; retort pouch products such as curry, thick starch sauce coated food, soup; dairy products such as emulsions, dairy beverages, ice cream, cheese and yogurt; fermented products such as fermented soybean paste, yogurt, fermented beverage and kimchi; a bean product; a variety of confectionery products including biscuits, cookies, and the like; rock candy, chewing gum and soft sweets; cold desserts including pectin, caramel pudding and quick frozen desserts; instant foods such as instant soup bases and instant soybean soup bases; microwaveable food; etc. In addition, examples include health foods and beverages prepared in the form of powders, granules, tablets, capsules, liquids, pastes and pectins.

In some embodiments, the solution and/or dry form is added to a food or food supplement for an animal (including a human). Animals other than humans are not particularly limited, and the composition can be used for various livestock, poultry, pets, experimental animals, and the like. Specific examples of animals include pigs, cows, horses, sheep, goats, chickens, ducks, ostriches, turkeys, dogs, cats, rabbits, hamsters, mice, rats, monkeys, etc., but these animals are not limited thereto.

Therapeutic compositions

In some embodiments, the solutions and/or dry forms provided herein are formulated into a therapeutic composition.

In certain embodiments, provided herein are therapeutic compositions comprising the solutions and/or dried forms described herein. In some embodiments, the therapeutic compositions comprise a solution and/or dry form provided herein and a pharmaceutically acceptable carrier. In some embodiments, the therapeutic composition comprises pharmaceutically acceptable excipients, such as glidants, lubricants, and/or diluents.

In certain aspects, provided herein are therapeutic compositions comprising a bacterial-derived prasuvorexant EV that are useful for treating and/or preventing diseases (e.g., immune disorders (e.g., autoimmune diseases, inflammatory diseases, allergies), dysbacterioses, or metabolic diseases), as well as methods of making and/or identifying such EVs, and methods of using such therapeutic compositions (e.g., alone or in combination with other therapeutic agents for treating immune disorders (e.g., autoimmune diseases, inflammatory diseases, allergies), dysbacterioses, or metabolic diseases). In some embodiments, the therapeutic composition comprises EV and intact bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria). In some embodiments, the therapeutic composition comprises an EV in the absence of bacteria (e.g., at least about 85%, at least about 90%, at least about 95%, or at least about 99% free of bacteria). In some embodiments, the therapeutic composition comprises EV and/or bacteria from one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) bacteria of the taxonomic group. In some embodiments, the therapeutic composition comprises EV and/or bacteria from one or more bacterial strains. In some embodiments, the therapeutic composition comprises EV and/or bacteria from one bacteria of the taxonomic group. In some embodiments, the therapeutic composition comprises EV and/or bacteria from one of the bacterial strains or species.

In certain aspects, therapeutic compositions for administration to a subject (e.g., a human subject) are provided. In some embodiments, the therapeutic composition is combined with additional active and/or inert materials to produce the final product, which may be in single dose units or multi-dose forms. In some embodiments, the therapeutic composition is combined with an adjuvant, such as an immunoadjuvant (e.g., STING agonist, TLR agonist, or NOD agonist).

In some embodiments, the therapeutic composition comprises at least one carbohydrate.

In some embodiments, the therapeutic composition comprises at least one lipid. In some embodiments, the lipid comprises at least one fatty acid selected from the group consisting of: lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), margaric acid (17:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), stearidonic acid (18:4), arachic acid (20:0), eicosenoic acid (20:1), eicosadienoic acid (20:2), eicosotenic acid (20:4), eicosapentaenoic acid (20:5) (EPA), docosylic acid (22:0), docosylic acid (22:1), docosylic acid (22:5), docosylic acid (22:6) (DHA) and tetracosylic acid (24:0).

In some embodiments, the therapeutic composition comprises at least one supplemental mineral or mineral source. Examples of minerals include, but are not limited to: 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, sparingly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals (e.g., carbonyl minerals and reduced minerals), and combinations thereof.

In some embodiments, the therapeutic composition comprises at least one supplemental vitamin. At least one vitamin may be a fat-soluble or water-soluble vitamin. Suitable vitamins include, but are not limited to, vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin (niacin), vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin. Suitable forms of any of the foregoing are vitamin salts, vitamin derivatives, compounds having the same or similar activity as vitamins, and vitamin metabolites.

In some embodiments, the therapeutic composition comprises an excipient. Non-limiting examples of suitable excipients include buffers, preservatives, stabilizers, binders, compactors, lubricants, dispersion enhancers, disintegrants, flavoring agents, sweeteners and colorants.

In some embodiments, the excipient is a buffer. Non-limiting examples of suitable buffers include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.

In some embodiments, the excipient comprises a preservative. Non-limiting examples of suitable preservatives include antioxidants (e.g., alpha-tocopherol and ascorbate) and antimicrobial agents (e.g., parabens, chlorobutanol, and phenol).

In some embodiments, the therapeutic composition comprises a binder as an excipient. Non-limiting examples of suitable binders include starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyloxazolidone, polyvinyl alcohol, C ₁₂ -C ₁₈ Fatty acid alcohols, polyethylene glycols, polyols, sugars, oligosaccharides, and combinations thereof.

In some embodiments, the therapeutic composition comprises a lubricant as an excipient. Non-limiting examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oil, refined hydrogenated vegetable oil (sterotex), polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil.

In some embodiments, the therapeutic composition comprises a dispersion enhancing agent as an excipient. Non-limiting examples of suitable dispersants include starch, alginic acid, polyvinylpyrrolidone, guar gum, kaolin, bentonite, purified lignocellulose, sodium starch glycolate, isomorphous silicate, and microcrystalline cellulose (as high HLB emulsifier surfactants).

In some embodiments, the therapeutic composition comprises a disintegrant as an excipient. In some embodiments, the disintegrant is a non-effervescent disintegrant. Non-limiting examples of suitable non-effervescent disintegrants include starches (e.g., corn starch, potato starch, pregelatinized and modified starches thereof), sweeteners, clays (e.g., bentonite), microcrystalline cellulose, alginates, sodium starch glycolate, gums (e.g., agar, guar gum, locust bean gum, karaya gum, pectin, and tragacanth gum). In some embodiments, the disintegrant is an effervescent disintegrant. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.

In some embodiments, the therapeutic composition is a food (e.g., a food or beverage), such as a healthy food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, elderly people, or other specific populations, a functional food, beverage, a food or beverage for a specified healthy application, a dietary supplement, a food or beverage for patients, or an animal feed. Specific examples of foods and beverages include various beverages such as fruit juice, soft drink, tea drink, beverage preparation, jelly drink, and functional drink; alcoholic beverages, such as beer; carbohydrate-containing foods such as rice foods, noodles, bread and dough; paste products, such as fish ham, sausage, seafood paste products; retort pouch products such as curry, thick starch sauce coated food products, and Chinese stews; soup; dairy products such as emulsions, dairy beverages, ice cream, cheese and yogurt; fermented products such as fermented soybean paste, yogurt, fermented beverage and kimchi; a bean product; a variety of confectionery products including biscuits, cookies, and the like; rock candy, chewing gum and soft sweets; cold desserts including pectin, caramel pudding and quick frozen desserts; instant foods such as instant soup bases and instant soybean soup bases; microwaveable food; etc. In addition, examples include health foods and beverages prepared in the form of powders, granules, tablets, capsules, liquids, pastes and pectins.

In some embodiments, the therapeutic composition is a food product for animals (including humans). Animals other than humans are not particularly limited, and the composition can be used for various livestock, poultry, pets, experimental animals, and the like. Specific examples of the animals include pigs, cows, horses, sheep, goats, chickens, wild ducks, ostriches, ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, etc., but the animals are not limited thereto.

Dosage form

In some embodiments, the therapeutic composition comprising the dry form is formulated as a solid dosage form (also referred to as a "solid dosage form"), for example for oral administration. In some embodiments, the solid dosage form comprises one or more excipients, such as pharmaceutically acceptable excipients, in addition to the dry form. The dry form in the solid dosage form contains isolated Prevotella EV, a tissue of interest. Optionally, gamma irradiation is performed on Prevotella EV, a perchloric tissue in the solid dosage form. In some embodiments, the solid dosage form comprises a tablet, a minitablet, a capsule, or a powder; or a combination of these forms (e.g., miniature tablets contained in a capsule).

The solid dosage forms described herein may be, for example, capsules. The solid dosage forms described herein may be, for example, tablets or minitablets. Further, a plurality of miniature tablets may be in (e.g., enclosed in) a capsule.

In some embodiments, the solid dosage form comprises a capsule. In some embodiments, the capsule is a number 00, number 0, number 1, number 2, number 3, number 4, or number 5 capsule. In some embodiments, the capsule is capsule No. 0. As used herein, the size of a capsule refers to the size of the tablet prior to application of the enteric coating. In some embodiments, the capsule is banded after loading (and prior to enteric coating the capsule). In some embodiments, the capsule is banded with an HPMC-based edging solution.

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

In some embodiments, the solid dosage form comprises a minitablet. The size of the miniature tablets ranges from 1mm to 4mm. For example, the minitablets may be 1mm minitablets, 1.5mm minitablets, 2mm minitablets, 3mm minitablets or 4mm minitablets. As known in the art, size refers to the diameter of the miniature tablet. As used herein, the size of the miniature tablet refers to the size of the miniature tablet prior to application of the enteric coating.

The miniature tablets may be in capsules. The capsule may be a number 00, number 0, number 1, number 2, number 3, number 4 or number 5 capsule. The capsule containing the minitablets may comprise hydroxypropyl methylcellulose (HPMC) or gelatin. Miniature tablets may be placed within the capsule: the number of miniature tablets within the capsule will depend on the size of the capsule and the size of the miniature tablets. For example, capsule number 0 may contain 31-35 (average 33) 3mm miniature tablets. In some embodiments, the capsule is trimmed after loading. In some embodiments, the capsule is banded with an HPMC-based edging solution.

Therapeutic compositions comprising solutions and/or powders (e.g., comprising EV and filler) may be formulated as suspensions (e.g., powders may be reconstituted; solutions may be diluted), for example, for oral administration or for injection. Injection administration includes Intravenous (IV), intramuscular (IM) and Subcutaneous (SC) administration. For suspensions, the EV may be in a buffer, for example a pharmaceutically acceptable buffer, for example physiological saline or PBS. The suspension may comprise one or more excipients, for example pharmaceutically acceptable excipients. The suspension may comprise, for example, sucrose or glucose. The EV in the solution or powder (e.g., comprising EV and filler) may be an isolated EV. Optionally, the EV in suspension may be gamma irradiated.

Coating layer

The solid dosage forms (e.g., capsules, tablets, or minitablets) described herein may be enteric coated, for example, with one enteric coating or two enteric coatings (e.g., an inner enteric coating and an outer enteric coating). The inner and outer enteric coatings are not the same (e.g., the inner and outer enteric coatings do not contain the same components in the same amounts). The enteric coating allows for release of the therapeutic agent (e.g., prevotella denticola EV, its dried form, and/or solid dosage form), e.g., in the small intestine.

The release of the therapeutic agent in the small intestine allows the therapeutic agent to target and affect cells (e.g., epithelial cells and/or immune cells) located at these specific locations, which may, for example, cause local effects in the gastrointestinal tract and/or cause systemic effects (e.g., parenteral effects).

EUDRAGIT is a brand name for a wide variety of polymethacrylate-based copolymers. It includes anionic, cationic and neutral copolymers based on methacrylic acid and methacrylic acid/acrylic esters or derivatives thereof.

Can be used for enteric coating (e.g., examples of other materials for the enteric coating or the inner enteric coating and/or the outer enteric coating include Cellulose Acetate Phthalate (CAP), cellulose Acetate Trimellitate (CAT), polyvinyl acetate phthalate (PVAP), hydroxypropyl methylcellulose phthalate (HPMCP), fatty acids, waxes, shellac (esters of eleostearic acid), plastics, vegetable fibers, zein, polyethylene terephthalate (HPMCP), (alcohol-free aqueous zein formulation), amylose, starch derivatives, dextrin, methyl acrylate-methacrylic acid copolymer, cellulose acetate succinate, hydroxypropyl methyl cellulose acetate succinate (hydroxypropyl methylcellulose acetate succinate), methyl methacrylate-methacrylic acid copolymer, and/or sodium alginate.

The enteric coating (e.g., a layer of enteric coating or an inner enteric coating and/or an outer enteric coating) may include an ethyl Methacrylate (MAE) copolymer (1:1).

One enteric coating may comprise an ethyl Methacrylate (MAE) copolymer (1:1) (e.g., kollicoat MAE 100P).

An enteric coating may include an Eudragit copolymer, such as Eudragit L (e.g., eudragit L100-55; eudragit L30D-55), eudragit S, eudragit RL, eudragit RS, eudragit E, or Eudragit FS (e.g., eudragit FS 30D).

Other examples of materials that may be used in the enteric coating (e.g., a layer of enteric coating or an inner enteric coating and/or an outer enteric coating) include those described below, e.g., U.S.6312728; U.S.6623759; U.S.4775536; U.S.5047258; U.S. 529522; 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.

See also, e.g., U.S. 923074, which provides pH-dependent enteric polymers that can be used with the solid dosage forms provided herein, including methacrylic acid copolymers, poly (vinyl acetate phthalate), hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose phthalate, and cellulose acetate phthalate; suitable methacrylic acid copolymers include: poly (methacrylic acid, methyl methacrylate) 1:1 solids, such as sold under the trade name eudragit L100; poly (methacrylic acid, ethyl acrylate) 1:1 solids, such as sold under the trade name Uttky L100-55; partially neutralized poly (methacrylic acid, ethyl acrylate) 1:1 solids, such as sold under the trade name Kollicoat MAE-100P; and poly (methacrylic acid, methyl methacrylate) 1:2 solids, such as sold under the trade name Uttky S100.

Solid dosage forms, such as capsules, may comprise a single coating, such as a non-enteric coating, e.g., HPMC (hydroxypropyl methylcellulose) or gelatin.

Method for producing solutions and dry forms

The present disclosure also provides a method of preparing a solution of Prevotella denticola EV and an excipient (which comprises a filler). For example, in some embodiments, the bulking agent comprises mannitol, sucrose, polyethylene glycol (PEG, e.g., PEG 6000), cyclodextrin, maltodextrin, dextran, ficoll, or PVP-K30. In some embodiments, the excipient comprises a lyoprotectant. In some embodiments, the excipient optionally includes additional components, such as trehalose, mannitol, sucrose, sorbitol, dextran, poloxamer 188, maltodextrin, PVP-K30, ficoll, citrate, arginine, and/or hydroxypropyl-B-cyclodextrin. For example, in some embodiments, a liquid formulation of Prevotella denticola EV and an excipient comprising a filler are combined to prepare a solution. For example, in some embodiments, a liquid formulation of prasuvorexant EV (e.g., obtained by separating the EV from a bacterial culture (e.g., supernatant or retentate)) and an excipient comprising a bulking agent (e.g., excipient stock of the formulation provided in one of tables A, B, C, D, K or P) are combined to prepare a solution. For example, in some embodiments, a liquid formulation containing a prasuvorexant EV of the tissue (e.g., obtained by separating the EV from a bacterial culture (e.g., supernatant or retentate)) is combined with an excipient comprising a bulking agent, e.g., a liquid formulation containing a prasuvorexant EV of the tissue (e.g., obtained by separating the EV from a bacterial culture (e.g., supernatant or retentate) is combined with an excipient comprising a bulking agent (e.g., mannitol) or an excipient stock of the formulation provided in one of tables A, B, C, D, K or P, to prepare a solution.

The present disclosure also provides methods of preparing a dried form of Prevotella denticola EV. For example, in some embodiments, the method is used to prepare a lyophilizate such as a lyophilized powder and/or a lyophilized cake. For example, in some embodiments, the method is used to prepare a powder such as a lyophilized powder and/or a spray dried powder. In some embodiments, the excipient comprises a filler. For example, in some embodiments, the bulking agent comprises mannitol, sucrose, polyethylene glycol (PEG, e.g., PEG 6000), cyclodextrin, maltodextrin, dextran, ficoll, or PVP-K30. In some embodiments, the excipient comprises a lyoprotectant. In some embodiments, the excipient is optionalIncluding additional components such as trehalose, mannitol, sucrose, sorbitol, dextran, poloxamer 188, maltodextrin, PVP-K30, ficoll, citrate, arginine and/or hydroxypropyl-B-cyclodextrin. For example, in some embodiments, a liquid formulation containing a prasuvorexant EV of the tissue (e.g., obtained by separating the EV from a bacterial culture (e.g., supernatant or retentate)) is combined with an excipient comprising a bulking agent (e.g., mannitol) or an excipient stock of the formulation provided in one of tables A, B, C, D, K or P; and dried (e.g., by lyophilization or spray drying) to produce a dry form. In some embodiments, the dry form has a moisture content of less than about 6%, less than about 5%, less than about 4%, between about 0.5% and about 5%, between about 1% and about 4%, between about 1.5% and about 4%, between about 2% and about 4%, or between about 2% and about 3% (e.g., as determined by karl fischer titration). In some embodiments, the dry form has about 10% to about 80% (by weight) of an excipient, such as an excipient comprising a filler. In some embodiments, the dry form has about 10% to about 80% (by weight) of excipients, for example, excipients from stock solutions of the formulations provided in one of tables A, B, C, D, K or P. In some embodiments, the prasuvorexant EV tissue comprises from about 1% to about 99% of the total solids by weight of the dry form. In some embodiments, the dry form has at least about 1e10 particles of prasugrel tissue per mg of dry form (e.g., as determined by particles/mg, e.g., by NTA). In some embodiments, the particles in dry form have a hydrodynamic diameter (zeaverage, Z) of about 130nm to about 300nm after being resuspended from dry form (e.g., resuspended in deionized water) _ave ) (e.g., as determined by dynamic light scattering).

