WO2023114300A1 - Préparations de vésicules extracellulaires de bactéries fournierella massiliensis - Google Patents

Préparations de vésicules extracellulaires de bactéries fournierella massiliensis Download PDF

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Publication number
WO2023114300A1
WO2023114300A1 PCT/US2022/052846 US2022052846W WO2023114300A1 WO 2023114300 A1 WO2023114300 A1 WO 2023114300A1 US 2022052846 W US2022052846 W US 2022052846W WO 2023114300 A1 WO2023114300 A1 WO 2023114300A1
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Prior art keywords
evs
massiliensis
solution
excipient
dried form
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PCT/US2022/052846
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English (en)
Inventor
Derek DORMAN
Collin MCKENNA
Bill Wang
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Evelo Biosciences, Inc.
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Publication of WO2023114300A1 publication Critical patent/WO2023114300A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • A61K9/1623—Sugars or sugar alcohols, e.g. lactose; Derivatives thereof; Homeopathic globules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions

Definitions

  • compositions comprising extracellular vesicles (EVs), such as EVs obtained from bacteria, have therapeutic effects and are useful for the treatment and/or prevention of disease and/or health disorders.
  • EVs extracellular vesicles
  • the compositions comprise extracellular vesicles obtained from Fournierella massiliensis strain A (ATCC Deposit Number PTA-126696).
  • F. massiliensis EVs are prepared as solutions and as dried forms.
  • the solutions and dried forms are for use in preparing therapeutic compositions comprising F. massiliensis EVs.
  • the dried forms comprising F. massiliensis EVs (for example, prepared using the excipients and/or methods described herein) have a moisture content of below about 6% upon completion of drying. In some embodiments, dried forms having a moisture content below about 6% are better suited for downstream processing. In some embodiments, dried forms having a moisture content below about 6% have improved stability. In some embodiments, the solutions comprising the F.
  • the massiliensis EVs also comprise an excipient that contains a bulking agent, and optionally comprises one or more additional ingredients, such as a lyoprotectant.
  • the solutions comprising the F. massiliensis EVs also comprise an excipient that contains a lyoprotectant, and optionally comprises one or more additional ingredients, such as a bulking agent.
  • the dried forms comprising the F. massiliensis EVs also comprise an excipient that contains a bulking agent, and that optionally comprises one or more additional ingredients, such as a lyoprotectant.
  • the dried forms comprising the F. massiliensis EVs also comprise an excipient that contains a lyoprotectant, and optionally comprise one or more additional ingredients, such as a bulking agent.
  • Bulking agents and/or lyoprotectants are used when preparing the F. massiliensis EVs for drying, such as freeze drying and spray drying.
  • bulking agents including but not limited to sucrose, mannitol, polyethylene glycol (PEG, such as PEG 6000), cyclodextrin, maltodextrin, and dextran (such as dextran 40k), make dried forms (such as powders and/or lyophilates) easier to handle after drying.
  • bulking agents improve the properties of a dried form.
  • lyoprotectants including but not limited to trehalose, sucrose, and lactose protect the EVs during drying, such as freeze-drying or spray drying.
  • the excipient functions to decrease drying cycle time. In some embodiments, the excipient functions to maintain therapeutic efficacy of the EVs.
  • F. massiliensis EVs have therapeutic effects and are useful for the treatment and/or prevention of disease and/or health disorders.
  • therapeutic compositions of the solutions and dried forms containing F. massiliensis EVs are prepared.
  • the disclosure provides a lyophilate comprising F. massiliensis EVs, wherein the lyophilate has a moisture content (e.g., as determined by the Karl Fischer method) of below about 6%.
  • the lyophilate has a moisture content (e.g., as determined by the Karl Fischer method) of below about 5%.
  • the lyophilate has a moisture content (e.g., as determined by the Karl Fischer method) of below about 4%.
  • the lyophilate has a moisture content (e.g., as determined by the Karl Fischer method) of between about 1% to about 4%.
  • the lyophilate has a moisture content (e.g., as determined by the Karl Fischer method) of between about 2% to about 3%.
  • the lyophilate has a moisture content (e.g., as determined by the Karl Fischer method) of between about 1.5% to about 7%.
  • the disclosure provides a lyophilate comprising F. massiliensis EVs, wherein the lyophilate has a particle numeration of about 6.24e9 to about 2.89el0 particles/mg lyophilate, e.g., after the lyophilate (for example, lyophilized powder) is resuspended, such as in deionized water.
  • the disclosure provides a lyophilate comprising F.
  • massiliensis EVs wherein the particles have a charge of about -32 to about -25.3 mV, as measured by DLS of the charge of the most dominant DLS integrated peak of particles, e.g., after the lyophilate (for example, lyophilized powder) is resuspended, such as in deionized water or in a buffer such as PBS (for example, 0. IX PBS).
  • lyophilate for example, lyophilized powder
  • PBS for example, 0. IX PBS
  • the disclosure provides a lyophilate comprising F. massiliensis EVs, wherein the particles have a hydrodynamic diameter (Z average, Zave) of about 132 nm to about 315.2 nm, e.g., after the lyophilate (for example, lyophilized powder) is resuspended, such as in deionized water or in a buffer such as PBS (for example, 0.1X PBS).
  • lyophilate for example, lyophilized powder
  • PBS for example, 0.1X PBS
  • the disclosure provides a lyophilate comprising F. massiliensis EVs, wherein the particles have a mean size of the most dominant DLS integrated peak of between about 43.72 nm to about 79.18 nm, e.g., after the lyophilate (for example, lyophilized powder) is resuspended, such as in deionized water or in a buffer such as PBS (for example, 0.1X PBS).
  • lyophilate for example, lyophilized powder
  • PBS for example, 0.1X PBS
  • the disclosure provides a lyophilate comprising F. massiliensis EVs bacteria and an excipient, wherein the excipient comprises about 95% to about 99% of the total mass of the lyophilate.
  • the disclosure provides a lyophilate comprising F. massiliensis EVs and an excipient, wherein the EVs comprise about 2% to about 6% of the total mass of the lyophilate.
  • the lyophilate comprises a lyophilized powder.
  • the lyophilate comprises a lyophilized cake.
  • the disclosure provides a powder comprising F. massiliensis EVs, wherein the powder has a moisture content (e.g., as determined by the Karl Fischer method) of below about 6%.
  • the powder has a moisture content (e.g., as determined by the Karl Fischer method) of below about 5%.
  • the powder has a moisture content (e.g., as determined by the Karl Fischer method) of below about 4%. [23] In some embodiments, the powder has a moisture content (e.g., as determined by the Karl Fischer method) of between about 1% to about 4%.
  • the powder has a moisture content (e.g., as determined by the Karl Fischer method) of between about 2% to about 3%.
  • the powder has a moisture content (e.g., as determined by the Karl Fischer method) of between about 1.5% to about 7%.
  • the disclosure provides a powder comprising F. massiliensis EVs, wherein the powder has a particle numeration of about 6.24e9 to about 2.89el0 particles/mg powder, e.g., after the powder is resuspended, such as in deionized water.
  • the disclosure provides a powder comprising F. massiliensis EVs, wherein the particles have a charge of about -32 to about -25.3 mV, as measured by DLS of the charge of the most dominant DLS integrated peak of particles, e.g., after the powder is resuspended, such as in deionized water or in a buffer such as PBS (for example, 0. IX PBS).
  • a powder comprising F. massiliensis EVs, wherein the particles have a charge of about -32 to about -25.3 mV, as measured by DLS of the charge of the most dominant DLS integrated peak of particles, e.g., after the powder is resuspended, such as in deionized water or in a buffer such as PBS (for example, 0. IX PBS).
  • the disclosure provides a powder comprising F. massiliensis EVs, wherein the particles have a hydrodynamic diameter (Z average, Zave) of about 132 nm to about 315.2 nm, e.g., after the powder is resuspended, such as in deionized water or in a buffer such as PBS (for example, 0. IX PBS).
  • Z average, Zave hydrodynamic diameter
  • the disclosure provides a powder comprising F. massiliensis EVs, wherein the particles have a mean size of the most dominant DLS integrated peak of between about 43.72 nm to about 79.18 nm, e.g., after the powder is resuspended, such as in deionized water or in a buffer such as PBS (for example, 0. IX PBS).
  • a buffer such as PBS (for example, 0. IX PBS).
  • the disclosure provides a powder comprising F. massiliensis EVs and an excipient, wherein the excipient comprises about 95% to about 99% of the total mass of the powder.
  • the disclosure provides a powder comprising F. massiliensis EVs and an excipient, wherein the EVs comprise about 2% to about 6% of the total mass of the powder.
  • the powder comprises a lyophilized powder.
  • the powder comprises a spray-dried powder.
  • the disclosure provides a dried form comprising F. massiliensis EVs, wherein the dried form has a moisture content (e.g., as determined by the Karl Fischer method) of below about 6%.
  • the dried form provided herein has a moisture content (e.g., as determined by the Karl Fischer method) of below about 5%.
  • the dried form provided herein has a moisture content (e.g., as determined by the Karl Fischer method) of below about 4%.
  • the dried form provided herein has a moisture content (e.g., as determined by the Karl Fischer method) of between about 1% to about 4%.
  • the dried form provided herein has a moisture content (e.g., as determined by the Karl Fischer method) of between about 2% to about 3%.
  • the dried form has a moisture content (e.g., as determined by the Karl Fischer method) of between about 1.5% to about 7%.
  • the disclosure provides a dried form comprising F. massiliensis EVs, wherein the dried form has a particle numeration of about 6.24e9 to about 2.89el0 particles/mg dried form, e.g., after the dried form is resuspended, such as in deionized water.
  • the disclosure provides a dried form comprising F. massiliensis EVs, wherein the particles have a charge of about -32 to about -25.3 mV, as measured by DLS of the charge of the most dominant DLS integrated peak of particles, e.g., after the dried form is resuspended, such as in deionized water or in a buffer such as PBS (for example, 0. IX PBS).
  • a buffer such as PBS (for example, 0. IX PBS).
  • the disclosure provides a dried form comprising F. massiliensis EVs, wherein the particles have a hydrodynamic diameter (Z average, Zave) of about 132 nm to about 315.2 nm, e.g., after the dried form is resuspended, such as in deionized water or in a buffer such as PBS (for example, 0.1X PBS).
  • Z average, Zave hydrodynamic diameter
  • the disclosure provides a dried form comprising F. massiliensis EVs, wherein the particles have a mean size of the most dominant DLS integrated peak of between about 43.72 nm to about 79.18 nm, e.g., after the dried form is resuspended, such as in deionized water or in a buffer such as PBS (for example, 0. IX PBS).
  • a buffer such as PBS (for example, 0. IX PBS).
  • the disclosure provides a dried form comprising F. massiliensis EVs and an excipient, wherein the excipient comprises about 95% to about 99% of the total mass of the dried form.
  • the disclosure provides a dried form comprising F. massiliensis EVs and an excipient, wherein the EVs comprise about 2% to about 6% of the total mass of the dried form.
  • the dried form comprises a powder.
  • the powder comprises a lyophilized powder.
  • the powder comprises a spray-dried powder.
  • the dried form comprises a lyophilate.
  • the lyophilate comprises a lyophilized powder.
  • the lyophilate comprises a lyophilized cake.
  • the disclosure provides a solution comprising F. massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a solution consisting essentially of F. massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a solution comprising F. massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a solution consisting essentially of F. massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a solution comprising F. massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a solution consisting essentially of F. massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a therapeutic composition comprising the solution, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a dried form comprising F. massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a dried form consisting essentially of F. massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a dried form comprising F. massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a dried form consisting essentially of F. massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant. [59] In some aspects, the disclosure provides a dried form comprising F. massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a dried form consisting essentially of F. massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a therapeutic composition comprising the dried form, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a powder comprising F. massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a powder consisting essentially of F. massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a powder comprising F. massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a powder consisting essentially of F. massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a powder comprising F. massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a powder consisting essentially of F. massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a therapeutic composition comprising the powder, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a spray-dried powder comprising F. massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a spray-dried powder consisting essentially of F. massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a spray-dried powder comprising F. massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a spray-dried powder consisting essentially of F. massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a spray-dried powder comprising F. massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a spray-dried powder consisting essentially of F. massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a therapeutic composition comprising the spray-dried powder, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilate comprising F. massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a lyophilate consisting essentially of F. massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a lyophilate comprising F. massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a lyophilate consisting essentially of F. massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a lyophilate comprising F. massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a lyophilate consisting essentially of F. massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a therapeutic composition comprising the lyophilate, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilized powder comprising F. massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a lyophilized powder consisting essentially of F. massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a lyophilized powder comprising F. massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a lyophilized powder consisting essentially of F. massiliensis EVs and from an excipient that comprises a bulking agent and a lyoprotectant. [87] In some aspects, the disclosure provides a lyophilized powder comprising F. massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a lyophilized powder consisting essentially of F. massiliensis EVs and from an excipient that comprises a lyoprotectant.