In some embodiments, the dry form is a lyophilisate. In some embodiments, the lyophilisate is a lyophilized powder or a lyophilized cake. In some embodiments, the dry form is a powder. In some embodiments, the powder is a lyophilized powder or a spray dried powder.

In some embodiments, a method of preparing a solution comprising Extracellular Vesicles (EVs) from Prevotella denticola bacteria comprises:

a liquid formulation comprising EV from prasugrel bacteria of the tissue is combined with an excipient comprising a bulking agent, thereby preparing a solution.

a liquid formulation comprising EV from prasugrel bacteria of the tissue is combined with an excipient comprising a bulking agent and a lyoprotectant to prepare a solution.

a liquid formulation comprising EV from prasugrel bacteria of the tissue is combined with an excipient comprising a lyoprotectant, thereby preparing a solution.

The solution is dried, thereby preparing the dried form.

Drying the solution to prepare a cake, and

the cake is milled (e.g., ground) to produce the dry form.

The solution is dried, thereby preparing the dried form.

drying the solution to prepare a cake, and

the cake is milled (e.g., ground) to produce the dry form.

The solution is dried, thereby preparing the dried form.

drying the solution to prepare a cake, and

the cake is milled (e.g., ground) to produce the dry form.

In some embodiments, drying comprises lyophilization.

In some embodiments, drying comprises spray drying.

In some embodiments, the method further comprises combining the dried form with additional ingredients. In some embodiments, the additional ingredients comprise excipients, such as glidants, lubricants, and/or diluents.

The solution was dried to prepare the powder.

drying the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the powder.

The solution was dried to prepare the powder.

Drying the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the powder.

The solution was dried to prepare the powder.

drying the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the powder.

In some embodiments, drying comprises lyophilization.

In some embodiments, drying comprises spray drying.

In some embodiments, the method further comprises combining the powder with additional ingredients. In some embodiments, the additional ingredients comprise excipients, such as glidants, lubricants, and/or diluents.

Spray drying the solution to produce the spray dried powder.

In some embodiments, the method further comprises combining the spray-dried powder with additional ingredients. In some embodiments, the additional ingredients comprise excipients, such as glidants, lubricants, and/or diluents.

freeze drying (lyophilization) the solution to prepare a cake, and

The cake is milled (e.g., ground) to prepare the lyophilizate.

freeze drying (lyophilization) the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the lyophilizate.

freeze drying (lyophilization) the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the lyophilizate.

In some embodiments, the method further comprises combining the lyophilizate with additional ingredients. In some embodiments, the additional ingredients comprise excipients, such as glidants, lubricants, and/or diluents.

freeze drying (lyophilization) the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the lyophilized powder.

Freeze drying (lyophilization) the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the lyophilized powder.

freeze drying (lyophilization) the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the lyophilized powder.

In some embodiments, the method further comprises combining the lyophilized powder with additional ingredients. In some embodiments, the additional ingredients comprise excipients, such as glidants, lubricants, and/or diluents.

The solution is dried, thereby preparing the dried form.

drying the solution to prepare a cake, and

the cake is milled (e.g., ground) to produce the dry form.

In some embodiments, drying comprises lyophilization.

In some embodiments, drying comprises spray drying.

The solution was dried to prepare the powder.

drying the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the powder.

In some embodiments, drying comprises lyophilization.

In some embodiments, drying comprises spray drying.

Spray drying the solution to produce the spray dried powder.

Freeze drying (lyophilization) the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the lyophilizate.

Freeze drying (lyophilization) the solution to prepare a cake, and

the cake is milled (e.g., ground) to prepare the lyophilized powder.

Method for preparing therapeutic compositions

The present disclosure also provides methods of preparing therapeutic compositions. In some embodiments, the method comprises combining a solution or dry form as described herein with a pharmaceutically acceptable excipient (e.g., glidant, lubricant, and/or diluent) to produce a therapeutic composition.

The present disclosure also provides methods of preparing therapeutic compositions (e.g., solid dosage forms) comprising the dry forms described herein. In some embodiments, the solid dosage form is a capsule, tablet, or minitablet.

The present disclosure also provides methods of manufacturing solid dosage forms comprising a dry form (e.g., for oral administration) (e.g., for pharmaceutical use). In some embodiments, the dry form comprises Prevotella denticola Extracellular Vesicles (EV) and an excipient comprising a filler. In some embodiments, the dry form comprises Prevotella denticola Extracellular Vesicles (EV) and an excipient comprising a lyoprotectant. In some embodiments, the dry form comprises Prevotella denticola Extracellular Vesicles (EV) and an excipient comprising a bulking agent and a lyoprotectant. In some embodiments, the dry form further comprises one or more additional components. In some embodiments, the dry form is combined with one or more pharmaceutically acceptable excipients. In some embodiments, the solid dosage form is enteric coated, e.g., with a coating as described herein.

In some aspects, a method of manufacturing a solid dosage form comprises:

encapsulating a dry form comprising Prevotella denticola Extracellular Vesicles (EV) to produce a capsule, thereby producing a solid dosage form;

Optionally combining the dried form with a pharmaceutically acceptable excipient prior to encapsulation; and/or

Optionally edging the capsule after filling (e.g., optionally edging the capsule after filling).

In some aspects, a method of manufacturing a solid dosage form comprises:

compressing a dry form comprising the Prevotella denticola Extracellular Vesicles (EV) described herein into a minitablet, thereby producing a solid dosage form;

optionally combining the dried form with a pharmaceutically acceptable excipient prior to compression;

the capsule is optionally filled with a plurality of enteric coated mini-tablets.

In some aspects, a method of manufacturing a solid dosage form comprises:

compressing a dry form comprising the Prevotella denticola Extracellular Vesicles (EV) described herein into a tablet, thereby preparing a solid dosage form;

the dry form is optionally combined with a pharmaceutically acceptable excipient prior to compression.

In certain embodiments, the method comprises wet granulating the powder prior to combining the powder with one or more (e.g., one, two, or three) excipients into a therapeutic composition, such as a solid dosage form. In some embodiments, wet granulation includes (i) mixing the powder with a granulation fluid (e.g., water, ethanol, or isopropanol, alone or in combination). In some embodiments, wet granulation includes mixing the powder with water. In some embodiments, wet granulation comprises (ii) drying the mixed powder and granulation fluid (e.g., drying on a fluid bed dryer). In some embodiments, wet granulation comprises (iii) milling (e.g., grinding) the dried powder and granulating fluid. The milled (e.g., ground) powder and granulation fluid are then combined with one or more (e.g., one, two, or three) excipients to prepare a therapeutic composition, such as a solid dosage form. In some embodiments, the powder is a lyophilized powder. In some embodiments, the powder is a spray-dried powder.

In some embodiments, the dry forms described herein are reconstituted in a liquid (e.g., buffer, juice, or water) to prepare a therapeutic composition.

In some embodiments, the solution is resuspended (e.g., diluted) in a liquid (e.g., buffer, juice, or water) to prepare the therapeutic composition.

In some embodiments, a therapeutic composition comprising a dry form as described herein is reconstituted in a liquid (e.g., buffer, juice, or water) to prepare a suspension.

In some embodiments, the therapeutic composition comprising the solution is resuspended (e.g., diluted) in a liquid (e.g., buffer, juice, or water) to prepare a suspension.

Gamma irradiation

The powder (e.g., powder from EV of prasuvorexa bacteria of the tissue genus) may be gamma irradiated at ambient temperature in 17.5kGy irradiation units.

The frozen biomass (e.g., frozen biomass from EV of prasuvorexant bacteria, a tissue-dwelling) may be gamma irradiated in the presence of dry ice in 25kGy irradiation units.

Additional therapeutic agents

In certain aspects, methods provided herein comprise administering to a subject a therapeutic composition described herein, alone or in combination with an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an immunosuppressant, an anti-inflammatory agent, and/or a steroid.

In some embodiments, the therapeutic composition comprising an EV from prasugrel bacteria at the tissue is administered to the subject prior to administration of the additional therapeutic agent (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days prior to or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours prior to administration of the additional therapeutic agent. In some embodiments, the therapeutic composition comprising an EV from prasugrel bacteria is administered to the subject after at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or after at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after administration of the additional therapeutic agent. In some embodiments, the therapeutic composition comprising EV from prasugrel bacteria of the tissue and the additional therapeutic agent are administered to the subject simultaneously or nearly simultaneously (e.g., administration occurs within one hour of each other).

In some embodiments, the antibiotic is administered to the subject prior to (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours prior to or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days prior to) administering to the subject a therapeutic composition comprising an EV from a prasuvorexa bacteria. In some embodiments, the antibiotic is administered to the subject after (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after) administering to the subject a therapeutic composition comprising an EV from a prasuvorexa bacteria. In some embodiments, the therapeutic composition comprising EV from prasugrel bacteria, a tissue of interest, and the antibiotic are administered to the subject simultaneously or nearly simultaneously (e.g., administration occurs within one hour of each other).

In some embodiments, the methods provided herein comprise administering a therapeutic composition described herein in combination with one or more additional therapeutic agents. In some embodiments, the methods disclosed herein comprise administering two therapeutic agents.

In some embodiments, the therapeutic agent is an antibiotic. "antibiotic" refers broadly to a compound capable of inhibiting or preventing a bacterial infection. Antibiotics can be classified in a number of ways, including according to their use for a particular infection, their mechanism of action, their bioavailability, or their target microorganism range (e.g., gram negative bacteria versus gram positive bacteria, aerobic bacteria versus anaerobic bacteria, etc.), and can be used to kill particular bacteria in a particular area of the host ("niche") (Leekha et al, 2011.General Principles of Antimicrobial Therapy [ general principles of antimicrobial therapy ]. Mayo Clin Proc. [ journal of plum European Hospital ]86 (2): 156-167). In certain embodiments, antibiotics may be used to selectively target bacteria of a particular niche. In some embodiments, the antibiotic is administered after a therapeutic composition comprising an EV from a prasuvorexa bacteria of the tissue. In some embodiments, the antibiotic is administered prior to the therapeutic composition comprising an EV from a prasuvorexant bacterium of the tissue.

In some aspects, the antibiotic may be selected based on bactericidal or bacteriostatic properties. The bactericidal antibiotics comprise mechanisms of action that disrupt cell walls (e.g., beta-lactams), cell membranes (e.g., daptomycin) or bacterial DNA (e.g., fluoroquinolones). Bacterial inhibitors inhibit bacterial replication and contain sulfonamides, tetracyclines (tetracyclic lactones) and act by inhibiting protein synthesis. In addition, while some drugs may be bactericidal in certain organisms and bacteriostatic in other organisms, the knowledge of the target organism allows one skilled in the art to select antibiotics with the appropriate properties. In certain therapeutic conditions, the bacterial inhibition antibiotic inhibits the activity of a bactericidal antibiotic. Thus, in certain embodiments, the bactericidal antibiotic and the bacterial inhibiting antibiotic are not combined.

Antibiotics include, but are not limited to, aminoglycosides, ansamycins, carbacephems, carbapenems, cephalosporins, glycopeptides, lincosamide, lipopeptides, macrolides, monoamides, nitrofurans, oxazolidinones, penicillins, polypeptide antibiotics, quinolones, fluoroquinolones, sulfonamides, tetracyclines, and antimycobacterial compounds, and combinations thereof.

Aminoglycosides include, but are not limited to, amikacin (Amikacin), gentamicin (Gentamicin), kanamycin (Kanamycin), neomycin (Neomycin), netilmicin (Netilmicin), tobramycin (Tobramycin), paromomycin (Paromomycin), and Spectinomycin (Spctinomycin). Aminoglycosides are effective against, for example, gram-negative bacteria such as e.coli, klebsiella, pseudomonas aeruginosa (Pseudomonas aeruginosa) and franciscensis (Francisella tularensis) and against certain aerobic bacteria, but are less effective against obligate/facultative anaerobes. It is believed that aminoglycosides bind to bacterial 30S or 50S ribosomal subunits, thereby inhibiting bacterial protein synthesis.

Ansamycins include, but are not limited to, geldanamycin (Geldanamycin), herbimycin (Herbimycin), rifamycin (Rifamycin), and strepavidin (strepavidin). Geldanamycin and herbimycin are believed to inhibit or alter the function of heat shock protein 90.

Carbacephem includes, but is not limited to, chlorocarba-cephem (Loracarbef). It is believed that carbacephem inhibits bacterial cell wall synthesis.

Carbapenems include, but are not limited to, ertapenem (Ertapenem), doripenem (doripeem), imipenem (Imipenem)/Cilastatin (Cilastatin), and Meropenem (Meropenem). Carbapenems are bactericidal against both gram-positive and gram-negative bacteria as broad spectrum antibiotics. Carbapenems are believed to inhibit bacterial cell wall synthesis.

Cephalosporins include, but are not limited to, cefadroxil (Cefadroxil), cefazolin (Cefazolin), cefalotin (cefaloxin), cefaloxin (cefaloxin), cefaclor (Cefaclor), cefamandole (Cefamandole), cefoxitin (cefoxil), cefprozil (Cefprozil), cefuroxime (Cefuroxime), cefixime (Cefixime), cefdinir (Cefdinir), cefditoren (Cefditoren), cefpirome (Cefprozil), cefprozil (Cefprozil), ceftizoxime (Cefprozil), cefpodoxime (Cefpodoxime), cefpodoxime (ceffpodime), ceftazil (Ceftizoxime), ceftriaxone (Ceftriaxone), ceftriaxone (ceftixel), ceftizoxime (Ceftriaxone) and Ceftriaxone (Ceftriaxone) 62. The cephalosporins selected are effective against, for example, gram-negative bacteria and gram-positive bacteria including Pseudomonas, and some cephalosporins are effective against methicillin-resistant Staphylococcus aureus (Staphylococcus aureus) (MRSA). Cephalosporins are believed to inhibit bacterial cell wall synthesis by disrupting the synthesis of peptidoglycan layers of the bacterial cell wall.

Glycopeptides include, but are not limited to, teicoplanin (Teicoplanin), vancomycin (Vancomycin), and Telavancin (Telavancin). Glycopeptides are effective against, for example, aerobic and anaerobic gram positive bacteria including MRSA and clostridium difficile. It is believed that glycopeptides inhibit bacterial cell wall synthesis by disrupting the synthesis of peptidoglycan layers of the bacterial cell wall.

Lincomides include, but are not limited to, clindamycin (Clindamycin) and Lincomycin (Lincomycin). Lincomamides are effective against, for example, anaerobic bacteria, staphylococci (Staphylococcus) and streptococci (Streptococcus). It is believed that lincoamide binds to bacterial 50S ribosomal subunits, thereby inhibiting bacterial protein synthesis.

Lipopeptides include, but are not limited to, daptomycin. Lipopeptides are effective against, for example, gram-positive bacteria. Lipopeptides are believed to bind to bacterial membranes and cause rapid depolarization.

The macrolides include, but are not limited to, azithromycin (Azithromycin), clarithromycin (Clarithromycin), dirithromycin (Dirithromycin), erythromycin (Erythromycin), roxithromycin (Roxithromycin), acorn (Trolley mycin), telithromycin (Telithromycin) and Spiramycin (Spiramycin). The macrocyclic lactones are effective against, for example, streptococcus and Mycoplasma (Mycoplasma). It is believed that the macrolide binds to bacteria or 50S ribosomal subunits, thereby inhibiting bacterial protein synthesis.

Monoamide antibiotics include, but are not limited to, aztreonam (Aztreonam). Monoamide antibiotics are effective against, for example, gram-negative bacteria. It is believed that monoamide mycotoxins inhibit bacterial cell wall synthesis by disrupting the synthesis of peptidoglycan layers of bacterial cell walls.

Nitrofurans include, but are not limited to, furazolidone (Furazolidone) and Nitrofurantoin (Nitrofurantoin).

Oxazolidinones include, but are not limited to, linezolid (Linezolid), prednisozolid (Posizolid), radzolid (radzolid), and tedizolid (Torezolid). Oxazolidinones are believed to be inhibitors of protein synthesis.

Penicillin includes, but is not limited to, amoxicillin (Amoxicillin), ampicillin (Ampicillin), azlocillin (Azlocillin), carbenicillin (Carbenicillin), chlorthiacillin (cloxacins), dichlorthiacillin (dichlorxacins), flucloxacillin (flucloxacins), mezlocillin (Mezlocillin), methicillin, nafcillin (nafcilin), oxacillin (oxacilin), penicillin G, penicillin V, piperacillin (piperacilin), temoxicillin (Temocillin) and Ticarcillin (Ticarcillin). Penicillin is effective against, for example, gram-positive bacteria, facultative anaerobes (e.g., streptococcus, borrelia (Borrelia), treponema). Penicillin is believed to inhibit bacterial cell wall synthesis by disrupting the synthesis of peptidoglycan layers of the bacterial cell wall.

Penicillin combinations include, but are not limited to, amoxicillin/clavulanate (clavulanate), ampicillin/sulbactam (sulbactam), piperacillin/tazobactam (tazobactam), and ticarcillin/clavulanate.

Polypeptide antibiotics include, but are not limited to Bacitracin (Bacitracin), colistin (Colistin), and polymyxins (Polymyxin) B and E. Polypeptide antibiotics are effective against, for example, gram-negative bacteria. It is believed that certain polypeptide antibiotics inhibit the synthesis of prenyl pyrophosphate involving the peptidoglycan layer of the bacterial cell wall, while others destabilize the bacterial outer membrane by displacing bacterial counter ions.

Quinolones and fluoroquinolones include, but are not limited to, ciprofloxacin (Ciprofloxacin), enoxacin (Enoxacin), gatifloxacin (Gatifloxacin), gemifloxacin (Gemifloxacin), levofloxacin (Levofloxacin), lomefloxacin (Lomefloxacin), moxifloxacin (Moxifloxacin), nalidixic acid (Nalidixic acid), norfloxacin (Norfloxacin), ofloxacin (Ofloxacin), trovafloxacin (Trovafloxacin), glafloxacin (greepafloxacin), sparfloxacin (Sparfloxacin), and Temafloxacin (Temafloxacin). Quinolones/fluoroquinolones are effective against, for example, streptococcus and Neisseria (Neisseria). It is believed that the quinolone/fluoroquinolone inhibits bacterial DNA gyrase or topoisomerase IV, thereby inhibiting DNA replication and transcription.

Sulfonamides include, but are not limited to, sulfamilone (Mafenide), sulfacetamide, sulfadiazine (Sulfadiazine), silver Sulfadiazine, sulfadimine (Sulfadimethoxine), sulfamethiodizole (Sulfamethizole), sulfamethimazole (Sulfamethoxazole), sulfaimido (sulfanilamide), sulfasalazine (Sulfasalazine), sulfaisozole (sulfafisoxazole), trimethoprim-Sulfamethoxazole (Trimethoprim-Sulfamethoxazole), and sulfonamide Ke Yiting (sulfanamichloride). It is believed that sulfonamide inhibits folate synthesis by competitively inhibiting dihydropteroate synthase, thereby inhibiting nucleic acid synthesis.

Tetracyclines include, but are not limited to, demeclocycline (Doxycycline), doxycycline (Doxycycline), minocycline (Minocycline), oxytetracycline (Oxytetracycline), and tetracycline. Tetracyclines are effective against, for example, gram-negative bacteria. It is believed that tetracycline binds to bacterial 30S ribosomal subunits, thereby inhibiting bacterial protein synthesis.

Antimycobacterial compounds include, but are not limited to, clofazimine (Clofazimine), dapsone (Dapsone), calicheamicin (Capreomycin), cycloserine (Cycloserine), ethambutol (Ethamatol), ethionamide (Ethionamide), isoniazid isonicotinate (Isoniazid), pyrazinamide (Pyrazinamide), rifampicin (Rifampicin), rifabutin (Rifabutin), rifapentine (Rifapentine), and Streptomycin (Streptomyces).

Suitable antibiotics also include arsenicol (arshenamine), chloramphenicol (chloramphenicol), fosfomycin (fosfomycin), fusidic acid (fusidic acid), metronidazole (metamycin), mupirocin (mupirocin), plamycins (platenmycin), quinioprene (quinupristin)/dalfopritin (dazoprin), tigecycline (tigecycline), tinidazole (tinidazole), trimethoprim-amoxicillin (trimethoprim amoxicillin)/clavulanate, ampicillin/sulbactam, ambroxycycline-rithromycin (amphomycin ristocetin), azithromycin, bacitracin, bo Fulin (buformin) II, carbomycin (carbomycin), cecropin (cecropin) Pl, clarithromycin, erythromycin, furazolidone Fulvidic acid, sodium fusidate, gramicidin (gramicidin), imipenem, indolomycin (indomycin), crosamycin (josamycin), ma Gaina Ni (magainin) II, metronidazole (azozole), nitroimidazole, mi Kamei (mikamycin), mutacin (mutacin) B-Ny266, mutacin B-JHl, mutacin J-T8, nisin (nisin), nisin A, novobiocin (novobiocin), hypogamycin (oleuromycin), piraxomycin/tazobactam, pristinamycin (priminamycin), ramoplanin (ramoplanin), bullfrog skin antibacterial peptide (ranalexin), reuterin), rifaximin (rifaximin), rosamycin (rosamicin), luo Shami star (rosaracin), spectinomycin, spiramycin, glucomycin (staphy), streptocidin (streptograin), streptocidin a, synergistic mycin (synegitin), taurolidine (taurinine), teicoplanin, telithromycin, ticarcillin/clavulanic acid (clavulanic acid), triacetyl hypocrellin (triacylglycerol), tylosin (tyrosin), gramicidin (tyrothricin), vancomycin (velocidin), and virginiamycin (virginiamycin).

In some embodiments, the additional therapeutic agent is an immunosuppressant, DMARD, analgesic, steroid, non-steroidal anti-inflammatory drug (NSAID), or cytokine antagonist, and combinations thereof. Representative agents include, but are not limited to, cyclosporine, retinoids, corticosteroids, propionic acid derivatives, acetic acid derivatives, enolic acid derivatives, fenamic acid derivatives, cox-2 inhibitors, lumiracoxib (lumiracoxib), ibuprofen (ibuprophen), choline magnesium salicylate (cholin magnesium salicylate), fenoprofen (fenoprofen), salsalate (salsate), diflunisal (difucal), tolmetin (tolmetin), ketoprofen (ketoprofen), flurbiprofen (flurbiprofen), oxaprozin (oxaprozin), indomethacin (indomethacin), sulindac (sulindac), etodolac (etodolac), ketorolac (ketotorac), nabumetone (nabumetone), naproxen (naproxen), valdecoxib (valdecoxib), etoricoxib (etoricoxib), and MK 66; rofecoxib, acetaminophen, celecoxib, diclofenac (Dicl)ofenac), tramadol (tramadol), piroxicam (piroxicam), meloxicam (meloxicam), tenoxicam (tenoxicam), droxicam (droxicam), lornoxicam (lornoxicam), isoxicam (isoxicam), mefenamic acid (mefenamic acid), meclofenamic acid (meclofenamic acid), flufenamic acid (flufenamic acid), tolfenamic acid (tolfenamic), valdecoxib (valdecoxib), parecoxib (parecoxib), etodolac (etodolac), indomethacin (indomethacin), aspirin (aspirin), ibuprofen, non-Luo Kaoxi (firocoxicab), methotrexate (methotrexate (MTX)), antimalarial drugs (e.g., hydroxychloroquine (chloroquine), sulfasalazine, leflunomide (Leflunomide), azathioprine (azathioprine), cyclosporin (cycloporin), gold salts (gold salt), minocycline (minocycline), cyclophosphamide (cyclophosphamide), D-penicillamine (D-pencilamine), minocycline (minocycline), aurofine (aurofin), tacrolimus (tacrolimus), gold sodium thiobenzoate (mycrilin), chlorambucil (chloramamide), tnfα antagonists (e.g., tnfα antagonists or tnfα receptor antagonists), e.g., adalimumab Etanercept->Infliximab (++>TA-650), polyethylene glycol cetuximab (>CDP 870), golimumab (>CNTO 148), anakinraRituximab->Arbazedox->Tozumazumab (Roactmura/->) Integrin antagonists (+)>(natalizumab)), IL-1 antagonist (ACZ 885 (Ilaris)), anakinra ++>) CD4 antagonists, IL-23 antagonists, IL-20 antagonists, IL-6 antagonists, BLyS antagonists (e.g., asenapine, & lt + & gt> (belimumab)), p38 inhibitor, CD20 antagonist (Ocreelizumab), ofamumab ∈>) An interferon gamma antagonist (rituximab), prednisolone (prednisolone), prednisone (Prednisone), dexamethasone (dexamethazone), cortisol (cortisone), cortisone (cortisone), hydrocortisone (hydrocortisone), methylprednisolone (methylprednisolone), betamethasone, triamcinolone, beclomethasone (beclomethasone), triamcinolone (beclomethasone) fludrocortisone (fludrocortisone), deoxycorticosterone (deoxyoxycodone), aldosterone (aldosterone), doxycycline (Doxycycline), vancomycin (vancomycin), pioglitazone (pioglitazone), SBI-087, SCIO-469, cura-100, oncoxin+Viusid, twHF, methoxsalen (Methoxsalen), vitamin D-ergocalciferol (Vitamin D-ergalciferol), milnaciprol Lorently (Milnacipran), paclitaxel (Paclitaxel), rosiglitazone (rosiglitazone), tacrolimus (Tacrolimus) are added to the composition>Radaol, lapachone, rapamycin, fosamitinib, fentanyl, XOMA 052, fosamitinib disodium (Fostamatinib disodium), rosiglitazone, curcumin (Curcumin) (Longvida ^TM ) Rosuvastatin (Rosuvastatin), maraviroc (Maraviroc), ramipril (ramipnl), milnacipran (Milnacipran), cobiprostone (Cobiprostone), growth hormone (somaropin), tgAAC94 gene therapy vector, MK0359, GW856553, esomeprazole (esomeprazole), everolimus (everolimus), trastuzumab, JAKl and JAK2 inhibitors, ubiquitin inhibitors such as tetracyclic pyridone 6 (P6), 325, PF-956980, diels-6 antagonist, CD20 antagonist, CTLA4 antagonist, IL-8 antagonist, IL-21 antagonist, IL-22 antagonist, integrin antagonist (", j-2 inhibitor, difenoki)>(natalizumab)), VGEF antagonists, CXCL antagonists, MMP antagonists, defensin antagonists, IL-1 antagonists (including IL-1 beta antagonists), and IL-23 antagonists (e.g., receptor traps, antagonistic antibodies, etc.). / >

In some embodiments, the additional therapeutic agent is an immunosuppressant. Examples of immunosuppressants include, but are not limited to, corticosteroids, melazine, mersalamine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporin a, mercaptopurine, azathioprine (azathioprine), prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine, theophylline, sodium cromoglycate, anti-leukotrienes, anticholinergic agents for rhinitis, TLR antagonists, inflammatory inhibitors, anticholinergic decongestants, mast cell stabilizers, monoclonal anti-IgE antibodies, vaccines (e.g., vaccines for vaccination in which the amount of allergen is gradually increased), cytokine inhibitors (e.g., anti-IL-6 antibodies), TNF inhibitors (e.g., infliximab, adalimumab, polyethylene glycol cetuximab, golimumab, or etanercept), and combinations thereof.

Application of

In certain aspects, provided herein are methods of delivering a therapeutic composition described herein (e.g., a therapeutic composition from EV of prasuvorexant bacteria, a tissue of interest) to a subject. In some embodiments of the methods provided herein, the therapeutic composition is administered in combination with administration of an additional therapeutic agent. In some embodiments, the therapeutic composition comprises an EV from a prasuvorexant bacterium of the tissue that is co-formulated with an additional therapeutic agent. In some embodiments, a therapeutic composition comprising EV from prasugrel bacteria of the tissue is co-administered with an additional therapeutic agent. In some embodiments, the additional therapeutic agent is administered to the subject prior to administration of the therapeutic composition comprising an EV from a prasugrel bacteria of the tissue (e.g., prior to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 minutes, prior to 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, or prior to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days). In some embodiments, the additional therapeutic agent is administered to the subject after administration of the therapeutic composition comprising an EV from a prasugrel bacteria of the tissue (e.g., after 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, or after about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days). In some embodiments, the same delivery pattern is used to deliver a therapeutic composition comprising EV from prasuvorexant bacteria of the tissue of interest and an additional therapeutic agent. In some embodiments, different modes of delivery are used to administer a therapeutic composition comprising EV from prasuvorexant bacteria of the tissue of interest and an additional therapeutic agent. For example, in some embodiments, a therapeutic composition comprising EV from prasuvorexant bacteria is administered orally, while additional therapeutic agents are administered via injection (e.g., intravenous and/or intramuscular injection).

In some embodiments, the therapeutic compositions described herein are administered once daily. In some embodiments, the therapeutic compositions described herein are administered twice daily. In some embodiments, the therapeutic compositions described herein are formulated as daily doses. In some embodiments, the therapeutic compositions described herein are formulated as twice daily doses, wherein each dose is half of a daily dose.

The dosage regimen can be any of a variety of methods and amounts, and can be determined by one of skill in the art based on known clinical factors. As is known in the medical arts, the dosage of any patient may depend on a number of factors including the subject species, size, body surface area, age, sex, immune activity and general health, the particular microorganism to be administered, the duration and route of administration, the type and stage of disease and other compounds (e.g., drugs administered simultaneously or near simultaneously). In addition to the factors described above, such levels may be affected by microbial infectivity and microbial properties, as may be determined by one of skill in the art. In the methods of the invention, the appropriate minimum dosage level of the microorganism may be a level sufficient to allow the microorganism to survive, grow and replicate. The dosage of the therapeutic composition comprising EV from prasuvorexant bacteria of the tissue of interest described herein may be appropriately set or adjusted according to the dosage form, the route of administration, the extent or stage of the target disease, etc. For example, a typical effective dosage range of the agent may be 0.01mg/kg body weight/day to 1000mg/kg body weight/day, 0.1mg/kg body weight/day to 1000mg/kg body weight/day, 0.5mg/kg body weight/day to 500mg/kg body weight/day, 1mg/kg body weight/day to 100mg/kg body weight/day, or 5mg/kg body weight/day to 50mg/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 1000mg/kg body weight/day or more, but the dose is not limited thereto.

In some embodiments, the dose administered to the subject is sufficient to prevent a disease (e.g., an immune disorder (e.g., autoimmune disease, inflammatory disease, allergy), dysbacteriosis, or metabolic disease), delay its onset, or slow or stop its progression, or alleviate one or more symptoms of the disease. Those skilled in the art will recognize that the dosage will depend on a variety of factors, including the strength of the particular agent (e.g., therapeutic agent) employed, as well as the age, species, condition, and weight of the subject. Dose size is also determined according to the following factors: the route, timing and frequency of administration, and the presence, nature and extent of any adverse side effects that may accompany the administration of a particular therapeutic agent, and the desired physiological effect.

The appropriate dose and dosage regimen may be determined by conventional range detection techniques known to those skilled in the art. Typically, treatment is initiated with a smaller dose, which is less than the optimal dose of the compound. The dose is then increased in small increments until the optimal effect under the conditions is reached. Effective dosages and treatment regimens can be determined by conventional and routine means, for example, wherein the dosages are started at a low dose and then increased in laboratory animals, while monitoring the effect, and also systematically varying the dosage regimen. Animal studies are commonly used to determine the maximum tolerated dose ("MTD") of bioactive agent per kilogram weight. Those skilled in the art typically push out doses in other species (including humans) to achieve efficacy while avoiding toxicity.

In accordance with the above, in therapeutic applications, the dosage of therapeutic agent used in the present invention will vary depending upon, inter alia, the following factors, as compared to other factors affecting the selected dosage: the active agent, age, weight, and experience and judgment of the clinician or practitioner receiving the patient's clinical condition and administering the therapy. As another example, the dosage should be sufficient to result in slowing the progression of the disease being treated by the subject, preferably ameliorating one or more symptoms of the disease being treated by the subject.

Separate administrations may include any number of two or more administrations, including two, three, four, five or six administrations. The number of administrations or the desire to administer one or more additional administrations can be readily determined by those skilled in the art based on methods known in the art for monitoring therapeutic methods and other monitoring methods provided herein. Thus, the methods provided herein include methods of providing one or more administrations of a therapeutic composition to a subject, wherein the number of administrations can be determined by monitoring the subject and, based on the results of the monitoring, determining whether one or more additional administrations need to be provided. Whether one or more additional administrations need to be provided may be determined based on various monitoring results.

The period of time between administrations may be any of various periods of time. The period of time between administrations may vary with any of a variety of factors, including the monitoring step (as described with respect to the number of administrations), the period of time during which the subject establishes an immune response. In one example, the period of time may vary with the period of time in which the subject establishes an immune response; for example, the period of time may be greater than a period of time for which the subject establishes an immune response, such as greater than about one week, greater than about ten days, greater than about two weeks, or greater than about one month; in another example, the period of time may be less than a period of time for which the subject establishes an immune response, such as less than about one week, less than about ten days, less than about two weeks, or less than about one month.

In some embodiments, delivery of the combination of the additional therapeutic agent with the therapeutic compositions described herein reduces adverse effects of the additional therapeutic agent and/or improves the efficacy of the additional therapeutic agent.

An effective dose of an additional therapeutic agent described herein is an amount of the additional therapeutic agent effective to achieve the desired therapeutic agent response and minimal toxicity to the subject for the particular subject, composition, and mode of administration. The methods described herein can be used to identify effective dosage levels and will depend on a variety of pharmacokinetic factors including the activity of the particular composition or agent being 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 composition being 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 the additional therapeutic agent will be the amount of the additional therapeutic agent, which is the lowest dose effective to produce a therapeutic effect. Typically such effective dosages will depend on these factors as described above.

Toxicity of an additional therapeutic agent is the degree of adverse effect a subject experiences during and after treatment. Adverse events associated with additional therapeutic toxicity may include, but are not limited to, abdominal pain, acid dyspepsia, acid reflux, anaphylaxis, alopecia, systemic anaphylaxis, anemia, anxiety, anorexia, joint pain, weakness, movement disorders, azotemia, imbalance, bone pain, hemorrhage, blood clots, hypotension, elevated blood pressure, dyspnea, bronchitis, congestion, decreased white blood cell count, decreased red blood cell count, decreased platelet count, cardiotoxicity, cystitis, hemorrhagic cystitis, arrhythmia, heart valve disease, cardiomyopathy, coronary artery disease, cataracts, central neurotoxicity, cognitive dysfunction, confusion, conjunctivitis, constipation, cough, cramps, cystitis, deep venous embolism, dehydration, depression, diarrhea, dizziness (dimziness), dry mouth, dry skin, dyspepsia dyspnea (dysphaea), edema, electrolyte imbalance, esophagitis, fatigue, fertility loss, fever, flatulence, flushing, gastric reflux, gastroesophageal reflux disease, genital pain, granulocytopenia, gynecomastia, glaucoma, alopecia, hand-foot syndrome (hand-foot syndrome), headache, hearing loss, heart failure, palpitations, heartburn, hematoma, hemorrhagic cystitis, hepatotoxicity, hyperamylase, hypercalcemia, hyperchlorhydria, hyperglycosemia, hyperkalemia, hyperlipidemia, hypermagnesium, hypernatremia, hyperphosphatemia, pigmentation, gao Gansan oleo-ester syndrome, hyperuricemia, hypoalbuminemia, hypocalcemia, hypochloremia, hypoglycemia, hypokalemia, hypomagnesium, hyponatremia, hypophosphorus, impotence, infection, injection site reaction, insomnia, iron deficiency, itching, joint pain, renal failure, leukopenia, liver dysfunction, memory loss, amenorrhea, aphtha, mucositis, muscle pain, myalgia, bone marrow suppression, myocarditis, neutropenia fever, nausea, nephrotoxicity, neutropenia, nose bleeding, numbness, ototoxicity, pain, hand and foot syndrome (palmar-plantar erythrodysesthesia), various types of cytopenias, pericarditis, peripheral neuropathy, pharyngitis, photophobia, pneumonia (pneumonitia), pneumonia (pneumonitis), proteinuria, pulmonary embolism, pulmonary fibrosis, pulmonary toxicity, rash, acceleration of heart beat, rectal bleeding, restlessness, rhinitis, epilepsy, shortness of breath, sinusitis, thrombocytopenia, tinnitus, urinary tract infection, vaginal bleeding, vaginal dryness, dizziness (verigo), water retention (wall retention), weakness, weight loss, increased oral dryness (body weight loss). In general, toxicity is acceptable if the subject benefit achieved via therapy outweighs the adverse event experienced by the subject as a result of the therapy.