  • the disclosure provides a therapeutic composition comprising the lyophilized powder, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilized cake comprising F. massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a lyophilized cake consisting essentially of F. massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a lyophilized cake comprising F. massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a lyophilized cake consisting essentially of F. massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a lyophilized cake comprising F. massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a lyophilized cake consisting essentially of F. massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a therapeutic composition comprising F. massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a therapeutic composition consisting essentially of F. massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a therapeutic composition comprising F. massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a therapeutic composition consisting essentially of F. massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a therapeutic composition comprising F. massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a therapeutic composition consisting essentially of F. massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a solution comprising F. massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F.
  • the disclosure provides a solution consisting essentially of F. massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F.
  • the disclosure provides a therapeutic composition comprising such solution, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a dried form comprising F. massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F.
  • the disclosure provides a dried form consisting essentially of F. massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F.
  • the disclosure provides a therapeutic composition comprising such dried form, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a powder comprising F. massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F.
  • the disclosure provides a powder consisting essentially of F. massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F.
  • the disclosure provides a therapeutic composition comprising such powder, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a spray-dried powder comprising F. massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F.
  • the disclosure provides a spray-dried powder consisting essentially of F. massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F.
  • the disclosure provides a therapeutic composition comprising such spray-dried powder, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilate comprising F. massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F.
  • the disclosure provides a lyophilate consisting essentially of F. massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F.
  • the disclosure provides a therapeutic composition comprising such lyophilate, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilized powder comprising F. massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F.
  • the disclosure provides a lyophilized powder consisting essentially of F. massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F.
  • the disclosure provides a therapeutic composition comprising such lyophilized powder, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilized cake comprising F. massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F.
  • the disclosure provides a lyophilized cake consisting essentially of F. massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F.
  • the disclosure provides a therapeutic composition comprising such lyophilized cake, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a method of treating a subject (for example, human) (for example, a subject in need of treatment), the method comprising:
  • a solution, dried form, or therapeutic composition provided herein is for use in treating a subject (for example, human) (for example, a subject in need of treatment).
  • the disclosure provides use of a solution, dried form, or therapeutic composition provided herein for the preparation of a medicament for treating a subject (for example, human) (for example, a subject in need of treatment).
  • solution, dried form, therapeutic composition, or use provided herein the solution, dried form, or therapeutic composition is orally administered (for example, is for oral administration).
  • compositions e.g. solutions, dried forms or therapeutic compositions descried herein are useful for inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell and/or a decrease in expression of a pro-inflammatory cytokine by an immune cell).
  • an immune effect e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell and/or a decrease in expression of a pro-inflammatory cytokine by an immune cell.
  • methods of using such F massiliensis EVs compositions e.g., for inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell)).
  • a method of inducing an immune effect in a subject comprising administering (e.g., orally, rectally, or vaginally) to the subject an effective amount of F. massiliensis EVs in a composition described herein.
  • administering e.g., orally, rectally, or vaginally
  • an effective amount of F. massiliensis EVs in a composition described herein is hereby incorporated by reference in its entirety.
  • the disclosure provides a method of treating a subject (for example, human) (for example, a subject in need of treatment), the method comprising:
  • a solution, dried form, or therapeutic composition provided herein is for use in treating a subject (for example, human) (for example, a subject in need of treatment).
  • the disclosure provides use of a solution, dried form, or therapeutic composition provided herein for the preparation of a medicament for treating a subject (for example, human) (for example, a subject in need of treatment).
  • solution, dried form, therapeutic composition, or use provided herein the solution, dried form, or therapeutic composition is orally administered (for example, is for oral administration).
  • the subject is in need of treatment (and/or prevention) of a cancer.
  • the subject is in need of treatment (and/or prevention) of an autoimmune disease.
  • the subject is in need of treatment (and/or prevention) of an inflammatory disease.
  • the subject is in need of treatment (and/or prevention) of a metabolic disease.
  • the subject is in need of treatment (and/or prevention) of dysbiosis.
  • solution, dried form, therapeutic composition, or use provided herein the solution, dried form, or therapeutic composition is administered in combination with an additional therapeutic agent.
  • the dried form is a powder.
  • the powder is a lyophilized powder.
  • the powder is a spray-dried powder.
  • the dried form is a lyophilate.
  • the lyophilate is a lyophilized powder.
  • the lyophilate is a lyophilized cake.
  • the disclosure provides a method of preparing a solution that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent, thereby preparing the solution.
  • the disclosure provides a method of preparing a solution that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant, thereby preparing the solution.
  • the disclosure provides a method of preparing a solution that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant, thereby preparing the solution.
  • the disclosure provides a solution prepared by a method described herein.
  • the disclosure provides a method of preparing a dried form that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; and drying the solution, thereby preparing the dried form.
  • the disclosure provides a method of preparing a dried form that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; drying the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the dried form.
  • the disclosure provides a method of preparing a dried form that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; and drying the solution, thereby preparing the dried form.
  • the disclosure provides a method of preparing a dried form that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; drying the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the dried form.
  • the disclosure provides a method of preparing a dried form that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; and drying the solution, thereby preparing the dried form.
  • the disclosure provides a method of preparing a dried form that comprises F. massiliensis EVs, the method comprising: combining a liquid preparationthat comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) lyoprotectant to prepare a solution; drying the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the dried form.
  • the drying comprises lyophilization.
  • the drying comprises spray drying.
  • the method further comprises combining the dried form with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a dried form prepared by a method described herein.
  • the disclosure provides a method of preparing a powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; and drying the solution, thereby preparing the powder.
  • the disclosure provides a method of preparing a powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; drying the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the powder.
  • the disclosure provides a method of preparing a powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; and drying the solution, thereby preparing the powder.
  • the disclosure provides a method of preparing a powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; drying the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the powder.
  • the disclosure provides a method of preparing a powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; and drying the solution, thereby preparing the powder.
  • the disclosure provides a method of preparing a powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; drying the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the powder.
  • the drying comprises lyophilization.
  • the drying comprises spray drying.
  • the method further comprises combining the powder with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a powder prepared by a method described herein.
  • the disclosure provides a method of preparing a spray-dried powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; and spray drying the solution, thereby preparing the spray-dried powder.
  • the disclosure provides a method of preparing a spray-dried powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; and spray drying the solution, thereby preparing the spray-dried powder.
  • the disclosure provides a method of preparing a spray-dried powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; and spray drying the solution, thereby preparing the spray-dried powder.
  • the method further comprises combining the spray-dried powder with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a spray-dried powder prepared by a method described herein.
  • the disclosure provides a method of preparing a lyophilate that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilate.
  • the disclosure provides a method of preparing a lyophilate that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; freeze drying (lyophilizing) the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the lyophilate.
  • the disclosure provides a method of preparing a lyophilate that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilate.
  • the disclosure provides a method of preparing a lyophilate that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; freeze drying (lyophilizing) the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the lyophilate.
  • the disclosure provides a method of preparing a lyophilate that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilate.
  • the disclosure provides a method of preparing a lyophilate that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; freeze drying (lyophilizing) the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the lyophilate.
  • the method further comprises combining the lyophilate with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilate prepared by a method described herein.
  • the disclosure provides a method of preparing a lyophilized powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilized powder.
  • the disclosure provides a method of preparing a lyophilized powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; freeze drying (lyophilizing) the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the lyophilized powder.
  • the disclosure provides a method of preparing a lyophilized powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilized powder.
  • the disclosure provides a method of preparing a lyophilized powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; freeze drying (lyophilizing) the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the lyophilized powder.
  • the disclosure provides a method of preparing a lyophilized powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilized powder.
  • the disclosure provides a method of preparing a lyophilized powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; freeze drying (lyophilizing) the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the lyophilized powder.
  • the method further comprises combining the lyophilized powder with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilized powder prepared by a method described herein.
  • the disclosure provides a method of preparing a lyophilized cake that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilized cake.
  • the disclosure provides a method of preparing a lyophilized cake that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilized cake.
  • the disclosure provides a method of preparing a lyophilized cake that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilized cake.
  • the disclosure provides a lyophilized cake prepared by a method described herein.
  • the disclosure provides a method of preparing a solution that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises composition comprising F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution.
  • the disclosure provides a solution prepared by a method described herein.
  • the disclosure provides a method of preparing a dried form that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises composition comprising F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; and drying the solution, thereby preparing the dried form.
  • the disclosure provides a method of preparing a dried form that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises composition comprising F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; drying the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the dried form.
  • the drying comprises lyophilization.
  • the drying comprises spray drying.
  • the method further comprises combining the dried form with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a dried form prepared by a method described herein.
  • the disclosure provides a method of preparing a powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; and drying the solution, thereby preparing the powder.
  • the disclosure provides a method of preparing a powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; drying the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the powder.
  • the drying comprises lyophilization.
  • the drying comprises spray drying.
  • the method further comprises combining the powder with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a powder prepared by a method described herein.
  • the disclosure provides a method of preparing a spray-dried powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; and spray drying the solution, thereby preparing the spray-dried powder.
  • the method further comprises combining the spray-dried powder with an excipient, for example, a glidant, lubricant, and/or diluent.
  • an excipient for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a spray-dried powder prepared by a method described herein.
  • the disclosure provides a method of preparing a lyophilate that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilate.
  • the disclosure provides a method of preparing a lyophilate that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; freeze drying (lyophilizing) the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the lyophilate.
  • the method further comprises combining the lyophilate with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilate prepared by a method described herein.
  • the disclosure provides a method of preparing a lyophilized powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilized powder.
  • the disclosure provides a method of preparing a lyophilized powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, preparing a solution; freeze drying (lyophilizing) the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the lyophilized powder.
  • the method further comprises combining the lyophilized powder with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilized powder prepared by a method described herein.
  • the disclosure provides a method of preparing a lyophilized cake that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; and freeze drying (lyophilizing) the solution, thereby preparing a lyophilized cake.
  • the disclosure provides a lyophilized cake prepared by a method described herein.
  • the freeze drying comprises primary drying and secondary drying.
  • primary drying is performed at a temperature between about -35°C to about -20°C.
  • primary drying is performed at a temperature of about -20°C, about -25 °C, about -30°C or about -35°C.
  • secondary drying is performed at a temperature between about +20°C to about +30°C.
  • secondary drying is performed at a temperature of about +25°C.
  • the bulking agent comprises mannitol, sucrose, maltodextrin, dextran, Ficoll, polyethylene glycol (PEG, such as PEG 6000), cyclodextrin, or PVP-K30.
  • the bulking agent comprises mannitol.
  • the excipient comprises an additional ingredient.
  • the additional ingredient comprises trehalose, mannitol, sucrose, sorbitol, dextran, poloxamer 188, maltodextrin, PVP-K30, Ficoll, citrate, arginine, and/or hydroxypropyl-B-cyclodextrin.
  • the excipient comprises mannitol and trehalose.
  • the excipient consists essentially of mannitol and trehalose.
  • the excipient comprises mannitol, trehalose, and sorbitol.
  • the excipient consists essentially of mannitol, trehalose, and sorbitol.
  • the excipient comprises trehalose.
  • the excipient consists essentially of trehalose.
  • the excipient is from a stock comprising one or more excipients, wherein the stock comprises a formula provided in provided in Tables A or F.
  • the dried form is a powder.
  • the powder is a lyophilized powder.
  • the powder is a spray-dried powder.
  • the dried form is a lyophilate.
  • the lyophilate is a lyophilized powder.
  • the lyophilate is a lyophilized cake.
  • the excipient solution comprises mannitol and trehalose, wherein the mannitol and the trehalose are not present in equal amounts (for example, the mannitol and the trehalose are present in unequal amounts; for example, on a weight basis or a weight percent basis).
  • the excipient solution comprises more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution comprises at least two-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution comprises at least three-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient of the solution or dried form comprises mannitol and trehalose, wherein the mannitol and the trehalose are not present in equal amounts (for example, the mannitol and the trehalose are present in unequal amounts; for example, on a weight basis or a weight percent basis).
  • the excipient of the solution or dried form comprises more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient of the solution or dried form comprises at least two-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis. In some embodiments, the excipient of the solution or dried form comprises at least three-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution consists essentially of mannitol and trehalose.