Immune disorder

In some embodiments, the methods and therapeutic compositions described herein relate to treating or preventing a disease or disorder associated with a pathological immune response (e.g., an autoimmune disease, an allergic reaction, and/or an inflammatory disease). In some embodiments, the disease or disorder is inflammatory bowel disease (e.g., crohn's disease or ulcerative colitis). In some embodiments, the disease or disorder is psoriasis. In some embodiments, the disease or disorder is atopic dermatitis.

The methods described herein can be used to treat any subject in need thereof. As used herein, a "subject in need thereof" includes any subject having a disease or disorder associated with a pathological immune response (e.g., inflammatory bowel disease), as well as any subject having an increased likelihood of acquiring such a disease or disorder.

The therapeutic compositions described herein may be used, for example, as therapeutic (e.g., pharmaceutical) compositions for preventing or treating (partially or completely reducing the adverse effects of) autoimmune diseases, such as chronic inflammatory bowel disease, systemic lupus erythematosus, psoriasis, muesli-weber syndrome, rheumatoid arthritis, multiple sclerosis, or Hashimoto's disease; allergic diseases such as food allergy, pollinosis or asthma; infectious diseases such as clostridium difficile infection; inflammatory diseases such as TNF mediated inflammatory diseases (e.g., gastrointestinal inflammatory diseases such as pouchitis (pouchitis)), cardiovascular inflammatory diseases such as atherosclerosis, or inflammatory lung diseases such as chronic obstructive pulmonary disease; as a pharmaceutical composition for inhibiting rejection in organ transplantation or other conditions in which tissue rejection may occur; as a supplement, food or beverage for improving immune function; or as an agent for inhibiting proliferation or function of immune cells.

In some embodiments, the methods provided herein are useful for treating inflammation. In certain embodiments, inflammation of any tissue or organ of the body, including musculoskeletal inflammation, vascular inflammation, neuroinflammation, digestive system inflammation, ocular inflammation, reproductive system inflammation, and other inflammation, as discussed below. In some embodiments, the inflammation comprises Th 1-mediated inflammation. In some embodiments, the inflammation comprises Th 2-mediated inflammation (e.g., asthma or atopic dermatitis). In some embodiments, the inflammation comprises Th 17-mediated inflammation (e.g., psoriasis).

Immune disorders of the musculoskeletal system include, but are not limited to, those conditions that affect skeletal joints, including joints of the hand, wrist, elbow, shoulder, chin, spine, neck, hip, knee, ankle, and foot, and conditions that affect the tissue (e.g., tendons) that connect muscles to bones. Examples of such immune disorders that can be treated with the methods and compositions described herein include, but are not limited to, arthritis (including, for example, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acute and chronic infectious arthritis, arthritis associated with gout and pseudogout and juvenile idiopathic arthritis), tendinitis, synovitis, tenosynovitis, bursitis, fibrositis (fibromyalgia), epicondylitis, myositis, and osteositis (including, for example, paget's disease), pubic symphysis, and cystic fibrosis).

In some embodiments, the methods provided herein are suitable for treating psoriasis.

In some embodiments, the methods provided herein can be used to treat atopic dermatitis.

Ocular immune disorders refer to immune disorders affecting any structure of the eye, including the eyelid. Examples of ocular immune disorders that can be treated with the methods and compositions described herein include, but are not limited to, blepharitis, eyelid skin sagging, conjunctivitis, dacryocystitis, keratitis, keratoconjunctivitis sicca (dry eye), scleritis, trichiasis, and uveitis.

Examples of neurological immune disorders that can be treated with the methods and compositions described herein include, but are not limited to, encephalitis, guillain-Barre syndrome (Guillain-Barre syndrome), meningitis, neuromuscular rigidity, narcolepsy, multiple sclerosis, myelitis, and schizophrenia. Examples of vascular or lymphatic system inflammation that may be treated with the methods and compositions described herein include, but are not limited to, joint sclerosis, arthritis, phlebitis, vasculitis, and lymphangitis.

Examples of digestive system immune disorders that can be treated with the methods and therapeutic compositions described herein include, but are not limited to, cholangitis, cholecystitis, enteritis, enterocolitis, gastritis, gastroenteritis, inflammatory bowel disease, ileitis, and proctitis. Inflammatory bowel disease includes, for example, certain art-recognized forms of a group of related disorders. Several major forms of inflammatory bowel disease are known, with the most common of such disorders being crohn's disease (regional bowel disease, e.g., inactive and active forms) and ulcerative colitis (e.g., inactive and active forms). In addition, inflammatory bowel disease encompasses irritable bowel syndrome, microscopic colitis, lymphocytic-plasmacytic enteritis, celiac disease, collagenous colitis, lymphocytic colitis, and eosinophilic enterocolitis. Other unusual forms of IBD include non-established colitis, pseudomembranous colitis (necrotizing colitis), ischemic inflammatory bowel disease, behcet's disease, sarcoidosis, scleroderma, IBD-related dysplasia, dysplastic-related mass or lesions, and primary sclerosing cholangitis.

Examples of immune disorders of the reproductive system that may be treated with the methods and therapeutic compositions described herein include, but are not limited to, cervicitis, chorioamnion, endometritis, epididymitis, navel inflammation, oophoritis, orchitis, salpingitis, salpingo-ovarian abscess, urethritis, colpitis, vulvitis, and vulvodynia.

The methods and therapeutic compositions described herein are useful for treating autoimmune diseases having an inflammatory component. Such conditions include, but are not limited to, acute disseminated alopecia areata, behcet's disease, chagas's disease, chronic fatigue syndrome, autonomic dysfunction, encephalomyelitis, ankylosing spondylitis, aplastic anemia, suppurative sweat gland inflammation, autoimmune hepatitis, autoimmune ovaritis, celiac disease, crohn's disease, type 1 diabetes, giant cell arteritis, godpassis' Qius syndrome, graves 'disease, grin-Bali syndrome, hashimoto's disease, hun-Sjogren's purpura, kawasaki disease, lupus erythematosus, microscopic colitis microscopic polyarteritis, mixed connective tissue disease, murray-Weber syndrome, multiple sclerosis, myasthenia gravis, strabismus eye clonus syndrome, optic neuritis, orderthyroiditis, pemphigus, polyarteritis nodosa, polymyalgia, rheumatoid arthritis, lyter's syndrome, sjogren's syndrome, temporal arteritis, wegener's granulomatosis, warm autoimmune hemolytic anemia, interstitial cystitis, lyme disease, scleroderma, psoriasis, sarcoidosis, scleroderma, ulcerative colitis and vitiligo.

The methods and therapeutic compositions described herein are useful for treating T cell mediated hypersensitivity diseases having an inflammatory component. Such conditions include, but are not limited to, contact hypersensitivity, contact dermatitis (including those due to poison ivy), urticaria, skin allergies, respiratory allergies (hay fever, allergic rhinitis, house dust mite allergy), and gluten-sensitive bowel disease (celiac disease).

Other immune disorders that may be treated with the methods and therapeutic compositions include, for example, appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, hepatitis, hidradenitis suppurativa, iritis, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, pericarditis, peritonitis (perithoitis), pharyngitis, pleurisy, limiting pneumonia, prostatic hyperplasia (prostatis), pyelonephritis, and stomatitis (stomatidis), transplant rejection (involving organs such as the kidney, liver, heart, lung, pancreas (e.g., islet cells), bone marrow, cornea, small intestine, allogeneic skin grafts, skin allografts and heart valve xenografts, serum and graft versus host disease), acute pancreatitis, chronic pancreatitis, acute respiratory distress syndrome, szezary's syndrome, congenital adrenal hyperplasia, non-suppurative thyroiditis, hypercalcemia-associated cancer, pemphigus, bullous dermatitis, severe erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, seasonal or perennial allergic rhinitis, bronchial asthma, contact dermatitis, atopic dermatitis, drug hypersensitivity, allergic conjunctivitis, keratitis, ocular shingles, iritis and iridocyclitis, chorioretinitis, optic neuritis, sarcoidosis, fulminant or disseminated tuberculosis chemotherapy, adult idiopathic thrombocytopenic purpura, adult secondary thrombocytopenia, acquired (autoimmune) hemolytic anemia, adult leukemia, lymphoma, childhood acute leukemia, focal dermatitis, drug hypersensitivity, conjunctivitis, ocular inflammation, ocular herpes zoster, iritis, iridocyclitis, chorioretic thrombocytopenia, idiopathic thrombocytopenic purpura, adult idiopathic thrombocytopenia, adult hematoma, adult-type hematoma, and childhood, autoimmune vasculitis, multiple sclerosis, chronic obstructive pulmonary disease, rejection of solid organ transplants, sepsis. Preferred treatments include the following: graft rejection, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, type 1 diabetes, asthma, inflammatory bowel disease, systemic lupus erythematosus, psoriasis, chronic obstructive pulmonary disease, and inflammation associated with infectious conditions (e.g., sepsis).

Metabolic disorder

In some embodiments, the methods and therapeutic compositions described herein relate to treating or preventing metabolic diseases or disorders, such as type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, nonalcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, nonalcoholic steatohepatitis (NAFLD), nonalcoholic steatohepatitis (NASH), or related diseases. In some embodiments, the related disorder is cardiovascular disease, atherosclerosis, kidney disease, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, skin disease, dyspepsia, or edema. In some embodiments, the methods and therapeutic compositions described herein relate to the treatment of NAFLD and NASH.

The methods described herein can be used to treat any subject in need thereof. As used herein, a "subject in need thereof" includes any subject having a metabolic disease or disorder, as well as any subject having an increased likelihood of acquiring such a disease or disorder.

The therapeutic compositions described herein may be used, for example, to prevent or treat metabolic diseases (partially or completely reducing the adverse effects of metabolic diseases), such as type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, nonalcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, ketoacidosis, hypoglycemia, thrombotic diseases, dyslipidemia, NAFLD, NASH, or related diseases. In some embodiments, the related disorder is cardiovascular disease, atherosclerosis, kidney disease, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, skin disease, dyspepsia, or edema.

Other diseases and disorders

In some embodiments, the methods and therapeutic compositions described herein relate to the treatment of liver disease. Such diseases include, but are not limited to, alzheimer's Ji Erzeng syndrome (Alagille syndrome), alcohol-related liver disease, alpha-1 antitrypsin deficiency, autoimmune hepatitis, biliary closure, cirrhosis, galactosylation, gilbert's syndrome, hemochromatosis, hepatitis A, hepatitis B, hepatitis C, hepatic encephalopathy, intrahepatic Cholestasis of Pregnancy (ICP), lysosomal acid lipase deficiency (LAL-D), liver cyst, neonatal jaundice, primary cholangitis (PBC), primary Sclerosing Cholangitis (PSC), lei syndrome (Reyesyndom), type I glycogen storage disease, wilson disease.

The methods and therapeutic compositions described herein are useful for treating neurodegenerative and neurological diseases. In certain embodiments, the neurodegenerative and/or neurological disease is parkinson's disease, alzheimer's disease, prion disease, huntington's disease, motor Neuron Disease (MND), spinocerebellar ataxia, spinal muscular atrophy, dystonia, idiopathic intracranial hypertension, epilepsy, a neurological disease, a central nervous system disease, a movement disorder, multiple sclerosis, a brain disease, peripheral neuropathy, or post-operative cognitive dysfunction.

Dysbacteriosis

Intestinal microbiomes (also known as "intestinal microbiota") can have a significant effect on individual health by microbial activity on immune cells and other cells of the host as well as by effects (local and/or remote) on the host (Walker, w.a., dysbiosis [ dysbacteriosis ]. The Microbiota in Gastrointestinal Pathophysiology [ microorganisms in the pathophysiology of the gastrointestinal tract ]. Chapter 25. 2017; weiss and Thierry, mechanisms and consequences of intestinal Dysbiosis [ mechanisms and consequences of dysbacteriosis ]. Cellular and Molecular Life Sciences [ cell and molecular life sciences ] (2017) 74 (16): 2959-2977.Zurich Open Repository and Archive [ zurich open store and archive ], doi.org/10.1007/s 00018-017-2509-x)).

Healthy host intestinal microbiome homeostasis is sometimes referred to as "ecological balance" or "normal microbiome", while detrimental changes in the composition of the host microbiome and/or its diversity can lead to unhealthy imbalances in the microbiome, or "dysbacteriosis" (Hooks and O' malley. Dysbiosis and its discontents [ dysbacteriosis and dissatisfaction ]. American Society for Microbiology [ U.S. microbiology ].2017, 10 month, volume 8, 5, m bio 8: e01492-17.doi. Org/10.1128/m bio.01492-17). Dysbacteriosis and associated local or remote host inflammation or immune effects may occur when microbiome homeostasis is lost or attenuated, resulting in: increased sensitivity to pathogens; altered metabolic activity of the host bacterium; inducing pro-inflammatory activity in a host and/or reducing anti-inflammatory activity in a host. Such effects are mediated in part by interactions between host immune cells (e.g., T cells, dendritic cells, mast cells, NK cells, intestinal epithelial lymphocytes (IEC), macrophages and phagocytes) and cytokines, as well as other substances released by such cells and other host cells.

Dysbacteriosis may occur within the gastrointestinal tract ("dysbacteriosis of the gastrointestinal tract" or "dysbacteriosis of the intestinal tract"), or may occur outside the lumen of the gastrointestinal tract ("dysbacteriosis of the distal end). Gastrointestinal dysregulation is generally associated with decreased intestinal epithelial barrier integrity, decreased tight junction integrity, and increased intestinal permeability. Citi, S.Internal Barriers protect against disease [ intestinal barrier preventable disease ], science [ Science ]359:1098-99 (2018); srinivasan et al TEER measurement techniques for in vitro barrier model systems [ TEER measurement techniques for in vitro Barrier model System ]. J.Lab. Autom [ journal of laboratory Automation ].20:107-126 (2015). Dysbacteriosis in the gastrointestinal tract can produce physiological and immunological effects in the gastrointestinal tract.

The presence of dysbacteriosis can be associated with a variety of diseases and conditions, including: infection, cancer, autoimmune diseases (e.g., systemic Lupus Erythematosus (SLE)) or inflammatory diseases (e.g., functional gastrointestinal diseases such as Inflammatory Bowel Disease (IBD), ulcerative colitis, and crohn's disease), neuroinflammatory diseases (e.g., multiple sclerosis), transplant diseases (e.g., graft versus host disease), fatty liver disease, type I diabetes, rheumatoid arthritis, sjogren's syndrome, celiac disease, cystic fibrosis, chronic Obstructive Pulmonary Disease (COPD), and other diseases and conditions associated with immune dysfunction. Lynch et al The Human Microbiome in Health and Disease [ microbiome of humans in health and disease ], n.engl.j.med [ journal of new england medicine ].375:2369-79 (2016), carry et al Dysbiosis of the gut microbiota in disease [ dysbacteriosis of intestinal microorganisms in disease ]. Microb.ecl.health Dis [ microbial ecological and health disease ] (2015); 26:10:3402/mehd.v26.2619; levy et al, dysbiosis and the Immune System [ dysbacteriosis and immune system ], nature Reviews Immunology [ natural review immunology ]17:219 (month 4 of 2017).

In certain embodiments, the exemplary therapeutic compositions disclosed herein can treat dysbacteriosis and its effects by modifying the immune activity present at the site of dysbacteriosis. As described herein, such compositions can modify dysbacteriosis by action on host immune cells (resulting in, for example, increased secretion of anti-inflammatory cytokines and/or decreased secretion of pro-inflammatory cytokines, thereby alleviating inflammation in a subject) or by changes in metabolite production.

Exemplary therapeutic compositions disclosed herein useful for treating disorders associated with dysbacteriosis contain Prevotella EV, a tissue of interest. Such compositions are capable of affecting immune function of the recipient host in the gastrointestinal tract, and/or producing systemic effects at a distal site outside the gastrointestinal tract of the subject.

Exemplary therapeutic compositions disclosed herein useful for treating disorders associated with dysbacteriosis contain the Prevotella denticola EV population. Such compositions are capable of affecting immune function of the recipient host in the gastrointestinal tract, and/or producing systemic effects at a distal site outside the gastrointestinal tract of the subject.

In one embodiment, a therapeutic composition containing an isolated population of EVs derived from Prevotella denticola bacteria is administered (e.g., orally) to a mammalian recipient in an amount effective to treat dysbacteriosis and one or more effects thereof. The dysbacteriosis may be a gastrointestinal dysbacteriosis or a distal dysbacteriosis.

In another embodiment, the therapeutic compositions of the invention can treat gastrointestinal dysbacteriosis and one or more of its effects on host immune cells, resulting in increased secretion of anti-inflammatory cytokines and/or decreased secretion of pro-inflammatory cytokines, thereby reducing inflammation in a subject.

In another embodiment, the therapeutic composition may treat gastrointestinal dysbacteriosis and one or more of its effects by: the immune response of the recipient is modulated via cell and cytokine modulation to reduce intestinal permeability by increasing the integrity of the intestinal epithelial barrier.

In another embodiment, the therapeutic composition may treat a remote dysbacteriosis and one or more effects thereof by: the recipient immune response at the dysbacteriosis site is modulated by modulating host immune cells.