  • the excipient solution consists essentially of mannitol and trehalose, wherein the mannitol and the trehalose are not present in equal amounts (for example, the mannitol and the trehalose are present in unequal amounts; for example, on a weight basis or a weight percent basis).
  • the excipient solution consists essentially of mannitol and trehalose, wherein the excipient contains more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution consists essentially of mannitol and trehalose, wherein the excipient solution contains at least two-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis. In some embodiments, the excipient solution consists essentially of mannitol and trehalose, wherein the excipient solution contains at least three-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient of the solution or dried form consists essentially of mannitol and trehalose, wherein the excipient of the solution or dried form contains more mannitol than trehalose, for example, on a weight basis or weight percent basis. In some embodiments, the excipient of the solution or dried form consists essentially of mannitol and trehalose, wherein the excipient of the solution or dried form contains at least two-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient of the solution or dried form consists essentially of mannitol and trehalose, wherein the excipient of the solution or dried form contains at least three-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein neither the mannitol nor the trehalose is present in an amount of 5 mg/ml to 15 mg/ml.
  • the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein the mannitol is not present in an amount of 5 mg/ml to 15 mg/ml.
  • the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein the trehalose is not present in an amount of 5 mg/ml to 15 mg/ml.
  • the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein neither the mannitol nor the trehalose is present in an amount of 9 mg/ml.
  • the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein the mannitol is not present in an amount of 9 mg/ml.
  • the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein the trehalose is not present in an amount of 9 mg/ml.
  • the excipient comprises, or consists essentially of, mannitol and trehalose, and does not comprise methionine.
  • the dried form or therapeutic composition comprises, or consists essentially of, mannitol and trehalose, and the mannitol and the trehalose are not present in equal amounts (for example, the mannitol and the trehalose are present in unequal amounts, for example, on a weight basis or a weight percent basis) in the dried form or therapeutic composition.
  • At least about 10% (by weight) of the solution or dried form is excipient stock.
  • the solution, dried form, or therapeutic composition provided herein about 10% to about 80% (by weight) of the solution or dried form is excipient stock.
  • the EVs comprise at least about 1% of the total solids by weight of the dried form.
  • the EVs comprise about 1% to about 99% of the total solids by weight of the dried form.
  • the EVs comprise about 5% to about 90% of the total solids by weight of the dried form. In some embodiments of the dried form or therapeutic composition provided herein, the EVs comprise about 1% to about 60% of the total solids by weight of the dried form. In some embodiments of the dried form or therapeutic composition provided herein, the EVs comprise about 1% to about 20% of the total solids by weight of the powder or cake. In some embodiments of the dried form or therapeutic composition provided herein, the EVs comprise about 2% to about 10% of the total solids by weight of the dried form.
  • the EVs comprise about 2% to about 6% of the total solids by weight of the dried form. In some embodiments of the dried form or therapeutic composition provided herein, the dried form comprises a moisture content below about 6% (for example, as determined by Karl Fischer titration).
  • the dried form comprises a moisture content below about 5% (for example, as determined by Karl Fischer titration).
  • the dried form comprises a moisture content about 0.5% to about 5% (for example, as determined by Karl Fischer titration).
  • the dried form comprises a moisture content about 1% to about 5% (for example, as determined by Karl Fischer titration).
  • the dried form comprises a moisture content about 1% to about 4% (for example, as determined by Karl Fischer titration).
  • the dried form comprises a moisture content about 2% to about 5% (for example, as determined by Karl Fischer titration).
  • the dried form comprises a moisture content about 2% to about 4% (for example, as determined by Karl Fischer titration).
  • the dried form comprises a moisture content about 1.5% to about 7% (for example, as determined by Karl Fischer titration).
  • the dried form comprises at least 6.24e9 particles per mg of the dried form (for example, as determined by particles per mg, such as by NTA), e.g., after the dried form or therapeutic composition is resuspended, such as in deionized water.
  • the dried form comprises about 3el0 to about 8el0 particles per mg of the dried form (for example, as determined by particles per mg, such as by NTA), e.g., after the dried form or therapeutic composition is resuspended, such as in deionized water.
  • the dried form comprises about 6el0 to about 8el0 particles per mg of the dried form (for example, as determined by particles per mg, such as by NTA), e.g., after the dried form or therapeutic composition is resuspended, such as in deionized water.
  • the dried form comprises about 6e9 to about 3el0 particles/mg dried form), e.g., after the dried form or therapeutic composition is resuspended, such as in deionized water.
  • particle numeration is determined on the dried form (such as a lyophilate) resuspended in water, by NTA, with use of a Zetaview camera.
  • the particles have a hydrodynamic diameter (Z average, Zave) of about 132 nm to about 315.2 nm after resuspension from the dried form (for example, resuspension in deionized water) (for example, as determined by dynamic light scattering).
  • the particles have a hydrodynamic diameter (Z average, Zave) of about 200 nm after resuspension from the dried form (for example, resuspension in deionized water) (for example, as determined by dynamic light scattering).
  • the particles have a hydrodynamic diameter (Z average, Zave) of about 132 nm to about 315.2 nm.
  • DLS dynamic light scattering
  • PBS for example, 0. IX PBS
  • the particles have a mean size of the most dominant DLS integrated peak of between about 43.72 nm to about 79.18 nm, e.g., after the dried form or therapeutic composition is resuspended, such as in deionized water or in a buffer such as PBS (for example, 0. IX PBS).
  • a buffer such as PBS (for example, 0. IX PBS).
  • the particles have a charge (as measured by zeta potential (mV), for example, as measured by DLS of the charge of the most dominant DLS integrated peak of particles) of about -32 to about -25.3 mV, e.g., after the dried form or therapeutic composition is resuspended, such as in deionized water or in a buffer such as PBS (for example, 0.1X PBS).
  • zeta potential for example, as measured by DLS of the charge of the most dominant DLS integrated peak of particles
  • the F. massiliensis EVs are obtained from Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696).
  • the Fournierella massiliensis strain is a strain comprising at least at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Fournierella massiliensis strain A.
  • 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
  • the compositions described herein (e.g. a solution, dried form, or therapeutic composition) comprises EVs of one strain of F. massiliensis, wherein the one strain of F. massiliensis is a strain comprising at least 99.9% sequence identity to the nucleotide sequence of the Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696).
  • the compositions described herein comprises EVs of one strain of F. massiliensis, wherein the one strain of F. massiliensis is the Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696).
  • a solution, dried form, or therapeutic composition provided herein contains EVs from one or more F massiliensis strains. In some embodiments, a solution, dried form, or therapeutic composition provided herein contains EVs from one or more b F. massiliensis strain. In some embodiments, a solution, dried form, or therapeutic composition provided herein contains EVs from one F massiliensis strain. In some embodiments, the F massiliensis strain used as a source of EVs is selected based on the properties of the F massiliensis strain (for example, growth characteristics, yield, ability to modulate an immune response in an assay or a subject).
  • a solution, dried form, or therapeutic composition provided herein comprising F massiliensis EVs is used for the treatment or prevention of a disease and/or a health disorder, for example, in a subject (for example, human).
  • a dried form (or a therapeutic composition thereof) provided herein comprising F massiliensis EVs is prepared as a solid dose form, such as a tablet, a minitablet, a capsule, or a powder; or a combination of these forms (for example, minitablets comprised in a capsule).
  • the solid dose form comprises a coating (for example, enteric coating).
  • a dried form (or a therapeutic composition thereof) provided herein comprising F massiliensis EVs is reconstituted.
  • a solution (or a therapeutic composition thereof) provided herein comprising F massiliensis EVs is used as a suspension, for example, diluted to a suspension or used in undiluted form.
  • a therapeutic composition comprising a solution and/or dried form comprising F massiliensis EVs is prepared as provided herein.
  • the therapeutic composition comprising a dried form is formulated into a solid dose form, such as a tablet, a minitablet, a capsule, or a powder.
  • the therapeutic composition comprising a dried form is reconstituted in a suspension.
  • the therapeutic composition comprising a powder is formulated into a solid dose form, such as a tablet, a minitablet, a capsule, or a powder. In some embodiments, the therapeutic composition comprising a powder is reconstituted in a suspension.
  • a solution, dried form, or therapeutic composition provided herein comprises gamma irradiated F. massiliensis EVs. In some embodiments, the gamma irradiated F. massiliensis EVs are formulated into therapeutic composition. In some embodiments, the gamma irradiated F.
  • massiliensis EVs are formulated into a solid dose form, such as a tablet, a minitablet, a capsule, or a powder.
  • the gamma irradiated F. massiliensis EVs are formulated reconstituted in a suspension.
  • a solution, dried form, or therapeutic composition provided herein comprising F. massiliensis EVs is orally administered.
  • a solution, dried form, or therapeutic composition provided herein comprising F. massiliensis EVs is administered intranasally.
  • a solution, dried form, or therapeutic composition provided herein comprising F. massiliensis EVs is administered by inhalation.
  • a solution, dried form, or therapeutic composition provided herein comprising F. massiliensis EVs is administered intravenously.
  • a solution, dried form, or therapeutic composition provided herein comprising F. massiliensis EVs is administered by injection, for example, intratumorally or subtum orally, for example, to a subject who has a tumor.
  • a solution, dried form, or therapeutic composition provided herein comprising F. massiliensis EVs is administered topically.
  • compositions comprising solutions and/or dried forms comprising F. massiliensis EVs useful for the treatment and/or prevention of a disease or a health disorder (for example, adverse health disorders) (for example, a cancer, an autoimmune disease, an inflammatory disease, a dysbiosis, or a metabolic disease), as well as methods of making and/or identifying such solutions and/or dried forms and/or therapeutic compositions, and methods of using such solutions and/or dried forms, and/or therapeutic compositions thereof (for example, for the treatment of a cancer, an autoimmune disease, an inflammatory disease, a dysbiosis, or a metabolic disease, either alone or in combination with other therapeutics).
  • a health disorder for example, adverse health disorders
  • a cancer, an autoimmune disease, an inflammatory disease, a dysbiosis, or a metabolic disease for example, a cancer, an autoimmune disease, an inflammatory disease, a dysbiosis, or a metabolic disease, either alone or in combination with other therapeutics.
  • the therapeutic compositions comprise both massiliensis EVs and whole F. massiliensis bacteria, for example, F. massiliensis bacteria from which the EVs were obtained, such as live bacteria, killed bacteria, attenuated bacteria.
  • the therapeutic compositions comprise F. massiliensis EVs in the absence of the F. massiliensis bacteria from which they were obtained, such that over about 85%, over about 90%, or over about 95% (or over about 99%) of the bacteria-sourced content of the solutions and/or dried forms (such as powders) comprises EVs.
  • the massiliensis EVs used in a composition described herein are isolated EVs, for example, isolated by a method described herein.
  • the solution, dried form, or therapeutic composition comprises F. massiliensis EVs from one or more of the bacteria strains.
  • the solution, dried form, or therapeutic composition comprises isolated EVs (for example, from one or more strains of bacteria, preferably from one or more strains of F. massiliensis). In some embodiments, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the content (for example, of the content that does not exclude excipient) of the solution, dried form, or therapeutic composition, is isolated EVs from F. massiliensis.
  • the solution, dried form, or therapeutic composition comprises isolated F. massiliensis EVs (for example, from one strain of F. massiliensis).
  • isolated F. massiliensis EVs for example, from one strain of F. massiliensis.
  • 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 (for example, of the content that does not exclude excipient) of the solution and/or dried form is isolated EV of bacteria (for example, bacteria of interest, for example, bacteria disclosed herein).
  • the solution, dried form, or therapeutic composition comprises EVs from one strain of F. massiliensis bacteria.
  • the solution, dried form, or therapeutic composition comprises EVs from more than one strain of F. massiliensis bacteria.
  • the F. massiliensis EVs are lyophilized.
  • the F. massiliensis EVs are gamma irradiated.
  • the F. massiliensis EVs are UV irradiated.
  • the F. massiliensis EVs are heat inactivated (for example, at 50°C for two hours or at 90°C for two hours).
  • the F. massiliensis EVs are acid treated.
  • the F. massiliensis EVs are oxygen sparged (for example, at 0.1 vvm for two hours).
  • the F. massiliensis EVs are from engineered bacteria that are modified to enhance certain desirable properties.
  • the engineered bacteria are modified so that EVs produced therefrom will have reduced toxicity and adverse effects (for example, by removing or deleting lipopolysaccharide (LPS)), enhanced oral delivery (for example, by improving acid resistance, muco-adherence and/or penetration and/or resistance to bile acids, resistance to anti-microbial peptides and/or antibody neutralization), target desired cell types (for example, M-cells, goblet cells, enterocytes, dendritic cells, macrophages), improved bioavailability systemically or in an appropriate niche (for example, mesenteric lymph nodes, Peyer’s patches, lamina intestinal, tumor draining lymph nodes, and/or blood), enhanced immunomodulatory and/or therapeutic effect (for example, either alone or in combination with another therapeutic agent), enhanced immune activation, and/or improved manufacturing attributes (for example, growth characteristics, yield, greater stability, improved
  • solutions and/or dried forms comprising F. massiliensis EVs useful for the treatment and/or prevention of a disease or a health disorder (for example, a cancer, an autoimmune disease, an inflammatory disease, dysbiosis, or a metabolic disease), as well as methods of making and/or identifying such solutions and/or dried forms (or therapeutic compositions thereof), and methods of using such solutions and/or dried forms (for example, for the treatment of a cancer, an autoimmune disease, an inflammatory disease, dysbiosis, or a metabolic disease), either alone or in combination with one or more other therapeutics.
  • a disease or a health disorder for example, a cancer, an autoimmune disease, an inflammatory disease, dysbiosis, or a metabolic disease
  • a health disorder for example, a cancer, an autoimmune disease, an inflammatory disease, dysbiosis, or a metabolic disease
  • methods of making and/or identifying such solutions and/or dried forms (or therapeutic compositions thereof) for example, for the treatment of a cancer, an autoimmune disease
  • the gamma irradiated F. massiliensis EVs are formulated therapeutic compositions containing a solution and/or dried form (for example, lyophilate) and provide potency comparable to or greater than therapeutic compositions that contain the whole F. massiliensis bacteria from which the EVs were obtained.
  • a therapeutic composition containing solutions and/or dried forms such as powders
  • the gamma irradiated F are formulated therapeutic compositions containing a solution and/or dried form (for example, lyophilate) and provide potency comparable to or greater than therapeutic compositions that contain whole F. massiliensis bacteria of the same F. massiliensis strain from which the EVs were obtained.
  • massiliensis EVs are formulated such that solution- and/or dried form- (for example, lyophilate) containing therapeutic compositions allow the administration of higher doses and elicit a comparable or greater (for example, more effective) response than observed with a comparable therapeutic composition that contains whole F. massiliensis bacteria of the same F. massiliensis strain from which the EVs were obtained.
  • solution- and/or dried form- (for example, lyophilate) containing therapeutic compositions allow the administration of higher doses and elicit a comparable or greater (for example, more effective) response than observed with a comparable therapeutic composition that contains whole F. massiliensis bacteria of the same F. massiliensis strain from which the EVs were obtained.
  • the gamma irradiated F. massiliensis EVs are formulated at the same dose (for example, based on particle count or protein content), a therapeutic composition containing a solution and/or dried form (for example, lyophilate) contain less microbially-derived material (based on particle count or protein content), as compared to a therapeutic composition that contains the whole F. massiliensis bacteria of the same F. massiliensis strain from which the EVs were obtained, while providing an equivalent or greater therapeutic benefit to the subject receiving such therapeutic composition.
  • F. massiliensis EVs are administered at doses for example, of about IxlO 7 to about IxlO 15 particles, for example, as measured by NTA.
  • the dose of F. massiliensis EVs is about 1 x 10 5 to about 7 x 10 13 particles (for example, wherein particle count is determined by NTA (nanoparticle tracking analysis)).
  • the dose of F. massiliensis EVs is about I x lO 10 to about 7 x 10 13 particles (for example, wherein particle count is determined by NTA (nanoparticle tracking analysis)).
  • the dose of F. massiliensis EVs is about 1 x 10 9 to about 7 x IO 10 particles (for example, wherein particle count is determined by NTA (nanoparticle tracking analysis), such as NTA using Zetaview).
  • F. massiliensis EVs are administered at doses for example, of about 5 mg to about 900 mg total protein, for example, as measured by Bradford assay.
  • F. massiliensis EVs are administered at doses for example, of about 5 mg to about 900 mg total protein, for example, as measured by BCA assay.
  • provided herein are methods of treating a subject who has cancer comprising administering to the subject a therapeutic composition or a solution on- dried form described herein.
  • methods of treating a subject who has an immune disorder for example, an autoimmune disease, an inflammatory disease, an allergy
  • methods of treating a subject who has a metabolic disease comprising administering to the subject a therapeutic composition or a solution or dried form described herein.
  • provided herein are methods of treating a subject who has a dysbiosis comprising administering to the subject a therapeutic composition or a solution ordried form described herein. In certain embodiments, provided herein are methods of treating a subject who has a neurologic disease comprising administering to the subject a therapeutic composition or a solution or dried form described herein.
  • the method further comprises administering to the subject an antibiotic.
  • the method further comprises administering to the subject one or more other cancer therapies (for example, surgical removal of a tumor, the administration of a chemotherapeutic agent, the administration of radiation therapy, and/or the administration of a cancer immunotherapy, such as an immune checkpoint inhibitor, a cancer-specific antibody, a cancer vaccine, a primed antigen presenting cell, a cancer-specific T cell, a cancer-specific chimeric antigen receptor (CAR) T cell, an immune activating protein, and/or an adjuvant).
  • cancer therapies for example, surgical removal of a tumor, the administration of a chemotherapeutic agent, the administration of radiation therapy, and/or the administration of a cancer immunotherapy, such as an immune checkpoint inhibitor, a cancer-specific antibody, a cancer vaccine, a primed antigen presenting cell, a cancer-specific T cell, a cancer-specific chimeric antigen receptor (CAR) T cell, an immune activating protein, and/or an adjuvant).
  • the method further comprises the administration of another therapeutic bacterium and/or EVs from bacteria from one or more other bacterial strains (for example, therapeutic bacterium).
  • the method further comprises the administration of an immune suppressant and/or an anti-inflammatory agent.
  • the therapeutic composition or solution or dried form are for use in combination with one or more other immune effect modulators.
  • the method further comprises the administration of a metabolic disease therapeutic agent.
  • a therapeutic composition or a solution or dried form for use in the treatment and/or prevention of a disease (for example, a cancer, an autoimmune disease, an inflammatory disease, a dysbiosis, or a metabolic disease) or a health disorder, either alone or in combination with one or more other (e.g.., additional) therapeutic agent.
  • a disease for example, a cancer, an autoimmune disease, an inflammatory disease, a dysbiosis, or a metabolic disease
  • a health disorder either alone or in combination with one or more other (e.g.., additional) therapeutic agent.
  • a therapeutic composition or a solution ordried form for use in treating and/or preventing a cancer in a subject (for example, human).
  • the therapeutic composition or solution or dried form is used either alone or in combination with one or more other therapeutic agent for the treatment of the cancer.
  • a therapeutic composition or a solution or dried form for use in treating and/or preventing an immune disorder (for example, an autoimmune disease, an inflammatory disease, an allergy) in a subject (for example, human).
  • an immune disorder for example, an autoimmune disease, an inflammatory disease, an allergy
  • the therapeutic composition or solution or dried form is used either alone or in combination with one or more other therapeutic agent for the treatment of the immune disorder.
  • a therapeutic composition or a solution or dried form for use in treating and/or preventing a dysbiosis in a subject (for example, human).
  • the therapeutic composition or solution or dried form is used either alone or in combination with therapeutic agent for the treatment of the dysbiosis.
  • a therapeutic composition or a solution or dried form for use in treating and/or preventing a metabolic disease in a subject (for example, human).
  • the therapeutic composition or solution or dried form is used either alone or in combination with therapeutic agent for the treatment of the metabolic disease.
  • a therapeutic composition or a solution or dried form for use in treating and/or preventing a dysbiosis in a subject (for example, human).
  • the therapeutic composition or olution orr dried form is used either alone or in combination with therapeutic agent for the treatment of the dysbiosis.
  • a therapeutic composition or a solution ordried form for use in treating and/or preventing a neurologic disease in a subject (for example, human).
  • the therapeutic composition or solution ordried form is used either alone or in combination with one or more other therapeutic agent for treatment of the neurologic disorder.
  • the therapeutic composition or solution ordried form is for use in combination with an antibiotic.
  • the therapeutic composition or solution ordried form is for use in combination with one or more other cancer therapies (for example, surgical removal of a tumor, the use of a chemotherapeutic agent, the use of radiation therapy, and/or the use of a cancer immunotherapy, such as an immune checkpoint inhibitor, a cancer-specific antibody, a cancer vaccine, a primed antigen presenting cell, a cancer-specific T cell, a cancer-specific chimeric antigen receptor (CAR) T cell, an immune activating protein, and/or an adjuvant).
  • cancer therapies for example, surgical removal of a tumor, the use of a chemotherapeutic agent, the use of radiation therapy, and/or the use of a cancer immunotherapy, such as an immune checkpoint inhibitor, a cancer-specific antibody, a cancer vaccine, a primed antigen presenting cell, a cancer-specific T cell, a cancer-specific chimeric antigen receptor (CAR) T cell, an immune
  • the therapeutic composition or solution or dried form is for use in combination with another therapeutic bacterium and/or EVs obtained from one or more other bacterial strains (for example, therapeutic bacterium).
  • the therapeutic composition or solution ordried form is for use in combination with one or more immune suppressant(s) and/or an anti-inflammatory agent(s).
  • the therapeutic composition or solution or dried form is for use in combination with one or more other metabolic disease therapeutic agents.
  • a therapeutic composition or a solution orr dried form for the preparation of a medicament for the treatment and/or prevention of a disease (for example, a cancer, an autoimmune disease, an inflammatory disease, a dysbiosis, or a metabolic disease), either alone or in combination with another therapeutic agent.
  • a disease for example, a cancer, an autoimmune disease, an inflammatory disease, a dysbiosis, or a metabolic disease
  • the use is in combination with another therapeutic bacterium and/or EVs obtained from one or more other bacterial strains (for example, therapeutic bacterium).
  • a therapeutic composition or a solution ordried form for the preparation of a medicament for treating and/or preventing a cancer in a subject (for example, human).
  • the therapeutic composition or solution or dried form is for use either alone or in combination with another therapeutic agent for the cancer.
  • a therapeutic composition or a solution ordried form for the preparation of a medicament for treating and/or preventing an immune disorder (for example, an autoimmune disease, an inflammatory disease, an allergy) in a subject (for example, human).
  • an immune disorder for example, an autoimmune disease, an inflammatory disease, an allergy
  • the therapeutic composition or solution or dried form is for use either alone or in combination with another therapeutic agent for the immune disorder.
  • a therapeutic composition or a solution or dried form for the preparation of a medicament for treating and/or preventing a dysbiosis in a subject (for example, human).
  • the therapeutic composition or solution or dried form is for use either alone or in combination with another therapeutic agent for the dysbiosis.
  • a therapeutic composition or a solution or dried form for the preparation of a medicament for treating and/or preventing a metabolic disease in a subject (for example, human).
  • the therapeutic composition or solution ordried form is for use either alone or in combination with another therapeutic agent for the metabolic disease.
  • a therapeutic composition or solution or dried form for the preparation of a medicament for treating and/or preventing a dysbiosis in a subject (for example, human).
  • the therapeutic composition or olution or dried form is for use either alone or in combination with another therapeutic agent for the dysbiosis.
  • a therapeutic composition or a solution or dried form for the preparation of a medicament for treating and or preventing a neurologic disease in a subject (for example, human).
  • the therapeutic composition or solution or dried form is for use either alone or in combination with another therapeutic agent for the neurologic disorder.
  • the therapeutic composition or solution or dried form is for use in combination with an antibiotic.
  • the therapeutic composition or solution or dried form is use in combination with one or more other cancer therapies (for example, surgical removal of a tumor, the use of a chemotherapeutic agent, the use of radiation therapy, and/or the use of a cancer immunotherapy, such as an immune checkpoint inhibitor, a cancer-specific antibody, a cancer vaccine, a primed antigen presenting cell, a cancer-specific T cell, a cancer-specific chimeric antigen receptor (CAR) T cell, an immune activating protein, and/or an adjuvant).
  • cancer therapies for example, surgical removal of a tumor, the use of a chemotherapeutic agent, the use of radiation therapy, and/or the use of a cancer immunotherapy, such as an immune checkpoint inhibitor, a cancer-specific antibody, a cancer vaccine, a primed antigen presenting cell, a cancer-specific T cell, a cancer-specific chimeric antigen receptor (CAR) T cell, an immune activating protein
  • the therapeutic composition or solution ordried form is for use in combination with another therapeutic bacterium and/or EVs obtained from one or more other bacterial strains (for example, therapeutic bacterium).
  • the therapeutic composition or solution or dried form is for use in combination with one or more other immune suppressant(s) and/or an anti-inflammatory agent(s).
  • the therapeutic composition or solution or dried form is for use in combination with one or more other metabolic disease therapeutic agent(s).
  • a therapeutic composition or a solution or dried form, for example, as described herein, comprising F. massiliensis EVs provides a therapeutically effective amount of F. massiliensis EVs to a subject, for example, a human.