Other exemplary therapeutic compositions are useful for treating disorders associated with dysbacteriosis, which compositions contain one or more types of bacteria and/or EVs capable of altering the relative proportion of or function of a subpopulation of host immune cells (e.g., subpopulations of T cells, immune lymphoid cells, dendritic cells, NK cells, and other immune cells) in a recipient.

Other exemplary therapeutic compositions are useful for treating disorders associated with dysregulation of the flora, which compositions contain a population of Prevotella EV, a tissue that is capable of altering the relative proportion of immune cell subsets (e.g., T cell subsets, immune lymphoid cells, NK cells, and other immune cells) or their function in a recipient.

In one embodiment, the present invention provides a method of treating gastrointestinal dysbacteriosis and one or more effects thereof by: orally administering to a subject in need thereof a therapeutic composition that alters a microbiome population present at the site of dysbacteriosis. The therapeutic composition may contain Prevotella EV, a tissue of the genus Prevotella.

In one embodiment, the invention provides a method of treating a remote dysbacteriosis and one or more effects thereof by: orally administering to a subject in need thereof a therapeutic composition that alters an immune response outside the gastrointestinal tract of the subject. The therapeutic composition may contain one or more types of EV from immunomodulatory bacteria or a population of prasuvorexa bacteria EV on the tissue of interest.

In exemplary embodiments, therapeutic compositions useful for treating disorders associated with dysbacteriosis stimulate host immune cells to secrete one or more anti-inflammatory cytokines. Anti-inflammatory cytokines include, but are not limited to, IL-10, IL-13, IL-9, IL-4, IL-5, TGF beta, and combinations thereof. In other exemplary embodiments, therapeutic compositions useful for treating disorders associated with dysbacteriosis reduce (e.g., inhibit) secretion of one or more pro-inflammatory cytokines by host immune cells. Proinflammatory cytokines include, but are not limited to, IFNγ, IL-12p70, IL-1α, IL-6, IL-8, MCP1, MIP1 α, MIP1 β, TNF α, and combinations thereof. Other exemplary cytokines are known in the art and described herein.

In another aspect, the invention provides a method of treating or preventing a disorder associated with dysbacteriosis in a subject in need thereof, the method comprising administering (e.g., orally administering) to the subject a therapeutic composition in the form of a probiotic food or medical food comprising a microbiome of Prevotella EV in an amount sufficient to alter the site of dysbacteriosis, thereby treating the disorder associated with dysbacteriosis.

In another embodiment, the therapeutic composition of the invention in the form of a probiotic food or medical food may be used to prevent or delay the onset of dysbacteriosis in a subject at risk of developing dysbacteriosis.

Method for producing enhanced bacteria

In certain aspects, provided herein are methods of making an engineered bacterium for producing a prasuvorexant EV of tissue as described herein. In some embodiments, these 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 effects of the EV (e.g., alone or in combination with another therapeutic agent) to reduce toxicity and/or improve bacteria and/or EV manufacturing (e.g., higher oxygen tolerance, higher freeze-thaw resistance, shorter production time). Engineered bacteria may be produced using any technique known in the art, including, but not limited to, site-directed mutagenesis, transposon mutagenesis, knockout, knock-in, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet mutagenesis, transformation (chemical or by electroporation), phage transduction, directed evolution, CRISPR/Cas9, or any combination thereof.

In some embodiments of the methods provided herein, the bacteria are modified by directed evolution. In some embodiments, the directional evolution comprises exposing bacteria to environmental conditions and selecting bacteria with improved survival and/or growth under the environmental conditions. In some embodiments, the method comprises screening for mutagenized bacteria using an assay that recognizes the enhanced bacteria. In some embodiments, the method further comprises mutagenizing the bacteria (e.g., by exposure to a chemical mutagen and/or UV radiation), or exposing them to a therapeutic agent (e.g., an antibiotic), followed by analysis to detect bacteria having a desired phenotype (e.g., in vivo analysis, ex vivo analysis, or in vitro analysis).

Examples

Example 1: preparation of lyophilisates

Excipient stock solutions having the formulations provided in tables a-D were prepared as solutions (the amounts shown are percentages of the components in the formulation). The formulation of excipient stock is divided into two main categories: with and without polymer. The excipient stock solution is mixed with the liquid formulation of the extracellular vesicles. The resulting solution was freeze dried and analyzed.

In this example, the extracellular vesicles (smEV) used in the study were isolated from the tissue Prevotella strain (Prevotella strain B) (NRRL accession number B50329).

The data collected from lyophilization of these mixtures are provided in table E. All the samples measured had a residual moisture content of less than 5%. Some samples were additionally tested in vivo using Keyhole Limpet Hemocyanin (KLH) -specific inflammation in a delayed-type hypersensitivity (DTH) model. Samples tested in KLH-DTH showed efficacy.

Table a: a stock solution comprising an excipient for stabilizing extracellular vesicles during lyophilization. The values given are based on the weight percent in the solution.

Table B: a stock solution comprising an excipient comprising a polymer for stabilizing extracellular vesicles during lyophilization. The values given are based on the weight percent in the solution.

Formulation of	Sucrose	PVP-K30	Ficoll	Citrate salt	Arginine (Arg)
						6	20	78	1	1
14	20		78	1	1

Table C: a stock solution comprising an excipient comprising a polymer for stabilizing extracellular vesicles during lyophilization. The values given are based on the weight percent in the solution.

Table D: a stock solution comprising an excipient comprising a polymer for stabilizing extracellular vesicles during lyophilization. The values given are based on the weight percent in the solution.

Table E: analytical data obtained for excipient stock solutions for stabilization of extracellular vesicles. "% stabilizer" refers to the percentage by weight of the stock solution formulation added to the liquid formulation of the EV. "% moisture" was determined by karl fischer titration. Z is Z _ave Determined by Dynamic Light Scattering (DLS). For particle count/mass, particle count is determined by Z-view or NTA instruments; the mass (mg) was determined by analytical balance.

Lyophilization cycle for Extracellular Vesicles (EV)

The lyophilization cycle was optimized for each excipient formulation. The difference in the critical temperature and collapse temperature of the mixture means that the shelf temperature during lyophilization will be adjusted accordingly. The optimization process includes 3 steps: primary screening, primary drying optimization and secondary drying optimization. The last cycle was confirmed to be sufficient to dry the material to less than 5% residual moisture. In this example, the excipient formulation selected for optimization is excipient formulation 7.

Table F

Shelf temperature (. Degree. C.)	Sample temperature (. Degree. C.)
		-5	-17.9
-15	-23.6
		-20	-26.3
-25	-28.9

Table G


				-25	2.9	31.5	189
-20	2.8	28.2	215
				-15	3.3	20.8	224
-5	1.5	18.2	202

Table H

Primary drying	2.8	2
			Secondary drying	2.6	29

Table I: the final lyophilization cycle optimized extracellular vesicles stabilized with 47% (by volume) of excipient formulation 7.

Example 2: orally delivered microbial extracellular vesicles induce anti-inflammatory activity in mice

Significantly reduced ear swelling and inflammation was observed in the delayed-type hypersensitivity model, indicating that Extracellular Vesicles (EV) from prasuvorexant strain B (NRRL accession B50329) of the tissue regulate systemic inflammatory response. The activity of Prevotella EV, a percha tissue, depends on TLR2 signaling and the presence of localized immune cells. In vitro results show TLR2 agonism of EV and induction of anti-inflammatory cytokine responses in immune cells. These findings demonstrate the anti-inflammatory effect of orally delivered microbial extracellular vesicles. Prevotella EV, a perchloric tissue, induces extensive resolution of inflammation across a variety of pathways without systemic exposure through a novel systemic pharmacological mechanism. EV is particularly effective in causing host cells in the gut to participate in the regulation of distant inflammation. These data indicate that oral EV is a novel class of immunotherapeutic drugs.

Example 3: purification and preparation of Extracellular Vesicles (EV) from bacteria

Purification

Extracellular Vesicles (EV) were purified and prepared from bacterial cultures using methods known to those skilled in the art (S.Bin Park et al, PLoS ONE. [ public science library. Complex ]6 (3): e17629 (2011)).

For example, bacterial cultures are centrifuged at 10,000-15,500 Xg for 10-40 minutes at 4℃or room temperature to pellet the bacteria. The culture supernatant is then filtered to include material that is less than or equal to 0.22 μm (e.g., via a 0.22 μm or 0.45 μm filter) and to exclude intact bacterial cells. The filtered supernatant is concentrated using methods that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration. Briefly, for ammonium sulfate precipitation, 1.5 to 3M ammonium sulfate was slowly added to the filtered supernatant while stirring at 4 ℃. The pellet was incubated at 4℃for 8 to 48 hours and then centrifuged at 11,000Xg for 20 to 40 minutes at 4 ℃. The precipitate contains EV and other debris. Briefly, the filtered supernatant was centrifuged at 100,000 to 200,000Xg at 4℃for 1 to 16 hours using ultracentrifugation. This centrifuged pellet contains EV and other debris. Briefly, the supernatant was filtered using filtration techniques, using Amicon super spin filters or by tangential flow filtration in order to retain substances with molecular weights >50, 100, 300 or 500 kDa.

Alternatively, EVs are obtained continuously from bacterial cultures during growth (or at selected time points during growth) by connecting the bioreactor to an Alternating Tangential Flow (ATF) system (e.g., XCell ATF from Repligen) according to manufacturer's instructions. The ATF system retains intact cells (> 0.22 μm) in the bioreactor and allows smaller components (e.g., EV, free proteins) to pass through the filter for collection. For example, the system may be structured such that <0.22 μm filtrate is then passed through a 100kDa second filter, allowing collection of substances such as EVs between 0.22 μm and 100kDa, and pumping species less than 100kDa back into the bioreactor. Alternatively, the system may be structured to allow the medium in the bioreactor to be replenished and/or modified during the growth of the culture. The EVs collected by this method can be further purified and/or concentrated by ultracentrifugation or filtration as described above for the filtered supernatant.

The EV obtained by the method described above may be further purified by gradient ultracentrifugation using methods that may include, but are not limited to, the use of sucrose gradients or Optiprep gradients. Briefly, when using the sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation is used to concentrate the filtered supernatant, the precipitate is resuspended in 60% sucrose, 30mM pH 8.0 Tris. If filtration is used to concentrate the filtered supernatant, the concentrate buffer is exchanged into 60% sucrose, 30mM pH 8.0Tris using an Amicon Ultra column. Samples were applied to a 35% -60% discontinuous sucrose gradient and centrifuged at 200,000Xg at 4 ℃ for 3-24 hours. Briefly, when using the Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation is used to concentrate the filtered supernatant, the pellet is resuspended in 45% Optiprep in PBS. If filtration is used to concentrate the filtered supernatant, the concentrate is diluted to a final concentration of 45% Optiprep by using 60% Optiprep. Samples were applied to a 0-45% discontinuous sucrose gradient and centrifuged at 200,000Xg at 4 ℃ for 3-24 hours. Alternatively, high resolution density gradient fractionation may be used to separate EVs based on density.

Preparation

To confirm sterility and isolation of the EV formulation, EVs were serially diluted on agar medium (which was used for routine culture of the bacteria under test) and incubated using routine conditions. The unsterilized formulation was passed through a 0.22 μm filter to remove intact cells. To further increase purity, the isolated EV may be treated with DNase or proteinase K.

Alternatively, to prepare an EV for in vivo injection, the purified EV is treated as previously described (G.Norheim et al, PLoS ONE. [ public science library. Complex ]10 (9): e 0134553 (2015)). Briefly, after sucrose gradient centrifugation, the EV-containing bands were resuspended to a final concentration of 50 μg/mL in a solution containing 3% sucrose or other solutions known to those skilled in the art to be suitable for in vivo injection. The solution may also contain an adjuvant (e.g., aluminum hydroxide) at a concentration of 0-0.5% (w/v).

To make samples compatible with other tests (e.g., to remove sucrose prior to TEM imaging or in vitro analysis), samples were buffer exchanged into PBS or 30mm pH 8.0tris using the following: filtration (e.g., amicon Ultra column), dialysis, or ultracentrifugation (200,000 x g,1-3 hours, 4 ℃ C.) after 15-fold or more dilution with PBS and re-suspension in PBS.

For all these studies, EVs may be heated, irradiated, and/or lyophilized (as described herein) prior to administration.

Example 4: manipulation of bacteria by pressure to produce various amounts of EV and/or to alter the content of EV

Stress, and in particular outer membrane stress, has been shown to increase EV (I.MacDonald, M.Kuehn.J Bacteriol journal of bacteriology) 195 (13): doi: 10/1128/JB.02267-12) produced by some strains. To alter bacterial EV production, bacteria are pressurized using various methods.

The bacteria may be subjected to a single stress source or a combination of stress sources. The effect of different stressors on different bacteria was determined empirically by varying stress conditions and determining IC50 values (the conditions required to inhibit 50% of cell growth). EV purification, quantification and characterization take place. EV production is (1) in complex samples of bacteria and EV by Nanoparticle Tracking Analysis (NTA) or Transmission Electron Microscopy (TEM); or (2) after EV purification, quantification is performed by NTA, lipid quantification or protein quantification. The EV content was purified and then evaluated by the method described above.

Antibiotic stress

Bacteria were cultured under standard growth conditions with the addition of sublethal concentrations of antibiotics. This may include 0.1 to 1 μg/mL chloramphenicol, or 0.1 to 0.3 μg/mL gentamicin, or other antibiotics (e.g., ampicillin, polymyxin B) at similar concentrations. Host antibacterial products (e.g., lysozyme, defensin, and Reg protein) may be used in place of antibiotics. Antimicrobial peptides (including bacteriocins and microcins) produced by bacteria may also be used.

Temperature stress

Bacteria are grown under standard growth conditions, but at temperatures higher or lower than those typically used for their growth. Alternatively, the bacteria are grown under standard conditions and then subjected to cold shock or heat shock by short-term incubation at low or high temperatures, respectively. For example, bacteria grown at 37℃are incubated at 4℃to 18℃for 1 hour for cold shock or at 42℃to 50℃for 1 hour for heat shock.

Starvation and nutrient limitation

To induce nutritional stress, the bacteria are cultured under conditions in which one or more nutrients are limited. Bacteria may be subjected to nutritional stress or transferred from rich to lean media throughout the growth period. Some examples of limited media components are carbon, nitrogen, iron, and sulfur. An example medium is M9 minimal medium (Sigma Aldrich, aldrich) which contains low glucose as the sole carbon source. In particular for Prevotella, iron availability is altered by changing the concentration of hemin in the medium and/or by changing the type of porphyrin or other iron carrier present in the medium, as cells grown in hypohemin conditions were found to produce more EV (S.Stubbs et al Letters in Applied Microbiology [ applied microbiology report ]29:31-36 (1999.) Medium components were also manipulated by the addition of chelators such as EDTA and deferoxamine.

Saturation level

Bacteria were allowed to grow to saturation and incubated for various periods of time after the saturation point. Alternatively, conditioned media is used to simulate a saturated environment during exponential growth. Conditioned medium is prepared by centrifugation and filtration to remove intact cells from a saturated culture, and the conditioned medium may be further processed to concentrate or remove specific components.

Salt stress

Bacteria are cultured in or briefly exposed to a medium containing NaCl, bile salts or other salts.

UV stress

UV stress is achieved by culturing the bacteria under UV lamps or by exposing the bacteria to UV using an instrument such as Stratalinker (Agilent). UV may be applied during the entire culture period, during a short burst period or during a single defined period after growth.

Reactive oxygen stress

Bacteria are cultured in the presence of sublethal concentrations of hydrogen peroxide (250 to 1,000 μm) to induce stress in the form of reactive oxygen species. Anaerobic bacteria are cultured in or exposed to oxygen at concentrations toxic to them.

Detergent stress

Bacteria are cultured in or exposed to detergents such as sodium lauryl sulfate (SDS) or deoxycholate.

pH stress

Bacteria are cultured in or exposed to different pH media for a limited time.

Example 5: analysis of EV composition and content

EV may be characterized by any of a variety of methods including, but not limited to: nanoSight characterization, SDS-PAGE gel electrophoresis, western blotting, ELISA, liquid chromatography-mass spectrometry, dynamic light scattering, lipid level, total protein, lipid to protein ratio, nucleic acid analysis, and/or zeta potential.

NanoSight characterization of EV

Nanoparticle Tracking Analysis (NTA) was used to characterize the particle size distribution of the purified EV. Purified EV formulations were run on a NanoSight machine (malvern instruments (Malvern Instruments)) to evaluate EV size and concentration.

SDS-PAGE gel electrophoresis

To identify the protein component of the purified EV, the samples were run on a gel using standard techniques, such as Bolt Bis-Tris Plus 4% -12% gel (Siemens Feishmanic technologies (Thermo-Fisher Scientific)). The samples were boiled in 1x SDS sample buffer for 10 minutes, cooled to 4℃and then centrifuged at 16,000Xg for 1 minute. The samples were then run on SDS-PAGE gels and stained using any of several standard techniques (e.g., silver staining, coomassie blue, gel code blue) to visualize the bands.

Western blot analysis

To identify and quantify specific protein components of the purified EV, the EV proteins were separated by SDS-PAGE as described above and subjected to Western blot analysis (Cvjetkovic et al, sci. Rep. [ science report ]6,36338 (2016)) and quantified via ELISA.

EV proteomics and liquid chromatography-mass spectrometry (LC-MS/MS) and Mass Spectrometry (MS)

Proteins present in EVs are identified and quantified by mass spectrometry techniques. EV proteins can be prepared for LC-MS/MS using standard techniques including protein reduction using dithiothreitol solution (DTT) and protein digestion using enzymes (e.g., lysC and trypsin) (as described in Erickson et al, 2017 (Molecular Cell, volume 65, stage 2, pages 361-370, day 19 of 2017, 1)). Alternatively, peptides were prepared as described in Liu et al 2010 (JOURNAL OF BACTERIOLOGY [ journal of bacteriology ], 6 th 2010, pages 2852-2860, volume 192, 11 th edition), kieselbach and Oscasson 2017 (Data Brief [ Data Abstract ].2017, 2 nd; 10:426-431.), vildhele et al 2018 (Drug Metabolism and Disposition [ drug metabolism and treatment ]2018, 2 nd 8). Following digestion, the peptide preparation was run directly on liquid chromatography and mass spectrometry for identification of proteins in a single sample. Peptide digests from different samples were labeled with isobaric tags using the iTRAQ reagent-8 plex multiplex kit (applied biosystems (Applied Biosystems), foster city, california) or the TMT 10plex and 11plex labeling reagents (sammer feishier technologies (Thermo Fischer Scientific), san jose, california, usa) for relative quantification of proteins between samples. Each peptide digest was labeled with a different isobaric tag, and the labeled digests were combined into one sample mixture. The combined peptide mixture was analyzed by LC-MS/MS for identification and quantification. Database searches were performed using LC-MS/MS data to identify labeled peptides and corresponding proteins. In the case of isobaric labeling, the tag-attached fragments produce low molecular weight reporter ions that are used to obtain relative quantification of peptides and proteins present in each EV.

In addition, the metabolic content was determined using a combination of liquid chromatography and mass spectrometry. There are various techniques for determining the metabolic content of various samples and known to those skilled in the art, which involve solvent extraction, chromatographic separation, and various ionization techniques coupled to mass determination (Roberts et al 2012Targeted Metabolomics [ Targeted Metabolic groups ] Curr Protoc Mol Biol [ contemporary molecular biological protocols ]30:1-24; dettmer et al 2007,Mass spectrometry-based metabolomics [ mass spectrometry based Metabolic groups ] Mass Spectrom Rev (1): 51-78). As one non-limiting example, the LC-MS system includes a 4000QTRAP triple quadrupole mass spectrometer (AB SCIEX) combined with a 1100 series pump (Agilent) and an HTS PAL autosampler (Leap technologies (Leap Technologies)). The medium samples or other complex metabolic mixtures (about 10. Mu.L) were extracted with nine volumes of acetonitrile/methanol/formic acid containing stable isotope labeled internal standards (valine-d 8, isotec; and amphetamine-d 8, cambridge isotope laboratories (Cambridge Isotope Laboratories)). The standard may be adjusted or modified depending on the metabolite of interest. The samples were centrifuged (10 min, 9,000Xg, 4 ℃) and the supernatant (10. Mu.L) was presented to LCMS by injecting the solution onto an HILIC column (150X 2.1mm,3 μm particle size). The column was eluted by flowing 5% mobile phase [10mM ammonium formate, 0.1% formic acid in water ] at 250. Mu.L/min for 1 min followed by a linear gradient over 10 min to 40% mobile phase solution [ acetonitrile with 0.1% formic acid ]. The ion spray voltage was set to 4.5kV and the source temperature was 450 ℃.

The data were analyzed using commercially available software (such as Multiquant 1.2 from AB SCIEX) for mass spectrometry peak integration. The peak of interest should be manually controlled and compared to a standard to confirm the identity of the peak. Quantification is performed with appropriate standards to determine the amount of metabolite present after bacterial conditioning and in the initial medium. Non-targeted metabonomics methods may also be used for peak identification using a metabolite database (such as, but not limited to, the NIST database).

Dynamic Light Scattering (DLS)

DLS measurements (including the distribution of different sized particles in different EV formulations) were performed using instruments such as DynaPro NanoStar (Huai Ya trickplay company (Wyatt Technology)) and Zetasizer Nano ZS (malvern instruments).

Lipid levels

Lipid levels were quantified using FM4-64 (life technologies Co (Life Technologies)) by methods similar to those described by A.J. McBroom et al, J. Bacteriol [ J. Bacteriology ]188:5385-5392, and A.Frias et al, microb Ecol [ microbial ecology ].59:476-486 (2010). Samples were incubated with FM4-64 (3.3. Mu.g/mL in PBS, 10 minutes at 37℃in the dark). After excitation at 515nm, emission at 635nm was measured using a Spectramax M5 plate reader (molecular instruments (Molecular Devices)). Absolute concentrations are determined by comparing an unknown sample to a standard of known concentration, such as Palmitoyl Oleic Phosphatidylglycerol (POPG) vesicles. Lipidomics can be used to identify lipids present in EVs.

Total protein

Protein levels are quantified by standard assays (e.g., the Bradeford and BCA assays). These Brightness analyses were run according to the manufacturer's protocol using the Quick Start Brightness 1x dye reagent (Bio-Rad). BCA assays were run using the Pierce BCA protein assay kit (sameimers technologies). Absolute concentrations were determined by comparison with standard curves generated from BSA of known concentration. Alternatively, protein concentration may be calculated using the Beer-Lambert equation using absorbance at 280nm (a 280) of the sample as measured on a nanodrop spectrophotometer (sammer feichi technologies). Furthermore, proteomics can be used to identify proteins in a sample.

Lipid to protein ratio

Lipid to protein ratio is produced by dividing lipid concentration by protein concentration. Such provides a measure of the purity of the vesicles as compared to the free protein in each formulation.

Nucleic acid analysis

Nucleic acids were extracted from EVs and quantified using a Qubit fluorometer. Particle size distribution was assessed using a bioanalyzer and the material sequenced.

Zeta potential

Zeta potential of the various formulations was measured using an instrument such as Zetasizer ZS (malvern instruments).

Example 6: production conditions

Enrichment media is used to grow and prepare bacteria for in vitro and in vivo use, and ultimately for EV formulations. For example, the medium may contain sugar, yeast extract, plant-based peptone, buffers, salts, trace elements, surfactants, defoamers and vitamins. The composition of the complex components (e.g., yeast extract and peptone) may be undefined or partially defined (including approximate concentrations of amino acids, sugars, etc.). Microbial metabolism may depend on the availability of resources such as carbon and nitrogen. Various sugars or other carbon sources may be tested. Alternatively, a medium may be prepared and the selected bacteria grown, as shown by Saarela et al, J.applied Microbiology [ journal of applied Microbiology ].2005.99:1330-1339, which is hereby incorporated by reference. The effect of fermentation time, neutralization of cryoprotectants and cell concentrates on lyophilization survival, storage stability, and acid and bile exposure of selected bacteria without the production of milk-based components.

The culture medium was sterilized on a large scale. Sterilization may be accomplished by Ultra High Temperature (UHT) treatment. UHT treatment is carried out at extremely high temperatures for short periods of time. UHT can range from 135℃to 180 ℃. For example, the medium may be sterilized at 135℃for 10 to 30 seconds.

The inoculum can be prepared and growth monitored in flasks or smaller bioreactors. For example, the inoculum size may be about 0.5% to 3% of the total bioreactor volume. Depending on the application and material requirements, the bioreactor volume may be at least 2L, 10L, 80L, 100L, 250L, 1000L, 2500L, 5000L, 10,000L.

Prior to inoculation, the bioreactor is prepared using a medium at the desired pH, temperature, and oxygen concentration. The initial pH of the medium may be different from the treatment set point. pH stress can be detrimental at low cell concentrations; the initial pH may be between pH 7.5 and the process set point. For example, the pH may be set between 4.5 and 8.0. During fermentation, the pH can be controlled by using sodium hydroxide, potassium hydroxide or ammonium hydroxide. The temperature may be controlled between 25 ℃ and 45 ℃, for example at 37 ℃. Anaerobic conditions were created by reducing the oxygen content in the broth from about 8mg/L to 0 mg/L. For example, nitrogen or a gas mixture (N ₂ 、CO ₂ And H ₂ ) To establish anaerobic conditions. Alternatively, anaerobic conditions are established without the use of gas and by cells consuming the remaining oxygen from the medium. Depending on the strain and inoculum size, the bioreactor fermentation time may vary. For example, the fermentation time may vary from about 5 hours to 48 hours.

Recovery of bacteria from a frozen state may be of particular concern. The production medium may stress the cells after thawing; specific thawing media may be required to initiate strain culture throughout the thawed material. The kinetics of transfer or passage of the seed material to fresh medium may be affected by the current state of the bacteria (e.g., exponential growth, resting growth, no stress, stressed) for the purpose of increasing the seed volume or maintaining the state of microbial growth.

Inoculation of one or more production fermenters can affect growth kinetics and cell activity. The initial state of the bioreactor system must be optimized to promote successful and consistent production. The fraction (e.g., percentage) of the seed culture relative to the total medium has a significant effect on growth kinetics. The range may be 1% to 5% of the working volume of the fermenter. The initial pH of the medium may be different from the treatment set point. pH stress can be detrimental at low cell concentrations; the initial pH may be between pH 7.5 and the process set point. During inoculation, the agitation and gas flow into the system may be different from the process set point. At low cell concentrations, physical and chemical stress can be disadvantageous due to two conditions.

Treatment conditions and control settings can affect the kinetics of microbial growth and cellular activity. Variations in the processing conditions can alter membrane composition, metabolite production, growth rate, cell stress, and the like. The optimal temperature range for growth may vary with the strain. The range may be 20 ℃ to 40 ℃. The optimal pH for cell growth and downstream activity performance may vary with the strain. The range may be pH 5 to 8. The gas dissolved in the medium can be used by the cells for metabolism. It may be necessary to adjust O during the entire process ₂ 、CO ₂ N ₂ Concentration. The availability of nutrients can alter cell growth. Bacteria may have alternative kinetics when excess nutrients are available.

The status of bacteria at the end of fermentation and during harvest can affect cell survival and activity. Bacteria may be pre-treated shortly before harvesting to better prepare them for physical and chemical stresses involving isolation and downstream processing. When removed from the fermentor, changes in temperature (typically reduced to 20 ℃ to 5 ℃) can reduce cellular metabolism, slow growth (and/or death), and physiological changes. The effectiveness of the centrifugation concentration may be affected by the culture pH. The 1 to 2 point increase in pH may improve the effectiveness of the concentration but may also be detrimental to the cells. Bacteria may be stressed shortly before harvesting by increasing the concentration of salts and/or sugars in the medium. Cells stressed in this way can survive better in freezing and lyophilization during downstream periods.

Methods and techniques for isolation can affect the efficiency of bacteria isolation from a culture medium. Solids can be removed using centrifugation techniques. The effectiveness of the centrifugation concentration may be affected by the culture pH or by the use of a flocculant. The 1 to 2 point increase in pH may improve the effectiveness of the concentration but may also be detrimental to the cells. Bacteria may be stressed shortly before harvesting by increasing the concentration of salts and/or sugars in the medium. Cells stressed in this way can survive better in freezing and lyophilization during downstream periods. Alternatively, bacteria may be isolated by filtration. If the cells require an excess of g minutes to successfully centrifuge, filtration is preferred over centrifugation techniques for purification. Excipients may be added before and after isolation. Excipients may be added for cryoprotection or for protection during lyophilization. Excipients may include, but are not limited to, sucrose, trehalose, or lactose, and alternatively these excipients may be mixed with buffers and antioxidants. Prior to lyophilization, droplets of the cell pellet mixed with excipients were immersed in liquid nitrogen.

Harvesting may be performed by continuous centrifugation. The product may be resuspended to the desired final concentration with various excipients. Excipients may be added for cryoprotection or for protection during lyophilization. Excipients may include, but are not limited to, sucrose, trehalose, or lactose, and alternatively these excipients may be mixed with buffers and antioxidants. Prior to lyophilization, droplets of the cell pellet mixed with excipients were immersed in liquid nitrogen.

Lyophilization of materials (including live bacteria, vesicles, or other bacterial derivatives) includes freezing, primary drying, and secondary drying stages. Lyophilization begins with freezing. The product material may or may not be mixed with lyoprotectants or stabilizers prior to the freezing stage. The product may be frozen prior to loading in the lyophilizer or frozen under controlled conditions on shelves in the lyophilizer. In the next stage, the primary drying stage, ice is removed by sublimation. Here, a vacuum is created and an appropriate amount of heat is provided to the material. Ice will sublimate while maintaining the product temperature below the freezing point and below the critical temperature (T _c ). The temperature of the rack loaded with material and the vacuum of the chamber can be manipulated to achieve the desired product temperature. During the secondary drying period, water molecules bound to the product are removed. Here, the temperature is typically raised above the primary drying period to cleave any physico-chemical interactions that have formed between the water molecules and the product material. After the completion of the freeze-drying process,the chamber may be filled with an inert gas, such as nitrogen. The product may be sealed in a freeze-dryer under dry conditions, in a glass bottle or other similar container, to prevent exposure to atmospheric water and contaminants.

Example 7: preparation of smEV

Prevotella denticola smEV was prepared as follows.

smEV: immediately after harvesting in the bioreactor, downstream processing of the smEV was started. Centrifugation was performed at 20,000Xg to remove cells from the liquid medium. The resulting supernatant was clarified using a 0.22 μm filter. EV was concentrated and washed using Tangential Flow Filtration (TFF) and a flat plate cassette Ultrafiltration (UF) membrane with a Molecular Weight Cut Off (MWCO) of 100 kDa. Diafiltration (DF) was used to elute small molecules and small proteins using 5 volumes of Phosphate Buffered Solution (PBS). Retentate from TFF was centrifuged at 200,000x g in an ultracentrifuge for 1 hour to form EV-rich precipitate, known as High Speed Precipitate (HSP). The pellet was resuspended with minimal PBS and used with OptiPrep ^TM Density gradient media gradients were prepared and ultracentrifuged at 200,000Xg for 16 hours. In the fractions obtained, there are 2 intermediate bands containing EV. The fractions were washed with 15-fold PBS and the EVs were centrifuged at 200,000x g for 1 hour to yield fractionated HSP or fsp. They were then resuspended with minimal PBS, pooled, and analyzed for particle count/mL and protein content. Doses were prepared from particle count/mL counts to achieve the desired concentration. EV was characterized in the scattering mode of 532nm laser using NanoSight NS300 from Markov panaceae (Malvern Panalytical).

Example 8: EV separation and counting

The Sorvall RC-5C centrifuge used in EV separation comprises a SLA-3000 rotor; an Optima XE-90 ultracentrifuge with 45Ti rotor from Beckman Coulter; sorvall wX+ultra series centrifuges from Siemens technology; and a Fiberlite F37L-8x100 rotor.

Bacterial supernatant collection and filtration

To recover EV instead of bacteria, the bacteria must be precipitated and filtered from the supernatant.

A precipitated bacterial culture was produced by centrifugation using a Sorvall RC-5C centrifuge with an SLA-3000 rotor and at a rotational speed of at least 7,000rpm for at least 15 min. The supernatant was then poured into a new sterile container.

The supernatant was filtered through a 0.2 μm filter. For the less filterable supernatants (less than 300ml of supernatant passed through the filter), a 0.45 μm capsule filter was added before the 0.2 μm vacuum filter. The filtered supernatant was stored at 4 ℃. The filtered supernatant may then be concentrated using TFF.

EV separation using ultracentrifugation

The concentrated supernatant is centrifuged in an ultracentrifuge to precipitate the EV and separate the EV from the smaller biomolecules. The speed was 200,000Xg for 1 hour at a temperature of 4 ℃. When the rotor stopped, the tube was removed from the ultracentrifuge and the supernatant was gently decanted. More supernatant was added and the tube was again centrifuged. After centrifugation of all concentrated supernatants, the resulting precipitate was called "crude" EV precipitate. Sterile 1x PBS was added to the pellet placed in the container. The vessel was placed on a shaker at a speed of 70 c overnight or longer at 4 c. The EV pellet was resuspended with additional sterile 1x PBS. The resuspended EV crude sample was stored at 4℃or-80 ℃.

Purification of EV using density gradient method

The density gradient was used for EV purification. During ultracentrifugation, particles in a sample will move and separate in a gradient density medium according to their "buoyancy" density. In this way, the EV is separated from other particles (e.g., sugar, lipids, or other proteins) in the sample.

For EV purification, four different percentages of density medium (60% Optiprep) were used: 45%, 35%, 25% and 15% layers. This will create a hierarchical layer. A 0% layer was added on top, consisting of sterile 1x PBS. The 45% gradient layer should contain a crude EV sample. Add 5ml sample to 15ml OptiPrep ^TM Is a kind of medium. If the crude EV sample is less than 5ml, it is brought to volume using sterile 1 XPBS.

Using a serological pipette, a 45% gradient mixture was pipetted up and down for mixing. The samples were then pipetted into labeled clean sterile ultracentrifuge tubes. Next, 13ml of a 35% gradient mixture was slowly added with a 10ml serological pipette. 13ml of a 25% gradient mixture was then added followed by 13ml of a 15% mixture and finally 6ml of sterile 1 XPBS was added. Ultracentrifuge tubes were equilibrated with sterile 1x PBS. The gradient was carefully placed in the rotor and the ultracentrifuge was set at 200,000Xg and 4 ℃. Gradient centrifugation is performed for at least 16 hours.

One or more fractions of interest were removed using a clean pipette and added to a 15ml conical tube. These "purified" EV samples were stored at 4 ℃.

To clean and remove residual OptiPrep in EV ^TM To the purified EV was added 10 volumes of PBS. The ultracentrifuge was set at 200,000Xg and 4 ℃. Centrifuge and spin for 1 hour. The tube was carefully removed from the ultracentrifuge and the supernatant was decanted. The purified EV was washed until all samples were precipitated. 1 XPBS was added to the purified pellet placed in a container. The vessel was placed on a shaker at a speed of 70 c overnight or longer at 4 c. The "purified" EV pellet was resuspended with additional sterile 1 XPBS. The resuspended purified EV samples were stored at 4℃or-80 ℃.

Example 9: mechanism of action of Prevotella smEV

Some bacteria produce non-replicating forms of extracellular vesicles (smevs) that share a molecular content of about 1/1000 by volume with the parent bacteria in the particle.