  • a therapeutic composition or a solution or dried form, for example, as described herein, comprising F. massiliensis EVs provides a non-natural amount of the therapeutically effective components (for example, present in the EVs) to a subject, for example, a human.
  • a therapeutic composition or a solution ordried form, for example, as described herein, comprising F. massiliensis EVs provides unnatural quantity of the therapeutically effective components (for example, present in the EVs) to a subject, for example, a human.
  • a therapeutic composition or a solution or dried form, for example, as described herein, comprising F. massiliensis EVs brings about one or more changes to a subject, for example, human, for example, to treat or prevent a disease or a health disorder.
  • a therapeutic composition or a solution or dried form, for example, as described herein, comprising F. massiliensis EVs has potential for significant utility, for example, to affect a subject, for example, a human, for example, to treat or prevent a disease or a health disorder.
  • a stock comprising one or more excipients, wherein the stock comprises a bulking agent, wherein the stock is for use in combination with F. massiliensis EVs (for example, a liquid preparation thereof), for example, EVs from a source provided herein.
  • F. massiliensis EVs for example, a liquid preparation thereof
  • a stock comprising one or more excipients, wherein the stock comprises a bulking agent and a lyoprotectant, wherein the stock is for use in combination with F. massiliensis EVs (for example, a liquid preparation thereof), for example, EVs from a source provided herein.
  • F. massiliensis EVs for example, a liquid preparation thereof
  • a stock comprising one or more excipients, wherein the stock comprises a lyoprotectant, wherein the stock is for use in combination with F. massiliensis EVs (for example, a liquid preparation thereof), for example, EVs from a source provided herein.
  • F. massiliensis EVs for example, a liquid preparation thereof
  • the bulking agent comprises mannitol, sucrose, maltodextrin, dextran, Ficoll, or PVP-K30.
  • the bulking agent comprises mannitol.
  • the excipient solution comprises an additional ingredient.
  • the additional ingredient comprises trehalose, mannitol, sucrose, sorbitol, dextran, poloxamer 188, maltodextrin, PVP-K30, Ficoll, citrate, arginine, and/or hydroxypropyl-B-cyclodextrin.
  • the excipient solution comprises mannitol and trehalose.
  • the excipient solution consists essentially of mannitol and trehalose.
  • the excipient solution comprises mannitol, trehalose, and sorbitol.
  • the excipient solution consists essentially of mannitol, trehalose, and sorbitol.
  • the excipient solution comprises trehalose.
  • the excipient solution consists essentially of trehalose.
  • the excipient solution comprises mannitol and trehalose, wherein the mannitol and the trehalose are not present in equal amounts (for example, the mannitol and the trehalose are present in unequal amounts; for example, on a weight basis or a weight percent basis).
  • the excipient solution comprises more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution comprises at least two-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution comprises at least three-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient of the solution or dried form comprises mannitol and trehalose, wherein the mannitol and the trehalose are not present in equal amounts (for example, the mannitol and the trehalose are present in unequal amounts; for example, on a weight basis or a weight percent basis).
  • the excipient of the solution or dried form comprises more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient of the solution or dried form comprises at least two-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis. In some embodiments, the excipient of the solution or dried form comprises at least three-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • 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 the trehalose are not present in equal amounts (for example, the mannitol and the trehalose are present in unequal amounts; for example, 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, for example, on a weight basis or weight percent basis.
  • the excipient solution consists essentially of mannitol and trehalose, wherein the excipient solution contains at least two-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis. In some embodiments, the excipient solution consists essentially of mannitol and trehalose, wherein the excipient solution contains at least three-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient of the solution or dried form consists essentially of mannitol and trehalose, wherein the excipient of the solution or dried form contains more mannitol than trehalose, for example, on a weight basis or weight percent basis. In some embodiments, the excipient of the solution or dried form consists essentially of mannitol and trehalose, wherein the excipient of the solution or dried form contains at least two-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient of the solution or dried form consists essentially of mannitol and trehalose, wherein the excipient of the solution or dried form contains at least three-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein neither the mannitol nor the trehalose is present in an amount of 5 mg/ml to 15 mg/ml. In some embodiments, the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein the mannitol is not present in an amount of 5 mg/ml to 15 mg/ml. In some embodiments, the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein the trehalose is not present in an amount of 5 mg/ml to 15 mg/ml.
  • the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein neither the mannitol nor the 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 the 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 the trehalose is not present in an amount of 9 mg/ml.
  • the excipient solution comprises, or consists essentially of, mannitol and trehalose, and does not comprise methionine.
  • a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F.
  • a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, wherein the stock is for use in combination with F. massiliensis EVs (for example, a liquid preparation thereof), such as EVs from a source provided herein.
  • a liquid preparation comprises a cell culture supernatant, such as a bacterial cell culture supernatant, for example, as described herein.
  • the liquid preparation comprises a retentate, such as a concentrated retentate, for example, as described herein.
  • excipients are present in (for example, provided in) an excipient solution.
  • excipient solution include the stocks comprising one or more excipients provided in Tables A or F.
  • the dried forms provided herein contain excipients from the excipient solution (such as a stock) once the moisture has been removed, such as by drying.
  • a liquid preparation that comprises F. massiliensis EVs is combined with the stock of formula 7a (which comprises the excipients mannitol and trehalose) from Tables A or F to prepare a solution.
  • the solution is dried to prepare a dried form.
  • the dried form comprises F.
  • a “stock” refers to a solution comprising one or more excipients but no active ingredient (such as an extracellular vesicle).
  • a stock is used to introduce one or more excipients into a preparation (such as a liquid preparation) comprising EVs.
  • the stock is a concentrated solution comprising a known amount of one or more excipients.
  • the stock is combined with a preparation (such as a liquid preparation) that comprises EVs to prepare a solution or dried form provided herein.
  • Figure 1 is a graph showing moisture content of lyophilized EV powders.
  • Figure 2 is a graph showing particle count of lyophilized EV powders.
  • Figure 3 is a graph showing average particle size by DLS of lyophilized EV powders.
  • Figure 4 is a graph showing electrokinetic potential of the dominant subpopulation of lyophilized EV powder by DLS.
  • Figure 5 is a graph showing particle size of the dominant subpopulation of lyophilized EV powders.
  • Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696) is single strain of Fournierella massiliensis. Extracellular vesicles obtained from Fournierella massiliensis (F. massiliensis EVs) have been shown to have therapeutic effects, for example, as described in PCT/US21/36927 (WO 2021/252838), hereby incorporated by reference in its entirety.
  • F. massiliensis EVs are secreted (for example, produced) by bacterial cells in culture. Such secreted extracellular vesicles may be referred to as secreted microbial extracellular vesicles (smEVs).
  • smEVs secreted microbial extracellular vesicles
  • EVs are prepared (for example, artificially prepared) by processing bacterial cells, for example, by methods that disrupt the bacterial membrane, such as sonication. Such artificially prepared may be referred to as processed microbial extracellular vesicles (pmEVs).
  • a “dried form” that contains F. massiliensis EVs refers to the product resulting from drying a solution that contains EVs. In some embodiments, the drying is performed, for example, by freeze drying (lyophilization) or spray drying. In some embodiments, the dried form is a powder. As used herein, a powder refers to a type of dried form and includes a lyophilized powder, and a spray-dried powder.
  • the resulting dried form is a lyophilate.
  • the dried form is a lyophilate.
  • a lyophilate is a lyophilized powder or a lyophilized cake.
  • the lyophilized cake is milled to produce a lyophilized powder.
  • the solutions and dried forms that contain F. massiliensis EVs also comprise one or more excipients, such as a bulking agent, and/or a lyoprotectant.
  • bulking agents and lyoprotectants are used when preparing F. massiliensis EVs for freeze drying.
  • bulking agents including but not limited to sucrose, mannitol, polyethylene glycol (PEG, such as PEG 6000), cyclodextrin, maltodextrin, and dextran (such as dextran 40k), are added (for example, as a stock containing the same) to a liquid preparation of F.
  • massiliensis EVs for example, obtained by isolating the EVs from a bacterial culture
  • a dried form such as a lyophilate, making it easier to handle (and optionally, further formulate, for example, into a therapeutic composition) after drying.
  • lyoprotectants including but not limited to trehalose, sucrose, and lactose, are added (for example, as a stock containing the same) to a liquid preparation of F. massiliensis EVs (for example, obtained by isolating the EVs from a bacterial culture) to protect the F. massiliensis EVs while lyophilizing or spray drying.
  • a bulking agent and/or lyoprotectant is included from an excipient stock that is added to F. massiliensis EVs (for example, purified and/or concentrated F. massiliensis EVs) to produce a solution, and/or to produce a dried form upon subsequent drying, for example, of the solution.
  • F. massiliensis EVs for example, purified and/or concentrated F. massiliensis EVs
  • a dried form such as a lyophilate contains between about 5% and about 100% F. massiliensis EVs solids by weight.
  • the total solids, including F. massiliensis EVs and excipients are between about 2% and about 20% by weight.
  • the excipients make up about 95% to about 99% of the total mass of the powder or cake.
  • the F. massiliensis EVs make up about 2% to about 6% (for example, about 2% to about 5%, about 2% to about 3%, or about 3% to about 5%) of the total mass of the lyophilate.
  • the excipient functions to maintain F. massiliensis EV efficacy and/or decrease drying (for example, lyophilization) cycle time.
  • lyoprotectants protect F. massiliensis EVs (for example, protein components thereof) during the freeze-drying process.
  • bulking agents improve the lyophilate properties, for example, for further downstream processing (such as milling, blending, and/or preparing therapeutic compositions).
  • the length of the lyophilization cycle is important for cost considerations.
  • Critical temperature modifiers such as bulking agents and/or lyoprotectants can significantly reduce drying time.
  • an excipient stock containing one or more excipients (for example, that contain a bulking agent and/or lyoprotectant) is added to concentrated F. massiliensis EVs (for example, a liquid preparation thereof) to bring the total solids to between about 2% to about 20%.
  • the F. massiliensis EVs are concentrated to 5 to 100 times or volume concentration factors (VCF). Examples provided herein targeted about 10% total solids with actual dissolved solids ranging from about 6% to about 8%.
  • an excipient stock containing one or more excipients (for example, that contain a bulking agent and/or lyoprotectant) (for example, a stock comprising excipients of a formula provided in Tables A or F) is prepared as a stock solution in deionized water and sterile filtered with a 0.2 mm filter prior to use.
  • the stock solution is added to the concentrated F. massiliensis EVs, for example, based on weight up to 80%.
  • the percentage to add is based on the estimated solids contribution of EVs plus the dissolved solids of the excipient stock to achieve the desired total solids content prior to lyophilization.
  • the resulting lyophilate (for example, lyophilized cake) has a uniform appearance, and is a white to off-white.
  • the resulting lyophilate (for example, lyophilized cake) obtained after freeze-drying is a white to off-white, fine and smooth granular powder (for example, after milling (for example, grinding) the lyophilized cake).
  • DLS dynamic light scattering
  • Z average, Zave hydrodynamic diameter of particles present after the lyophilate (for example, lyophilized powder) is resuspended in deionized water or in a buffer such as PBS (for example, 0. IX PBS).
  • PBS for example, 0. IX PBS
  • the Zave is used to quantify the effectiveness of the stabilizer. For example, if the idealized Zave particle size is 200 nm; therefore, the resuspended EVs with the lowest Zave closest to this particle size is considered to be sufficiently stabilized.
  • the particle size ranges, for example, from 132 nm to 315.2 nm.
  • DLS dynamic light scattering
  • lyophilate for example, lyophilized powder
  • PBS for example, 0. IX PBS
  • the mean size of the particles is not necessarily identical to the mean size of the EVs prior to lyophilization.
  • the mean size of the particles after lyophilization (for example, after the lyophilate is resuspended in deionized water or in a buffer such as PBS (for example, 0.1X PBS)) is larger or smaller than the mean EV size prior to lyophilization, or the mean size after EV isolation or preparation from a bacterial culture (for example, the mean size after gradient purification of EVs from a bacterial culture).
  • PBS for example, 0.1X PBS
  • Particles in a lyophilate (after a solution containing EVs is lyophilized) contain EVs, and may also include other components from the culture media, such as cell debris, LPS, and/or proteins.
  • a lyophilate cake obtained after freeze-drying with the excipients and/or conditions provided herein does not have a porous sponge shape.
  • the lyophilate obtained after freeze-drying with the excipients and/or conditions provided herein is a white to off-white, fine and smooth granular lyophilate powder.
  • excipients allow for EVs to be freeze dried in less than 4000 minutes, for example, freeze dried in about 2800 to about 3200 minutes.