Preclinical pharmacological actions, mechanisms of action and biodistribution of orally administered prasuvorexa sm v formulations derived from a single gram-negative bacterial strain of the prasuvorexa family selected from EVs screened for anti-inflammatory pharmacology. Orally delivered prasuvorexa smEV is an enteric limiting bacterium EV that is effective in reducing inflammation in murine models of Th1 and Th17 inflammation.

In this example, the Prevotella extracellular vesicles (smEV) used in the study were isolated from Prevotella strain B (NRRL accession number B50329).

Orally administered prasuvorexa smEV requires multiple pathways directed against anti-inflammatory effects. Mice experiencing delayed hypersensitivity (DTH) to Keyhole Limpet Hemagglutinin (KLH) were dosed with 2E10 particles/dose of prasugrel EV by oral gavage on days 5-8. During the study, various mechanisms of action were explored by intraperitoneal injection of the indicated antibodies. Fig. 1A: the figure shows the change in ear thickness 24 hours after stimulation and blockade of TLR2 or IL-10R signaling with KLH protein. Fig. 1B: the graph shows the change in ear thickness 24 hours after stimulation and inhibition of lymphocyte intestinal homing (with anti-CD 62 and anti-LPAM-1 antibodies) with KLH protein. The dots represent individual mice and the lines represent the median change in ear thickness. Data represent 2 independent studies. Statistical analysis was performed using one-way ANOVA (relative to vehicle) or two-tailed unpaired t-test (isotype relative to treatment). Ns=not significant, < p <0.05, < p <0.01, < p <0.001, < p <0.0001.

The Prevotella smEV is localized to the gastrointestinal tract. 2E10 particles of Prevotella EV covalently labeled IRDye800 or dye-only control were administered intravenously or orally to mice. After 10 minutes, 1 hour, 6 hours or 24 hours, a small animal imaging system (Licor ) Fluorescence was measured in organs (spleen, liver, gastrointestinal tract, MLN (mesenteric lymph node), kidney, lung). The data are not shown. The results show that oral administration of Prevotella EV appears to be limited to the intestinal tract.

Prevotella smEV induces IL-10 release upon stimulation of TLR 2. Fig. 2: prevotella EV stimulates TLR1/2 and TLR2/6 heterodimers, with greater potency against TLR1/2 heterodimers. HEK293-SEAP reporter cells expressing a combination of human TLR1, TLR2 and TLR6 (Invivogen) were incubated with designated concentrations of Prevotella EV for 24 hours. Supernatants were collected and assayed for Secreted Embryonic Alkaline Phosphatase (SEAP) production to determine stimulation of TLR2 heterodimers. Fig. 3: the EV stimulated release of IL-10 from U937 cells by prasuvorexant is impaired by antibody-mediated blockade of TLR1 or TLR2 but not TLR 6. PMA differentiated human monocyte U937 cells were incubated with prevotella ev±2.5 μg/mL anti-TLR 1, TLR2, TLR6 or isotype control antibody for 24 hours. Supernatants were collected and analyzed for IL-10 response by MSD. Data represent 2 independent experiments.

Prevotella smEV induces IL-10 and IL-27 concentration dependent production of human PBMC. PBMCs were isolated from whole blood of six human donors, plated at 100,000 cells/well, left overnight, and then incubated with various concentrations of prasuvorexa EV for 24 hours. Supernatants were collected and IL-10 (FIG. 4A) and IL-27 (FIG. 4B) concentrations were determined by MSD. Data represent 2 independent experiments.

Conclusion: orally delivered microbial extracellular vesicles cause extensive resolution of inflammation, establishing a steady state inflammatory state. In addition to lymphocyte homing to intestinal lymphoid tissue, the efficacy of Prevotella EV is also dependent on stimulation of the TLR2 receptor and IL-10 receptor. Prevotella EV induces TLR 2-dependent release of IL-10. EV is an orally administered, enterally limited therapeutic agent that has no obvious safety or tolerability problems in animal models and has desirable therapeutic properties.

These data support the development of EV as a new class of immunotherapeutic drugs. They are particularly effective in participating in the small intestine axis, acting locally on host cells in the intestine to activate the far-end immune response. Prevotella EV is in preclinical development of inflammatory disorders involving aberrant Th1 and Th17 immune responses.

Example 10: a prenatal smEV lyophilizate: DTH efficacy

Female C57BL/6 mice were purchased 5 weeks old from Takara biosciences (Taconic Biosciences) and adapted for one week in a feeder box. Mice were primed with KLH and CFA (1:1) emulsion by subcutaneous immunization on day 0. From day 6-8, mice were either orally gavaged daily with Prevotella denticola smEV, or intraperitoneally with dexamethasone (positive control) at 1 mg/kg. After the administration on day 8, mice were anesthetized with isoflurane, basal measurements of the left ear were measured with Fowler calipers, and mice were challenged intradermally with KLH-containing physiological saline (10 μl) in the left ear, and ear thickness was measured at 24 hours.

In this example, the Prevotella extracellular vesicles (smEV) used in the study were isolated from Prevotella strain B (NRRL accession number B50329). The smEV was lyophilized in excipient formulation 7 a.

The 24 hour ear measurement results are shown in fig. 5. EV made from prasugrel tissue and lyophilized in the vehicle of formulation 7a was tested in one dose range study at four doses (2E 09, 2E07, 2E05, 2E 03) administered for three days. Except for the lowest dose (2E 03), all doses of prasuvorexant EV were effective in the vehicle and a trend in dose response was observed. As a negative control, only formulation 7a was used (the dose of excipient component corresponds to the amount present in the case of formulation 2e11 EV).

Example 11: additional stock solution for drying

EV from the tissue prasuvorexant bacteria is dried using one of the stock solutions provided in table K, for example by freeze-drying or spray-drying.

Table K: the stock solution contains excipients in relative concentrations (% w: w).

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The drying conditions in table L were used for lyophilization. Table L: general conservation freeze-drying cycle of EV.

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Example 12: spray-dried powder of Prevotella denticola smEV

In this example, the extracellular vesicles (smEV) used in the study were isolated from the serratia tissue strain B.

The smEV was spray dried as follows:

the EV retentate was mixed with one of the excipients provided in table P.

Table P: the stock solution contains excipients in relative concentration (% w: w)

Formulation of	Complete composition
		7a	80% mannitol, 20% trehalose
25	100% trehalose
		26	Maltodextrin-trehalose (20:60:20)
27	Maltodextrin-trehalose (70:30)
		28	PEG 6000-trehalose (70:30)
29	Mannitol-maltodextrin-trehalose (20:60:20)

Spray drying is carried out at 100℃or 130 ℃. The temperature is also included in table Q.

After spray drying, each powder was analyzed for Moisture Content (MC) (by karl fischer titration (KF)) and particle number (particle number/mg spray dried powder (p/mg)) using Zetaview quantification by nanoparticle follow-up analysis (NTA). The results are shown in Table Q. EXP7A is the stock solution of formula 7A.

Table Q: moisture content and particle content of spray dried samples

Man: mannitol; malt: maltodextrin; tre: trehalose.

Spray drying was also performed using a stock solution consisting of PEG 6000-mannitol-trehalose (60:20:20). However, the recovered dry product was less than other methods and was not analyzed further.

The tissue Prevotella smEV was spray-dried or freeze-dried in stock solutions of formula 7A (F7A) at two concentrations (25X and 500X), with an inlet temperature of 130 ℃.

The comparison of particle count/mg spray dried powder and size is shown in Table R. SD = spray drying; l0.47=lyophilization; 0.47 refers to the stock solution ratio used: 47g excipient per 100g retentate.

Particle packing and size of spray-dried and freeze-dried EVs are similar in both drying methods.

Table R

Sample of	Particle count/mg	CV	Size (nm)
				25X L0.47 F7A	3.35e+9	2.10％	172.7
25X SD F7A	3.95e+9	0.00％	164.8
				500X L0.47 F7A	5.50e+10	0.00％	165.1
500X SD F7A	4.88e+10	0.70％	163.8

Example 13: prevotella denticola smEV and anti-TNFa antibodies: DTH efficacy

Female C57BL/6 mice were purchased 5 weeks old from Takara biosciences (Taconic Biosciences) and adapted for one week in a feeder box. Mice were primed with KLH and CFA (1:1) emulsion by subcutaneous immunization on day 0. On days 0, 3 and 6, mice were injected intraperitoneally with 3mg/kg of anti-TNFα antibody (clone: XT3.11 was purchased from BioXcell) or equivalent isotype control (IgG 1 was also purchased from BioXcell). Starting on days 5-8, mice were daily gavaged orally with the tissue Prevotella extracellular vesicles (smEV) or intraperitoneally with 1mg/kg dexamethasone. After the 8 th day of administration, mice were anesthetized with isoflurane, basal measurements of the left ear were measured with Fowler calipers, and mice were challenged intradermally with KLH-containing physiological saline (10 μl) in the left ear. Ear thickness measurements were made at 24 hours.

The 24 hour ear measurement results are shown in fig. 6. smEV (2E 09 particles/dose) prepared from Prevotella denticola tissue (alone or in combination with 3mg/kg of anti-TNFα antibody or equivalent isotype control (IgG 1)) was tested. 3mg/kg of anti-TNFα antibody (alone or together with vehicle control) was also tested. The combination of Prevotella denticola smEV with anti-TNFα is more potent than Prevotella denticola smEV alone and anti-TNFα antibody alone.

Example 14: oral administration of Prevotella denticola smEV does not inhibit the immune system under non-inflammatory conditions

Oral administration before or after KLH DTH response: two groups of mice were used in this study: "preimmune" mice administered with a smEV or control prior to immunization, and "post-immune" mice administered with a smEV or control after immunization. From day-5, the "pre-immunized" mice were dosed daily with PBS vehicle PO, dexamethasone (1 mg/kg, IP (intraperitoneal)) or prasuvorexa smEV or smEV PO from another bacterial strain (oral) for 4 days. On day 0, all mice were immunized by subcutaneous injection of Complete Freund's Adjuvant (CFA) emulsified KLH. After 4 days of immunization with sucrose vehicle, prasugrel bacteria smEV or smEV PO or dexamethasone (1 mg/kg, IP) from another bacterial strain, the "post-immunization" mice were dosed daily starting on day 5. On day 8, baseline ear thickness was measured using calipers, and mice were challenged by intradermal ear injection of KLH. After 24 hours, the change in ear thickness was assessed and compared to baseline measurements. The Prevotella smEV and the smEV from another bacterial strain were used at 2E9 particles/dose. Other bacterial strains smEV reduce inflammation in DTH models based on them Is selected for study.

Results and conclusions: the results are shown in fig. 7. Treatment with dexamethasone reduced inflammation in the subsequent DTH response. Administration of a prasuvorexa smEV or a smEV from another bacterial strain prior to immunization did not reduce inflammation subsequently induced by intradermal ear challenge. Post-immunization administration of a prasuvorexa smEV or a smEV from another bacterial strain resulted in a reduction in post-challenge ear inflammation. These data show that administration of smEV in the absence of inflammation does not suppress the immune response, but rather regresses the persistent inflammatory response.

Example 15: antigen-specific response during inflammation induction was followed for resolution by Prevotella smEV Inflammation in KLH DTH reaction is not essential

Complete Freund's Adjuvant (CFA), which contains a TLR agonist and heat-inactivated Mycobacterium tuberculosis, and Incomplete Freund's Adjuvant (IFA), which contains only a TLR agonist, have been shown to drive Th1 and Th2 immune responses, respectively. In addition, IFA-PBS immunization induced an inflammatory response (absence of any antigen).

Post-inflammatory induction administration is sufficient to induce inflammation resolution in subsequent KLH DTH challenge : on day-9, the "PBS-IFA" and "PBS-CFA" mice were immunized by subcutaneous injection of PBS emulsified with complete or incomplete Freund's adjuvant (CFA or IFA, respectively). On day-5, mice were dosed daily with PBS vehicle PO or prasuvorexa smEV PO for 4 days. On day 0, mice were immunized with KLH emulsified with complete freund's adjuvant, but no smEV was administered after KLH-CFA immunization. On day 5, baseline ear thickness was measured using calipers, and mice were challenged by intradermal ear injection of KLH. After 24 hours, the change in ear thickness was assessed and compared to baseline measurements. Prevotella smEV was used at 2E9 particles/dose.

Results and conclusions: the results are shown in fig. 8. Administration of smEV only during inflammation induction with CFA or IFA prior to KLH sensitization can inhibit KLH DTH response after ear challenge. These data indicate that in the absence of the relevant antigen used in DTH (KLH), induction of systemic inflammation is sufficient to play a role in KLH DTH and is therefore an antigen-independent mechanism that can reduce antigen-specific inflammation.

Example 16: adoptive transfer of CD4+ T cells from KLH-CFA immunized mice from administered Prevotella smEV Immune resolution was conferred in recipient KLH-CFA immunized mice.

Transfer of CD4+ T cells adoptive cells from immunized mice orally administered with KLH-CFA to KLH-DTH immunoreceptor In mice: on day 0, "donor" mice were immunized by subcutaneous injection of KLH emulsified with complete freund's adjuvant. On day 5, the "recipient mice" were immunized by subcutaneous injection of KLH emulsified with complete freund's adjuvant, and the "donor mice" were dosed daily with PBS vehicle or prasuvorexa smEV or smEV PO from another strain for 4 days. On day 9, the humerus, axillary and inguinal lymph nodes and spleen were harvested from donor mice and enriched for cd4+ T cells by negative selection on magnetic beads. The enriched cells were then counted, washed with PBS (300 Xg, 10 min, 4 ℃) and washed at 5X 10 ⁷ Individual cells/mL were resuspended in PBS. After enrichment, 5X 10 was injected by IP ⁶ The cd4+ T cells were transferred into "recipient mice". On day 12, baseline ear thickness was measured using calipers, and then "recipient" mice were challenged by intradermal injection of KLH. After 24 hours, the change in ear thickness was assessed and compared to baseline measurements. The Prevotella smEV and the smEV from another bacterial strain were used at 2E9 particles/dose. Other bacterial strains smEV were selected for investigation based on their ability to reduce inflammation in the DTH model.

Results and conclusions: the results are shown in fig. 9. CD4+ T cells from mice treated with a smeV of Prevotella and from another bacterial strain were able to suppress the DTH response in recipient mice not treated with a smeV, CD4+ T cells from donor treated with PBS vehicle were ineffective in recipient mice. These data indicate that orally administered prasuvorexa smEV or a smEV from another bacterial strain produces a cd4+ T cell population that can resolve inflammation in inflamed mice that do not receive orally administered prasuvorexa smEV or a smEV from another bacterial strain.

Example 17: during oral administration of Prevotella smEV, TLR2 signaling is indicated for adoptable transfer to mediate Production of immunoresolved CD4+ T cells in the receptor KLH DTH model is essential

The small intestine contains a large number of Toll-like receptors (TLRs) that recognize a variety of microbial components. We have previously shown that oral administration of prasuvorexa smEV can produce a cd4+ T cell population that mediates immune regression upon adoptive transfer to the KLH DTH model.

Transfer of CD4+ T cell adoptive cells from orally administered KLH-CFA immunized mice treated with anti-TLR 2 In KLH-DTH immunoreceptor mice: on day 0, "donor mice" were immunized by subcutaneous injection of KLH emulsified with complete freund's adjuvant and IP treated with anti-TLR 2 or rat IgG2a isotype control antibodies (2 μg/mouse) on days 0, 3 and 6. On day 5, "recipient mice" were immunized by subcutaneous injection of KLH emulsified with complete freund's adjuvant, and "donor mice" were dosed daily with PBS vehicle or prasuvorexa smEV PO for 4 days. On day 9, the humerus, axillary and inguinal lymph nodes and spleen were harvested from donor mice and enriched for cd4+ T cells by negative selection on magnetic beads. The enriched cells were then counted, washed with PBS (300 Xg, 10 min, 4 ℃) and washed at 5X 10 ⁷ Individual cells/mL were resuspended in PBS. After enrichment, 5X 10 was injected by IP ⁶ The cd4+ T cells were transferred into "recipient mice". On day 12, baseline ear thickness was measured using calipers, and then "recipient" mice were challenged by intradermal injection of KLH. After 24 hours, the change in ear thickness was assessed and compared to baseline measurements. Use of Prevotella at 2E9 particles/dosesmEV。

Results and conclusions: the results are shown in fig. 10. CD4+ T cells isolated from mice treated with PBS vehicle and anti-TLR 2 blocking antibody did not inhibit DTH response to KLH compared to CD4+ T cells from mice treated with PBS alone or PBS with isotype control antibody. Cd4+ T cells from mice treated with prasuvorexa smEV and isotype control antibodies were potent in this model and were able to suppress ear inflammation following intradermal KLH challenge, as previously shown. However, in the presence of anti-TLR 2 antibodies, cd4+ T cells transferred from mice administered with prasuvorexa smEV were ineffective in recipient mice, and ear swelling was comparable to that of PBS-treated cd4+ T cells. These data indicate that TLR2 signaling is necessary for the generation of prevotella EV-induced immune-resolved cd4+ T cells, as antibody-mediated blockade of this receptor in donor mice abrogates the efficacy of cd4+ T cells transferred from the prevotella EV-dosed mice.

Example 18: adoptive transfer of CD8+ T cells from KLH-CFA immunized mice from administered Prevotella smEV Non-conferring immune resolution in recipient KLH-CFA immunized mice

We have previously shown that adoptive transfer of cd4+ T cells from mice administered with prasuvorexant EV has the ability to resolve inflammation in KLH-DTH receptors.