  • the freezing step is performed in less than 225 minutes, as opposed to 10 to 15 hours (600 to 900 minutes).
  • primary drying is performed at a temperature between about -35°C to about -20°C, for example, about -20°C, about -25 °C, about -30°C or about -35°C, as opposed to, for example, -50°C.
  • primary drying is performed for about 42 hours or less (for example, 2500 minutes or less), as opposed to, for example, 50-60 hours (3000 to 3600 minutes).
  • total dry times are, for example, about 72 hours or less, for example, about 48 to about 72 hours, for example, less than about 48 hours.
  • primary drying is performed for about 65 hours or less (for example, about 60 hours or less).
  • secondary drying is performed for about 12 hours or less (for example, about 10 to about 12 hours, about 5 to about 10 hours, about 10 hours or less, or about 5 hours or less).
  • secondary drying is performed at a temperature between about +20°C to about +30°C, for example, room temperature, for example, about +25°C, as opposed to, for example, -20°C.
  • use of shorter drying times and/or higher drying temperatures makes the lyophilization process for EVs more commercially feasible.
  • the lyophilates containing F. massiliensis EVs described herein are prepared to have a moisture content (for example, as determined by the Karl Fischer method) of below about 10% (for example, below about 9%, below about 8%, below about 7%, below about 6%, below about 5% or below about 4%, for example, about 1% to about 4%, about 1.5% to about 4%, about 2% to about 3%) upon completion of freeze drying.
  • a moisture content for example, as determined by the Karl Fischer method
  • the lyophilate are better suited for downstream processing, for example, for use in a therapeutic composition.
  • the lyophilate by preparing lyophilates to have a moisture content below about 6%, the lyophilate has improved stability, e.g., upon storage.
  • lyophilates containing EVs of the Fournierella massiliensis strain exemplified herein had a moisture content (determined by Karl Fischer) of between about 1.51% to about 7.01%.
  • An excipient can be selected and used in various quantities to obtain the desired moisture content.
  • the drying conditions can be selected to obtain the desired moisture content.
  • lyophilates containing EVs of the Fournierella massiliensis strain exemplified herein had particle numerations of between about 6.24e9 to about 2.89el0 particles/mg lyophilate.
  • particle numeration is determined, for example, on lyophilate resuspended in water and with use of a Zetaview camera. Components of the excipient can be selected to obtain the desired particle numeration. The drying conditions can be selected to obtain the desired particle numeration.
  • DLS is used to measure the charge of the most dominant DLS integrated peak of particles. In some embodiments, DLS is used to measure the charge of the total particles present in a lyophilate. Notably, the charge of the particles, whether measured for total particles or for the most dominant DLS integrated peak, is not necessarily identical to the charge of the EVs prior to lyophilization.
  • the charge of the particles after lyophilization (for example, after the lyophilate (for example, lyophilized powder) is resuspended in deionized water or in a buffer such as PBS (for example, 0.1X PBS)) is more or less negative than the charge of EVs prior to lyophilization, or the charge after EV isolation or preparation from a bacterial culture (for example, the charge after gradient purification of EVs from a bacterial culture).
  • PBS for example, 0.1X PBS
  • the particles in the lyophilates described herein are prepared to have a charge (as measured by zeta potential (mV), for example, as measured by DLS of the charge of the most dominant DLS integrated peak of particles) of about -32 to about - 25.3 mV.
  • a charge as measured by zeta potential (mV), for example, as measured by DLS of the charge of the most dominant DLS integrated peak of particles.
  • Components of the excipient can be selected to obtain the desired charge.
  • the drying conditions can be selected to obtain the desired charge.
  • the particles in the lyophilates (for example, lyophilized powders) described herein are prepared to have a hydrodynamic diameter (Z average, Zave) of about 132 nm to about 315.2 nm.
  • DLS dynamic light scattering
  • PBS buffer
  • the Zave of particles of lyophilates of the Fournierella massiliensis strain EVs exemplified was about 132 nm to about 315.2 nm (as measured by DLS as measured by DLS after the lyophilate was resuspended in
  • compositions of the excipient can be selected to obtain the desired Zave.
  • the drying conditions can be selected to obtain the desired Zave.
  • the particles in the lyophilates described herein are prepared to a mean size of the most dominant DLS integrated peak of between about 43.72 nm to about 79.18 nm.
  • DLS dynamic light scattering
  • the mean size of particles of lyophilates of the Fournierella massiliensis strain EVs exemplified herein was between about 43.72 nm to about 79.18 nm.
  • Components of the excipient can be selected to obtain the desired mean size.
  • the drying conditions can be selected to obtain the desired mean size.
  • lyophilates containing EVs have biological activity, for example, in a U937 cytokine secretion assay.
  • lyophilates of EVs prepared as described herein affect levels of secreted IL-10, IP-10, IL-10, TNF-a, and IL-6 levels from U937 cells, for example, as compared to control levels.
  • the spray-dried powders containing EVs described herein are prepared to have a moisture content (for example, as determined by the Karl Fischer method) of below about 10% (for example, below about 9%, below about 8%, below about 7%, below about 6%, below about 5% or below about 4%, for example, about 1% to about 4%, about 1.5% to about 4%, about 2% to about 3%) upon completion of spray drying.
  • a moisture content for example, as determined by the Karl Fischer method
  • the spray-dried powders are better suited for downstream processing, for example, for use in a therapeutic composition.
  • the spray- dried powder has improved stability, e.g., upon storage.
  • Components of the excipient can be selected to obtain the desired particle numeration.
  • the drying conditions can be selected to obtain the desired particle numeration.
  • the spray-dried powders containing EVs described herein are prepared to have a particle numeration of about 6.7e8 to about 2.55el0 particles/mg spray-dried powder.
  • particle numeration is determined, for example, by NTA.
  • Components of the excipient can be selected to obtain the desired particle numeration.
  • the drying conditions can be selected to obtain the desired particle numeration.
  • adjuvant or “Adjuvant therapy” broadly refers to an agent that affects an immunological or physiological response in a patient or subject (for example, human).
  • an adjuvant might increase the presence of an antigen over time or to an area of interest like a tumor, help absorb an antigen presenting cell antigen, activate macrophages and lymphocytes and support the production of cytokines.
  • an adjuvant might permit a smaller dose of an immune interacting agent to increase the effectiveness or safety of a particular dose of the immune interacting agent.
  • an adjuvant might prevent T cell exhaustion and thus increase the effectiveness or safety of a particular immune interacting agent.
  • administering broadly refers to a route of administration of a composition (for example, a pharmaceutical composition) to a subject.
  • routes of administration include oral administration, rectal administration, topical administration, inhalation (nasal) or injection.
  • Administration by injection includes intravenous (IV), intramuscular (IM), intratumoral (IT) and subcutaneous (SC) administration.
  • a therapeutic composition described herein is administered in any form by any effective route, including but not limited to intratumoral, oral, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal (for example, using any standard patch), intradermal, ophthalmic, (intra)nasally, local, non-oral, such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, (trans)rectal, vaginal, intra-arterial, and intrathecal, transmucosal (for example, sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (for example, trans- and perivaginally), implanted, intravesical, intrapulmonary, intraduodenal, intragastrical, and intrabronchial.
  • any effective route including but not limited to intratumoral, oral, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal (for example, using any standard patch), intradermal, ophthalmic
  • a therapeutic composition described herein is administered orally, rectally, intratumorally, topically, intravesically, by injection into or adjacent to a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously.
  • a therapeutic composition described herein is administered orally, intratumorally, or intravenously.
  • a therapeutic composition described herein is administered orally.
  • Cancer broadly refers to an uncontrolled, abnormal growth of a host’s own cells leading to invasion of surrounding tissue and potentially tissue distal to the initial site of abnormal cell growth in the host.
  • Major classes include carcinomas which are cancers of the epithelial tissue (for example, skin, squamous cells); sarcomas which are cancers of the connective tissue (for example, bone, cartilage, fat, muscle, blood vessels, etc.); leukemias which are cancers of blood forming tissue (for example, bone marrow tissue); lymphomas and myelomas which are cancers of immune cells; and central nervous system cancers which include cancers from brain and spinal tissue.
  • carcinomas which are cancers of the epithelial tissue (for example, skin, squamous cells)
  • sarcomas which are cancers of the connective tissue (for example, bone, cartilage, fat, muscle, blood vessels, etc.)
  • leukemias which are cancers of blood forming tissue (for example, bone marrow tissue)
  • cancer refers to all types of cancer or neoplasm or malignant tumors including leukemias, carcinomas and sarcomas, whether new or recurring. Specific examples of cancers are: carcinomas, sarcomas, myelomas, leukemias, lymphomas and mixed type tumors. Non-limiting examples of cancers are new or recurring cancers of the brain, melanoma, bladder, breast, cervix, colon, head and neck, kidney, lung, non-small cell lung, mesothelioma, ovary, prostate, sarcoma, stomach, uterus and medulloblastoma. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer comprises a metastasis.
  • a “carbohydrate” refers to a sugar or polymer of sugars.
  • saccharide a monosaccharide, a disaccharide, tri saccharide, oligosaccharide, or polysaccharide.
  • the most basic carbohydrate is a monosaccharide, such as glucose, galactose, mannose, ribose, arabinose, xylose, and fructose.
  • Disaccharides are two joined monosaccharides.
  • Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose.
  • an oligosaccharide includes between three and six monosaccharide units (for example, raffinose, stachyose), and polysaccharides include six or more monosaccharide units.
  • Exemplary polysaccharides include starch, glycogen, and cellulose.
  • Carbohydrates may contain modified saccharide units such as 2’- deoxyribose wherein a hydroxyl group is removed, 2’ -fluororibose wherein a hydroxyl group is replaced with a fluorine, or N-acetylglucosamine, a nitrogen-containing form of glucose (for example, 2’-fluororibose, deoxyribose, and hexose).
  • Carbohydrates may exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.
  • carcinoma refers to a malignant growth made up of epithelial cells tending to infiltrate the surrounding tissues, and/or resist physiological and non- physiological cell death signals and gives rise to metastases.
  • Cellular augmentation broadly refers to the influx of cells or expansion of cells in an environment that are not substantially present in the environment prior to administration of a composition and not present in the composition itself.
  • Cells that augment the environment include immune cells, stromal cells, bacterial and fungal cells. Environments of particular interest are the microenvironments where cancer cells reside or locate.
  • the microenvironment is a tumor microenvironment or a tumor draining lymph node.
  • the microenvironment is a pre-cancerous tissue site or the site of local administration of a composition or a site where the composition will accumulate after remote administration.
  • Clade refers to the OTUs or members of a phylogenetic tree that are downstream of a statistically valid node in a phylogenetic tree.
  • the clade comprises a set of terminal leaves in the phylogenetic tree that is a distinct monophyletic evolutionary unit and that share some extent of sequence similarity.
  • a “combination” can refer to EVs from one source strain with another agent, for example, another EV (for example, from another strain), with bacteria (for example, of the same or different strain that the EV was obtained from), or with another therapeutic agent.
  • the combination can be in physical co-existence, either in the same material or product or in physically connected products, as well as the temporal co-admini strati on or co-localization of the EVs and other agent.
  • the term “consists essentially of’ means limited to the recited elements and/or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.
  • Dysbiosis refers to a state of the microbiota or microbiome of the gut or other body area, including, for example, mucosal or skin surfaces (or any other microbiome niche) in which the normal diversity and/or function of the host gut microbiome ecological networks ( ’’microbiome”) are disrupted.
  • a state of dysbiosis may result in a diseased state, or it may be unhealthy under only certain conditions or only if present for a prolonged period.
  • Dysbiosis may be due to a variety of factors, including, environmental factors, infectious agents, host genotype, host diet and/or stress.
  • a dysbiosis may result in: a change (for example, increase or decrease) in the prevalence of one or more bacteria types (for example, anaerobic), species and/or strains, change (for example, increase or decrease) in diversity of the host microbiome population composition; a change (for example, increase or reduction) of one or more populations of symbiont organisms resulting in a reduction or loss of one or more beneficial effects; overgrowth of one or more populations of pathogens (for example, pathogenic bacteria); and/or the presence of, and/or overgrowth of, symbiotic organisms that cause disease only when certain conditions are present.
  • the term “decrease” or “deplete” means a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000 or undetectable after treatment when compared to a pre-treatment state.
  • Properties that may be decreased include the number of immune cells, bacterial cells, stromal cells, myeloid derived suppressor cells, fibroblasts, metabolites; the level of a cytokine; or another physical parameter (such as ear thickness (for example, in a DTH animal model) or tumor size (for example, in an animal tumor model)).
  • the term “effective dose” is the amount of the therapeutic composition that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, with the least toxicity to the subject.
  • engineered bacteria are any bacteria that have been genetically altered from their natural state by human activities, and the progeny of any such bacteria.
  • Engineered bacteria include, for example, the products of targeted genetic modification, the products of random mutagenesis screens and the products of directed evolution.
  • epitope means a protein determinant capable of specific binding to an antibody or T cell receptor.
  • Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. Certain epitopes can be defined by a particular sequence of amino acids to which an antibody is capable of binding.
  • EVs Extracellular vesicles
  • smEVs vesicles derived from bacteria
  • EVs are comprised of bacterial lipids and/or bacterial proteins and/or bacterial nucleic acids and/or bacterial carbohydrate moieties, and are isolated from culture supernatant.
  • the natural production of these vesicles can be artificially enhanced (for example, increased) or decreased through manipulation of the environment in which the bacterial cells are being cultured (for example, by media or temperature alterations).
  • EV compositions may be modified to reduce, increase, add, or remove bacterial components or foreign substances to alter efficacy, immune stimulation, stability, immune stimulatory capacity, stability, organ targeting (for example, lymph node), absorption (for example, gastrointestinal), and/or yield (for example, thereby altering the efficacy).
  • purified EV composition or “EV composition” refers to a preparation of EVs that have been separated from at least one associated substance found in a source material (for example, separated from at least one other bacterial component) or any material associated with the EVs in any process used to produce the preparation. It can also refer to a composition that has been significantly enriched for specific components.
  • Extracellular vesicles may also be obtained from mammalian cells and from can be obtained from microbes such as archaea, fungi, microscopic algae, protozoans, and parasites. Extracellular vesicles from any of these sources can be prepared into a solution and/or dried form as described herein.
  • Extracellular vesicles may be artificially-produced vesicles prepared from bacteria, such as pmEVs, for example, obtained by chemically disrupting (for example, by lysozyme and/or lysostaphin) and/or physically disrupting (for example, by mechanical force) bacterial cells and separating the bacterial membrane components from the intracellular components through centrifugation and/or ultracentrifugation, or other methods, can also be prepared into a solution and/or dried form as described herein.
  • bacteria such as pmEVs
  • lysozyme and/or lysostaphin obtained by chemically disrupting (for example, by lysozyme and/or lysostaphin) and/or physically disrupting (for example, by mechanical force) bacterial cells and separating the bacterial membrane components from the intracellular components through centrifugation and/or ultracentrifugation, or other methods, can also be prepared into a solution and/or dried form as described herein.
  • genomic is used broadly to refer to any nucleic acid associated with a biological function.
  • the term “gene” applies to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence.
  • “Identity” as between nucleic acid sequences of two nucleic acid molecules can be determined as a percentage of identity using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., et al., J Molec Biol 215:403 (1990); Guide to Huge Computers, Martin J.
  • the term “immune disorder” refers to any disease, disorder or disease symptom caused by an activity of the immune system, including autoimmune diseases, inflammatory diseases and allergies.
  • Immune disorders include, but are not limited to, autoimmune diseases (for example, psoriasis, atopic dermatitis, lupus, scleroderma, hemolytic anemia, vasculitis, type one diabetes, Grave’s disease, rheumatoid arthritis, multiple sclerosis, Goodpasture’s syndrome, pernicious anemia and/or myopathy), inflammatory diseases (for example, acne vulgaris, asthma, celiac disease, chronic prostatitis, glomerulonephritis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplant rejection, vasculitis and/or interstitial cystitis), and/or an allergies (for example, food allergies, drug allergies and/or environmental allergies).
  • autoimmune diseases for example,
  • Immunotherapy is treatment that uses a subject’s immune system to treat disease (for example, immune disease, inflammatory disease, metabolic disease, cancer) and includes, for example, checkpoint inhibitors, cancer vaccines, cytokines, cell therapy, CAR-T cells, and dendritic cell therapy.
  • disease for example, immune disease, inflammatory disease, metabolic disease, cancer
  • checkpoint inhibitors for example, checkpoint inhibitors, cancer vaccines, cytokines, cell therapy, CAR-T cells, and dendritic cell therapy.
  • the term “increase” means a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 4-fold, 10-fold, 100-fold, 10 A 3 fold, 10 A 4 fold, 10 A 5 fold, 10 A 6 fold, and/or 10 A 7 fold greater after treatment when compared to a pre-treatment state.
  • Properties that may be increased include the number of immune cells, bacterial cells, stromal cells, myeloid derived suppressor cells, fibroblasts, metabolites; the level of a cytokine; or another physical parameter (such as ear thickness (for example, in a DTH animal model) or tumor size (for example, in an animal tumor model).
  • “Innate immune agonists” or “immuno-adjuvants” are small molecules, proteins, or other agents that specifically target innate immune receptors including Toll-Like Receptors (TLR), NOD receptors, RLRs, C-type lectin receptors, STING-cGAS Pathway components, inflammasome complexes.
  • TLR Toll-Like Receptors
  • NOD receptors NOD receptors
  • RLRs C-type lectin receptors
  • STING-cGAS Pathway components inflammasome complexes.
  • LPS is a TLR-4 agonist that is bacterially derived or synthesized and aluminum can be used as an immune stimulating adjuvant
  • immuno-adjuvants are a specific class of broader adjuvant or adjuvant therapy.
  • STING agonists include, but are not limited to, 2'3'- cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, 2'2'-cGAMP, and 2'3'-cGAM(PS)2 (Rp/Sp) (Rp, Sp-isomers of the bis-phosphorothioate analog of 2'3'-cGAMP).
  • TLR agonists include, but are not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10 and TLR11.
  • NOD agonists include, but are not limited to, N-acetylmuramyl- L-alanyl-D-isoglutamine (muramyldipeptide (MDP)), gamma-D-glutamyl-meso- diaminopimelic acid (iE-DAP), and desmuramylpeptides (DMP).
  • MDP N-acetylmuramyl- L-alanyl-D-isoglutamine
  • iE-DAP gamma-D-glutamyl-meso- diaminopimelic acid
  • DMP desmuramylpeptides
  • ITS is a piece of non-functional RNA located between structural ribosomal RNAs (rRNA) on a common precursor transcript often used for identification of eukaryotic species in particular fungi.
  • rRNA structural ribosomal RNAs
  • the rRNA of fungi that forms the core of the ribosome is transcribed as a signal gene and consists of the 8S, 5.8S and 28 S regions with ITS4 and 5 between the 8S and 5.8S and 5.8S and 28 S regions, respectively.
  • isolated or “enriched” encompasses a microbe, an EV (such as a bacterial EV) or other entity or substance that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by the hand of man.
  • Isolated bacteria or EVs may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated.
  • isolated bacteria or EVs are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure, for example, substantially free of other components.
  • leukemia includes broadly progressive, malignant diseases of the hematopoietic organs/sy stems and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow.
  • lipid includes fats, oils, triglycerides, cholesterol, phospholipids, fatty acids in any form including free fatty acids. Fats, oils and fatty acids can be saturated, unsaturated (cis or trans) or partially unsaturated (cis or trans).
  • melanoma is taken to mean a tumor arising from the melanocytic system of the skin and other organs.
  • Microbiome broadly refers to the microbes residing on or in body site of a subject or patient. Microbes in a microbiome may include bacteria, viruses, eukaryotic microorganisms, and/or viruses.
  • microbiome may be metabolically active, dormant, latent, or exist as spores, may exist planktonically or in biofilms, or may be present in the microbiome in sustainable or transient manner.
  • the microbiome may be a commensal or healthy-state microbiome or a disease-state or dysbiotic microbiome.
  • the microbiome may be native to the subject or patient, or components of the microbiome may be modulated, introduced, or depleted due to changes in health state (for example, precancerous or cancerous state) or treatment conditions (for example, antibiotic treatment, exposure to different microbes).
  • the microbiome occurs at a mucosal surface.
  • the microbiome is a gut microbiome.
  • the microbiome is a tumor microbiome.
  • a “microbiome profile” or a “microbiome signature” of a tissue or sample refers to an at least partial characterization of the bacterial makeup of a microbiome.
  • a microbiome profile indicates whether at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bacterial strains are present or absent in a microbiome.
  • a microbiome profile indicates whether at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more cancer-associated bacterial strains are present in a sample.
  • the microbiome profile indicates the relative or absolute amount of each bacterial strain detected in the sample.
  • the microbiome profile is a cancer-associated microbiome profile.
  • a cancer-associated microbiome profile is a microbiome profile that occurs with greater frequency in a subject who has cancer than in the general population.
  • the cancer- associated microbiome profile comprises a greater number of or amount of cancer- associated bacteria than is normally present in a microbiome of an otherwise equivalent tissue or sample taken from an individual who does not have cancer.
  • “Modified” in reference to a bacteria broadly refers to a bacteria that has undergone a change from its wild-type form.
  • Bacterial modification can result from engineering bacteria. Examples of bacterial modifications include genetic modification, gene expression modification, phenotype modification, formulation modification, chemical modification, and dose or concentration. Examples of improved properties are described throughout this specification and include, for example, attenuation, auxotrophy, homing, or antigenicity.
  • Phenotype modification might include, by way of example, bacteria growth in media that modify the phenotype of a bacterium such that it increases or decreases virulence.
  • an “oncobiome” as used herein comprises tumorigenic and/or cancer-associated microbiota, wherein the microbiota comprises one or more of a virus, a bacterium, a fungus, a protist, a parasite, or another microbe.
  • Oncotrophic or “oncophilic” microbes and bacteria are microbes that are highly associated or present in a cancer microenvironment. They may be preferentially selected for within the environment, preferentially grow in a cancer microenvironment or hone to a said environment.
  • “Operational taxonomic units” and “OTU(s)” refer to a terminal leaf in a phylogenetic tree and is defined by a nucleic acid sequence, for example, the entire genome, or a specific genetic sequence, and all sequences that share sequence identity to this nucleic acid sequence at the level of species.
  • the specific genetic sequence may be the 16S sequence or a portion of the 16S sequence.
  • the entire genomes of two entities are sequenced and compared.
  • select regions such as multilocus sequence tags (MLST), specific genes, or sets of genes may be genetically compared.
  • OTUs that share > 97% average nucleotide identity across the entire 16S or some variable region of the 16S are considered the same OTU. See for example, Claesson MJ, Wang Q, O’Sullivan O, Greene-Diniz R, Cole JR, Ross RP, and O’Toole PW. 2010. Comparison of two nextgeneration sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Res 38: e200.
  • OTUs are frequently defined by comparing sequences between organisms. Generally, sequences with less than 95% sequence identity are not considered to form part of the same OTU. OTUs may also be characterized by any combination of nucleotide markers or genes, in particular highly conserved genes (for example, “housekeeping” genes), or a combination thereof. Operational Taxonomic Units (OTUs) with taxonomic assignments made to, for example, genus, species, and phylogenetic clade are provided herein.
  • a gene is “overexpressed” in a bacteria if it is expressed at a higher level in an engineered bacteria under at least some conditions than it is expressed by a wild-type bacteria of the same species under the same conditions.
  • a gene is “underexpressed” in a bacteria if it is expressed at a lower level in an engineered bacteria under at least some conditions than it is expressed by a wild-type bacteria of the same species under the same conditions.
  • polynucleotide and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), micro RNA (miRNA), silencing RNA (siRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • nucleotide structure may be imparted before or after assembly of the polymer.
  • a polynucleotide may be further modified, such as by conjugation with a labeling component.
  • U nucleotides are interchangeable with T nucleotides.
  • a substance is “pure” if it is substantially free of other components.
  • the terms “purify,” “purifying” and “purified” refer to an EV (such as an EV from bacteria) preparation or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (for example, whether in nature or in an experimental setting), or during any time after its initial production.
  • An EV preparation or compositions may be considered purified if it is isolated at or after production, such as from one or more other bacterial components, and a purified microbe or bacterial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “purified.”
  • purified EVs are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • EV compositions (or preparations) are, for example, purified from residual habitat products.
  • the term “purified EV composition” or “EV composition” refers to a preparation that includes EVs from bacteria that have been separated from at least one associated substance found in a source material (for example, separated from at least one other bacterial component) or any material associated with the EVs in any process used to produce the preparation. It also refers to a composition that has been significantly enriched or concentrated. In some embodiments, the EVs are concentrated by 2 fold, 3- fold, 4-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold or more than 10,000 fold.
  • “Residual habitat products” refers to material derived from the habitat for microbiota within or on a subject.
  • fermentation cultures of microbes can contain contaminants, for example, other microbe strains or forms (for example, bacteria, virus, mycoplasm, and/or fungus).
  • microbes live in feces in the gastrointestinal tract, on the skin itself, in saliva, mucus of the respiratory tract, or secretions of the genitourinary tract (i.e., biological matter associated with the microbial community).