Transfer of CD8+ T cells adoptive cells from immunized mice orally administered with KLH-CFA to KLH-DTH immunoreceptor mice In mice: on day 0, "donor" mice were immunized by subcutaneous injection of KLH emulsified with complete freund's adjuvant. On day 5, the "recipient mice" were immunized by subcutaneous injection of KLH emulsified with complete freund's adjuvant, and the "donor mice" QD were dosed with PBS or prasuvorexa smEV or smEV PO from another bacterial strain for 4 days. On day 9, the humerus, axillary and inguinal lymph nodes and spleen were harvested from donor mice and enriched for cd4+ T cells by negative selection on magnetic beads. ThenThe enriched cells were counted, washed with PBS (300 Xg, 10 min, 4 ℃) and washed at 5X 10 ⁷ Individual cells/mL were resuspended in PBS. After enrichment, 5X 10 was injected by IP ⁶ The cd8+ T cells were transferred into "recipient mice". On day 12, baseline ear thickness was measured using calipers, and then "recipient" mice were challenged by intradermal injection of KLH. After 24 hours, the change in ear thickness was assessed and compared to baseline measurements. The Prevotella smEV and the smEV from another bacterial strain were used at 2E9 particles/dose. Other bacterial strains smEV were selected for investigation based on their ability to reduce inflammation in the DTH model.

Results and conclusions: the results are shown in fig. 11. Unlike cd4+ T cells, transfer of cd8+ T cells from donor mice dosed with a prasuvorexa smEV and a smEV from another bacterial strain to KLH-CFA inflamed recipient mice did not confer immune regression of the KLH DTH model in the recipient mice. These data indicate that oral administration of a prasugrel smEV and a smEV from another bacterial strain induces an immunoregressing cd4+ T cell population, but not a cd8+ T cell population, that can inhibit the DTH response in recipient mice.

Example 19: preparation of solid dosage forms comprising Prevotella denticola smEV

Tablets of the formulation in table S were prepared:

table S: composition of active tablet (400 mg)

The Prevotella denticola smEV in Table S is from Prevotella denticola strain B50329 (NRRL accession number B50329).

The drug substance (powder) was prepared by lyophilization using excipient formulation 7 a. HSDS: high-strength crude drug. LSDS: low-strength raw materials. LSDS was prepared by diluting HSDS10x (using lyophilization excipients) prior to lyophilization.

To prepare a pharmaceutical composition tablet, a drug substance (pharmaceutical formulation) containing smEV was wet granulated. Mixing the crude drug (i) with water; (ii) drying on a fluid bed dryer; (iii) milling; (iv) then blending with pharmaceutical excipients provided in table S.

The tablet was 5.5mm x 15.8mm.

Example 20: preparation of solid dosage forms comprising Prevotella denticola smEV

Capsules of the formulation in table T were prepared:

table T: composition of active capsules

The Prevotella denticola smEV in Table T is from Prevotella denticola strain B50329 (NRRL accession number B50329).

The drug substance (powder) was prepared by lyophilization using excipient formulation 7 a. HSDS: high-strength crude drug. LSDS: low-strength raw materials. LSDS was prepared by diluting HSDS10x (using lyophilization excipients) prior to lyophilization. HSDS: high-strength crude drug. LSDS: low-strength raw materials.

LSDS was prepared by diluting HSDS10x (using lyophilization excipients) prior to lyophilization.

To prepare pharmaceutical composition capsules, a drug substance (pharmaceutical formulation) containing smEV was wet granulated. Mixing the crude drug (i) with water; (ii) drying on a fluid bed dryer; (iii) milling; (iv) then blending with pharmaceutical excipients provided in table T.

The capsule size was No. 0.

Example 21: testing of crude drugs

The Prevotella denticola smEV used for preparing the Drug Substance (DS) is derived from Prevotella denticola strain B50329 (NRRL accession number B50329).

The drug substance (powder) was prepared by lyophilization using excipient formulation 7a (80% mannitol; 20% trehalose). HSDS: high-strength crude drug. LSDS: low-strength raw materials. LSDS was prepared by diluting HSDS10x (using lyophilization excipients) prior to lyophilization.

HSDS and LSDS were evaluated in vitro assays performed in human Peripheral Blood Mononuclear Cells (PBMC), macrophages and dendritic cells. Obtaining cells from five donors; five donors were run repeatedly as organisms and data were expressed as average responses. Serial dilution was performed: evaluation 1X10 ³ 、1x10 ⁴ 、1x10 ⁵ 、1x10 ⁶ 、1x10 ⁷ 、1x10 ⁸ 、1x10 ⁹ And 1x10 ¹⁰ Effect of individual particles of HSDS and LSDS on cytokine secretion by PBMC, macrophages and dendritic cells. Similar trends were observed in all three cell populations. HSDS and LSDS are capable of inducing IL-10, IL-27, IL-6, IP-10 and TNFa from all three cell populations. Dendritic cells respond to both DS, producing low levels of IL-27 and IP-10.

HSDS and LSDS were evaluated in an in vitro assay performed in U937. Serial dilution was performed: evaluation 7.06x10 ³ 、2.12x10 ⁴ 、6.35x10 ⁴ 、1.91x10 ⁵ 、5.72x10 ⁵ 、1.71x10 ⁶ 、5.14x10 ⁶ 、1.54x10 ⁷ 、4.63x10 ⁷ 、1.39x10 ⁸ 、4.17x10 ⁸ And 1.25x10 ⁹ Effects of individual particles of HS DS and LS DS on cytokine secretion by U937 cells. HS DS and LS DS are capable of inducing IL-10, IL-27, IL-6, IP-10 and TNFa from U937 cells.

Incorporated by reference

All publications, patent applications, and articles 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 the event of a conflict, the present application, including any definitions herein, will control.

Equivalent forms

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 application described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method of activating TLR2 in a subject, the method comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasugrel strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising the extracellular vesicles.

2. A method of activating an anti-inflammatory cytokine response in a subject, the method comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of Extracellular Vesicles (EVs) from a prasugrel bacterial strain of tissue and/or compositions (e.g., solutions, dried forms, and/or therapeutic compositions) comprising the extracellular vesicles.

3. The method of claim 1 or 2, wherein the extracellular vesicles are from a prasuvorexa strain of tissue having 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) with the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of prasuvorexa strain B of tissue (NRRL accession No. B50329).

4. The method of any one of claims 1 to 3, wherein the prasuvorexa strain of tissue is prasuvorexa strain B of tissue (NRRL accession No. B50329).

5. The method of any one of claims 1-4, wherein the subject has an immune disorder.

6. The method according to claim 5, wherein the method comprises, wherein the immune disorder is joint sclerosis, arthritis, phlebitis, vasculitis and lymphangitis, cholangitis, cholecystitis, enteritis, enterocolitis, gastritis, gastroenteritis, inflammatory bowel disease, ileitis, proctitis, crohn's disease, ulcerative colitis, irritable bowel syndrome, microscopic colitis, lymphocytic-plasmacytoid enteritis, celiac disease, collagenous colitis, lymphocytic colitis, eosinophilic enterocolitis, indeterminate colitis, pseudomembranous colitis (necrotizing colitis), ischemic inflammatory bowel disease, behcet's disease, sarcoidosis, scleroderma, IBD-related dysplasia, dysplasia-related mass or lesions, primary sclerosing cholangitis, cervicitis, chorioamniosis, endometritis, epididymitis, navel inflammation, oophoritis, orchitis salpingitis, salpingemphraxis, urethritis, vaginitis, vulvitis, vulvodynia, acute disseminated alopecia universalis, behcet's disease, gauss's disease, chronic fatigue syndrome, autonomic imbalance, encephalomyelitis, ankylosing spondylitis, aplastic anemia, suppurative sweat gland inflammation, autoimmune hepatitis, autoimmune ovaritis, celiac disease, type 1 diabetes mellitus, giant cell arteritis, goldpasture's syndrome, graves ' disease, grin-Bali syndrome, hashimoto's disease, hun-Schoendo's purple, kawasaki disease, lupus erythematosus, microscopic colitis, polyarteritis under microscope, mixed connective tissue disease, murray-Wer's syndrome, multiple sclerosis, myasthenia gravis, strabismus myoclonus syndrome, optic neuritis, orde thyroiditis, multiple sclerosis, myasthenia gravis, pemphigus, polyarteritis nodosa, polymyalgia, rheumatoid arthritis, lattter's syndrome, sjogren's syndrome, temporal arteritis, wegener's granulomatosis, warm autoimmune hemolytic anemia, interstitial cystitis, lyme disease, scleroderma, psoriasis, sarcoidosis, scleroderma, contact hypersensitivity, contact dermatitis (including contact dermatitis due to Pueraria lobata), urticaria, skin allergy, airway allergy (hay fever, allergic rhinitis, house dust mite allergy), appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, suppurative sweat gland, iritis, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis, pleurisy, pneumonia, prostatitis, pyelonephritis, stomatitis, peritonitis, allergic rhinitis, allergic dermatitis, and the like graft rejection, acute pancreatitis, chronic pancreatitis, acute respiratory distress syndrome, cerclash syndrome, congenital adrenal hyperplasia, non-suppurative thyroiditis, cancer-related hypercalcemia, pemphigus, bullous dermatitis herpetiformis, severe erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, seasonal or perennial allergic rhinitis, bronchial asthma, contact dermatitis, atopic dermatitis, drug hypersensitivity, allergic conjunctivitis, keratitis, ocular shingles, iritis and iridocyclitis, chorioretinitis, optic neuritis, symptomatic sarcoidosis, fulminant or disseminated tuberculosis chemotherapy, adult idiopathic thrombocytopenic purpura, adult secondary thrombocytopenia, acquired (autoimmune) hemolytic anemia, adult leukemia and lymphoma, acute leukemia, regional enteritis, autoimmune vasculitis, chronic obstructive pulmonary disease, or sepsis in children.

7. The method of claim 6, wherein the subject has psoriasis.

8. The method of claim 6, wherein the subject has atopic dermatitis.

9. The method of any one of claims 1-4, wherein the subject has a Th 1-mediated inflammatory disease.

10. The method of any one of claims 1-4, wherein the subject has a Th2 mediated inflammatory disease.

11. The method of any one of claims 1-4, wherein the subject has a Th 17-mediated inflammatory disease.

12. The method of any one of claims 1-11, wherein the EVs are administered orally.

13. The method of any one of claims 1 to 12, wherein the dose is in the form of one or more capsules, optionally comprising an enteric coating (e.g., an enteric coated capsule).

14. The method of any one of claims 1 to 12, wherein the dose is in the form of one or more tablets, optionally comprising an enteric coating (e.g., an enteric coated tablet).

15. The method of any one of claims 1 to 12, wherein the dose is in the form of one or more miniature tablets.

16. The method of claim 15, wherein the miniature tablets are enteric coated miniature tablets.

17. The method of any one of claims 1 to 12, wherein the dose is in the form of a non-enteric coated capsule comprising enteric coated minitablets.

18. The method of any one of claims 1 to 17, wherein the dose is administered in combination with an additional therapeutic agent.

19. A solution comprising Extracellular Vesicles (EVs) from a strain of prevotella histolytica and an excipient comprising a bulking agent.

20. A therapeutic composition comprising the solution of claim 19, wherein the composition further comprises a pharmaceutically acceptable excipient.

21. The therapeutic composition of claim 20, wherein the pharmaceutically acceptable excipient comprises a glidant, a lubricant, and/or a diluent.

22. A dry form comprising Extracellular Vesicles (EVs) from a prasugrel strain of tissue, and an excipient comprising a bulking agent.

23. A therapeutic composition comprising the dry form of claim 22, wherein the composition further comprises a pharmaceutically acceptable excipient.

24. The therapeutic composition of claim 23, wherein the pharmaceutically acceptable excipient comprises a glidant, a lubricant, and/or a diluent.

25. A therapeutic composition comprising Extracellular Vesicles (EVs) from a prasugrel strain of tissue, and an excipient comprising a bulking agent.

26. A solution comprising Extracellular Vesicles (EVs) from a prasugrel strain of tissue and an excipient comprising a stock of one or more excipients, wherein the stock comprises the formulation provided in table A, B, C, D, K or P.

27. A therapeutic composition comprising the solution of claim 26, wherein the composition further comprises a pharmaceutically acceptable excipient.

28. The therapeutic composition of claim 27, wherein the pharmaceutically acceptable excipient comprises a glidant, a lubricant, and/or a diluent.

29. A dried form comprising Extracellular Vesicles (EVs) from a prasugrel strain of tissue and an excipient comprising a stock of one or more excipients, wherein the stock comprises the formulation provided in table A, B, C, D, K or P.

30. A therapeutic composition comprising the dry form of claim 29, wherein the composition further comprises a pharmaceutically acceptable excipient.

31. The therapeutic composition of claim 30, wherein the pharmaceutically acceptable excipient comprises a glidant, a lubricant, and/or a diluent.

32. A method of treating a subject (e.g., a subject in need of treatment), the method comprising:

administering to the subject the solution, dry form, or therapeutic composition of any one of claims 19 to 31 (e.g., a therapeutically effective amount thereof).

33. The solution, powder, or therapeutic composition of any one of claims 19 to 31 (e.g., a therapeutically effective amount thereof) for use in treating a subject (e.g., a subject in need of treatment).

34. Use of the solution, dry form, or therapeutic composition (e.g., a therapeutically effective amount thereof) of any one of claims 19 to 31 for the manufacture of a medicament for treating a subject (e.g., a human) (e.g., a subject in need of treatment).

35. The method/solution/powder/therapeutic composition/use of any one of claims 32 to 34, wherein the solution/powder/therapeutic composition is administered orally (e.g., for oral administration).

36. The method/solution/powder/therapeutic composition/use of any one of claims 32-35, wherein the subject has an immune disorder.

37. The method/solution/powder/therapeutic composition/use of claim 36, wherein the immune disorder is selected from the group consisting of arthritis, phlebitis, vasculitis, and lymphangitis, cholangitis, cholecystitis, enteritis, enterocolitis, gastritis, gastroenteritis, inflammatory bowel disease, ileitis, proctitis, crohn's disease, ulcerative colitis, irritable bowel syndrome, microscopic colitis, lymphocytic-plasma cell enteritis, celiac disease, collagenous colitis, inflammatory bowel disease, crohn's disease, ulcerative colitis, crohn's disease, crohn' lymphocytic colitis, eosinophilic enterocolitis, non-established colitis, pseudomembranous colitis (necrotizing colitis), ischemic inflammatory bowel disease, behcet's disease, sarcoidosis, scleroderma, IBD-related dysplasia, dysplasia-related mass or lesion, primary sclerosing cholangitis, cervicitis, chorioamnion, endometritis, epididymitis navel inflammation, oophoritis, orchitis, salpingitis, ovary abscess, urethritis, colpitis, vulvitis, vulvodynia, acute disseminated alopecia universalis, behcet's disease, chagas's disease, chronic fatigue syndrome, autonomic imbalance, encephalomyelitis, ankylosing spondylitis, aplastic anemia, suppurative sweat gland, autoimmune hepatitis, autoimmune oophoritis, celiac disease, type 1 diabetes, giant cell arteritis, godpasture's syndrome, graves 'disease, grin-Bali syndrome, hashimoto's disease, hun-Schenen's purpura, kawasaki disease, lupus erythematosus, microscopic colitis, microscopic polyarteritis, mixed connective tissue disease, muwei's syndrome, multiple sclerosis, myasthenia gravis, strabismus-eye myoclonus syndrome, optic neuritis, aldehydic thyroiditis, pemphigus, polyarteritis nodosa, polymyalgia, rheumatoid arthritis, lat's syndrome, sjogren's syndrome, temporal arteritis, wegener's granulomatosis, warm autoimmune hemolytic anemia, interstitial cystitis, lyme disease, scleroderma, psoriasis, sarcoidosis, scleroderma, contact hypersensitivity, contact dermatitis (including contact dermatitis due to Pueraria lobata), urticaria, skin allergies, respiratory allergies (hay fever, allergic rhinitis, house dust mite allergy), inflammation, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, suppurative sweat gland, iritis, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, pericarditis, peritonitis, ocular inflammation pleurisy, pneumonia, prostatitis, pyelonephritis, stomatitis, graft rejection, acute pancreatitis, chronic pancreatitis, acute respiratory distress syndrome, cerclash syndrome, congenital adrenal hyperplasia, non-suppurative thyroiditis, cancer-related hypercalcemia, pemphigus, bullous dermatitis herpetiformis, severe erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, seasonal or perennial allergic rhinitis, bronchial asthma, contact dermatitis, atopic dermatitis, drug hypersensitivity, allergic conjunctivitis, keratitis, ocular shingles, iritis and iridocyclitis, chorioretinitis, optic neuritis, symptomatic sarcoidosis, idiopathic or disseminated tuberculosis chemotherapy, adult idiopathic thrombocytopenic purpura, adult secondary thrombocytopenia, acquired (autoimmune) hemolytic anemia, adult leukemia and lymphoma, pediatric acute leukemia, regional enteritis, autoimmune vasculitis, chronic obstructive pulmonary disease, or sepsis.

38. The method/solution/powder/therapeutic composition/use of any one of claims 32-37, wherein the subject has psoriasis.

39. The method/solution/powder/therapeutic composition/use of any one of claims 32-37, wherein the subject has atopic dermatitis.

40. The method/solution/powder/therapeutic composition/use of any one of claims 32-35, wherein the subject has a Th1 mediated inflammatory disease.

41. The method/solution/powder/therapeutic composition/use of any one of claims 32-35, wherein the subject has a Th2 mediated inflammatory disease.

42. The method/solution/powder/therapeutic composition/use of any one of claims 32-35, wherein the subject has a Th 17-mediated inflammatory disease.

43. The method/solution/dry form/therapeutic composition/use of any one of claims 32 to 42, wherein the solution/powder/therapeutic composition is administered in combination with an additional therapeutic agent.

44. The method/solution/dry form/therapeutic composition/use of any one of claims 19 to 43, wherein the extracellular vesicles are from a prasuvorexa strain of tissue having 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 prasuvorexa strain B of tissue (NRRL accession No. B50329).

45. The method/solution/dry form/therapeutic composition/use of any one of claims 19 to 44, wherein the prasuvorexa tissue strain is prasuvorexa tissue strain B (NRRL accession No. B50329).

46. The method of any one of claims 1-18 or 32-44, wherein the subject is a human or non-human mammal.