  • Substantially free of residual habitat products means that the microbial composition no longer contains the biological matter associated with the microbial environment on or in the culture or human or animal subject and is 100% free, 99% free, 98% free, 97% free, 96% free, or 95% free of any contaminating biological matter associated with the microbial community.
  • Residual habitat products can include abiotic materials (including undigested food) or it can include unwanted microorganisms.
  • Substantially free of residual habitat products may also mean that the microbial composition contains no detectable cells from a culture contaminant or a human or animal and that only microbial cells are detectable.
  • substantially free of residual habitat products may also mean that the microbial composition contains no detectable viral (including bacteria, viruses (for example, phage)), fungal, mycoplasmal contaminants.
  • it means that fewer than lxl0' 2 %, lxl0' 3 %, 1x10" 4 %, lxl0' 5 %, lxl0' 6 %, lxl0' 7 %, lxl0' 8 % of the viable cells in the microbial composition are human or animal, as compared to microbial cells. There are multiple ways to accomplish this degree of purity, none of which are limiting.
  • contamination may be reduced by isolating desired constituents through multiple steps of streaking to single colonies on solid media until replicate (such as, but not limited to, two) streaks from serial single colonies have shown only a single colony morphology.
  • reduction of contamination can be accomplished by multiple rounds of serial dilutions to single desired cells (for example, a dilution of 10' 8 or 10' 9 ), such as through multiple 10- fold serial dilutions. This can further be confirmed by showing that multiple isolated colonies have similar cell shapes and Gram staining behavior.
  • Other methods for confirming adequate purity include genetic analysis (for example, PCR, DNA sequencing), serology and antigen analysis, enzymatic and metabolic analysis, and methods using instrumentation such as flow cytometry with reagents that distinguish desired constituents from contaminants.
  • sarcoma generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar, heterogeneous, or homogeneous substance.
  • specific binding refers to the ability of an antibody to bind to a predetermined antigen or the ability of a polypeptide to bind to its predetermined binding partner.
  • an antibody or polypeptide specifically binds to its predetermined antigen or binding partner with an affinity corresponding to a KD of about 10' 7 M or less, and binds to the predetermined antigen/binding partner with an affinity (as expressed by KD) that is at least 10-fold less, at least 100-fold less or at least 1000-fold less than its affinity for binding to a non-specific and unrelated antigen/binding partner (for example, BSA, casein).
  • specific binding applies more broadly to a two component system where one component is a protein, lipid, or carbohydrate or combination thereof and engages with the second component which is a protein, lipid, carbohydrate or combination thereof in a specific way.
  • Strain refers to a member of a bacterial species with a genetic signature such that it may be differentiated from closely-related members of the same bacterial species.
  • the genetic signature may be the absence of all or part of at least one gene, the absence of all or part of at least on regulatory region (for example, a promoter, a terminator, a riboswitch, a ribosome binding site), the absence (“curing”) of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one foreign gene (a gene derived from another species), the presence at least one mutated regulatory region (for example, a promoter, a terminator, a riboswitch, a ribosome binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof.
  • strains may be identified by PCR amplification optionally followed by DNA sequencing of the genomic region(s) of interest or of the whole genome.
  • strains may be differentiated by selection or counter- sei ection using an antibiotic or nutrient/metabolite, respectively.
  • subject refers to any mammal.
  • a subject or a patient described as “in need thereof’ refers to one in need of a treatment (or prevention) for a disease.
  • Mammals i.e., mammalian animals
  • mammals include humans, laboratory animals (for example, primates, rats, mice), livestock (for example, cows, sheep, goats, pigs), and household pets (for example, dogs, cats, rodents).
  • the subject may be a human.
  • the subject may be a non-human mammal including but not limited to of a dog, a cat, a cow, a horse, a pig, a donkey, a goat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, a gorilla, or a chimpanzee.
  • the subject may be healthy, or may be suffering from a cancer at any developmental stage, wherein any of the stages are either caused by or opportunistically supported of a cancer associated or causative pathogen, or may be at risk of developing a cancer, or transmitting to others a cancer associated or cancer causative pathogen.
  • a subject has lung cancer, bladder cancer, prostate cancer, plasmacytoma, colorectal cancer, rectal cancer, Merkel Cell carcinoma, salivary gland carcinoma, ovarian cancer, and/or melanoma.
  • the subject may have a tumor.
  • the subject may have a tumor that shows enhanced macropinocytosis with the underlying genomics of this process including Ras activation.
  • the subject has another cancer.
  • the subject has undergone a cancer therapy.
  • a therapeutic agent refers to an agent for therapeutic use.
  • a therapeutic agent is a composition comprising EVs (“an EV composition”) that can be used to treat and/or prevent a disease and/or condition.
  • the therapeutic agent is a pharmaceutical agent.
  • a medicinal product, medical food, a food product, or a dietary supplement comprises a therapeutic agent.
  • the therapeutic agent is in a solution, and in other embodiments, a dried form. The dried form embodiments may be produced, for example, by lyophilization or spray drying.
  • the dried form of the therapeutic agent is a lypholized cake or powder.
  • the dried form of the therapeutic agent is a spray-dried powder.
  • the term “therapeutic composition” or “pharmaceutical composition” refers to a composition that comprises a therapeutically effective amount of a therapeutic agent (for example an EV composition described herein).
  • the therapeutic composition is (or is present in) a medicinal product, medical food, a food product, or a dietary supplement.
  • the term “treating” a disease in a subject or “treating” a subject having or suspected of having a disease refers to administering to the subject to a pharmaceutical treatment, for example, the administration of one or more agents, such that at least one symptom of the disease is decreased or prevented from worsening.
  • “treating” refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof.
  • the term “preventing” a disease in a subject refers to administering to the subject to a pharmaceutical treatment, for example, the administration of one or more agents, such that onset of at least one symptom of the disease is delayed or prevented.
  • solutions and/or dried form, and therapeutic compositions that comprise extracellular vesicles (EVs).
  • solutions and/or dried form, and therapeutic compositions that comprise EVs obtained from Fournierella massiliensis bacteria.
  • Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696) is a single strain of Fournierella massiliensis. Extracellular vesicles obtained from Fournierella massiliensis (F. massiliensis EVs) have been shown to have therapeutic effects, for example, as described in PCT/US21/36927 (WO 2021/252838), hereby incorporated by reference in its entirety.
  • the Fournierella massiliensis is Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696) (also referred to as “Fournierella massiliensis Strain A”).
  • the Fournierella massiliensis strain is a strain comprising at least at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696).
  • sequence identity e.g., at least 99.5%
  • F. massiliensis Strain A can be cultured according to methods known in the art. For example, Fournierella massiliensis Strain A can be grown under anaerobic conditions in PM1 l+5g/L Na-L-lactate liquid medium supplemented with 0.05g/L FeSO4, and 0.5 g/L L-cysteine-HCL as reducing agent at 37°C. See also PCT/US21/36927 (WO 2021/252838).
  • the phase of growth can affect the amount or properties of bacteria and/or EVs produced by bacteria.
  • EVs can be isolated, for example, from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • F. massiliensis bacteria from which EVs are obtained are lyophilized.
  • F. massiliensis bacteria from which EVs are obtained are gamma irradiated (for example, at 17.5 or 25 kGy).
  • F. massiliensis bacteria from which EVs are obtained are UV irradiated.
  • F. massiliensis bacteria from which EVs are obtained are heat inactivated (for example, at 50°C for two hours or at 90°C for two hours).
  • F. massiliensis bacteria from which EVs are obtained are acid treated.
  • F. massiliensis bacteria from which EVs are obtained are oxygen sparged (for example, at 0.1 vvm for two hours).
  • the F. massiliensis EVs are lyophilized.
  • the F. massiliensis EVs are gamma irradiated (for example, at 17.5 or 25 kGy).
  • the F. massiliensis EVs are UV irradiated.
  • the F. massiliensis EVs are heat inactivated (for example, at 50°C for two hours or at 90°C for two hours).
  • the F. massiliensis EVs are acid treated.
  • the F. massiliensis EVs are oxygen sparged (for example, at 0.1 vvm for two hours).
  • the bacteria from which the EVs are obtained are modified (for example, engineered) to reduce toxicity or other adverse effects, to enhance delivery) (for example, oral delivery) of the EVs (for example, by improving acid resistance, muco- adherence and/or penetration and/or resistance to bile acids, digestive enzymes, resistance to anti-microbial peptides and/or antibody neutralization), to target desired cell types (for example, M-cells, goblet cells, enterocytes, dendritic cells, macrophages), to enhance their immunomodulatory and/or therapeutic effect of the EVs (for example, either alone or in combination with another therapeutic agent), and/or to enhance immune activation or suppression by the EVs (for example, through modified production of polysaccharides, pili, fimbriae, adhesins).
  • the engineered bacteria described herein are modified to improve EV manufacturing (for example, higher oxygen tolerance, stability, improved freeze-thaw tolerance, shorter generation times).
  • the engineered bacteria described include bacteria harboring one or more genetic changes, such change being an insertion, deletion, translocation, or substitution, or any combination thereof, of one or more nucleotides contained on the bacterial chromosome or endogenous plasmid and/or one or more foreign plasmids, wherein the genetic change may results in the overexpression and/or underexpression of one or more genes.
  • the engineered bacteria may be produced using any technique known in the art, including but not limited to site-directed mutagenesis, transposon mutagenesis, knockouts, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, or any combination thereof.
  • Modified EVs include site-directed mutagenesis, transposon mutagenesis, knockouts, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, or any combination thereof.
  • the massiliensis EVs described herein are modified such that they comprise, are linked to, and/or are bound by a therapeutic moiety.
  • the therapeutic moiety is a cancer-specific moiety.
  • the cancer-specific moiety has binding specificity for a cancer cell (for example, has binding specificity for a cancer-specific antigen).
  • the cancer-specific moiety comprises an antibody or antigen binding fragment thereof.
  • the cancer-specific moiety comprises a T cell receptor or a chimeric antigen receptor (CAR).
  • the cancer-specific moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof.
  • the cancer-specific moiety is a bipartite fusion protein that has two parts: a first part that binds to and/or is linked to the bacterium and a second part that is capable of binding to a cancer cell (for example, by having binding specificity for a cancer-specific antigen).
  • the first part is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP.
  • the first part has binding specificity for the EV (for example, by having binding specificity for a bacterial antigen).
  • the first and/or second part comprises an antibody or antigen binding fragment thereof.
  • the first and/or second part comprises a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the first and/or second part comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof. In certain embodiments, co-administration of the cancer-specific moiety with the EVs (either in combination or in separate administrations) increases the targeting of the EVs to the cancer cells.
  • CAR chimeric antigen receptor
  • the F. massiliensis EVs described herein are engineered such that they comprise, are linked to, and/or are bound by a magnetic and/or paramagnetic moiety (for example, a magnetic bead).
  • the magnetic and/or paramagnetic moiety is comprised by and/or directly linked to the bacteria.
  • the magnetic and/or paramagnetic moiety is linked to and/or a part of an EV-binding moiety that that binds to the EV.
  • the EV-binding moiety is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP.
  • the EV-binding moiety has binding specificity for the EV (for example, by having binding specificity for a bacterial antigen).
  • the EV-binding moiety comprises an antibody or antigen binding fragment thereof.
  • the EV-binding moiety comprises a T cell receptor or a chimeric antigen receptor (CAR).
  • the EV-binding moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof.
  • co-administration of the magnetic and/or paramagnetic moiety with the EVs can be used to increase the targeting of the EVs (for example, to cancer cells and/or a part of a subject where cancer cells are present.
  • F. massiliensis EVs such as secreted EVs (smEVs) described herein are prepared using any method known in the art.
  • the F. massiliensis EVs (such as secreted EVs (smEVs) are prepared without an EV purification step.
  • F. massiliensis bacteria described herein are killed using a method that leaves the EVs intact and the resulting bacterial components, including the EVs, are used in the methods and compositions described herein.
  • the bacteria are killed using an antibiotic (for example, using an antibiotic described herein).
  • the bacteria are killed using UV irradiation.
  • the bacteria are heat- killed.
  • the F. massiliensis EVs described herein are purified from one or more other bacterial components. Methods for purifying EVs from F. massiliensis bacteria are known in the art. In some embodiments, F. massiliensis EVs are prepared from bacterial cultures using methods described in S. Bin Park, et al. PLoS ONE.
  • the bacteria are cultured to high optical density and then centrifuged to pellet bacteria (for example, at 10,000 x g for 30 min at 4°C, at 15,500 x g for 15 min at 4°C).
  • the culture supernatants are then passed through filters to exclude intact bacterial cells (for example, a 0.22 pm filter).
  • the supernatants are then subjected to tangential flow filtration, during which the supernatant is concentrated, species smaller than 100 kDa are removed, and the media is partially exchanged with PBS.
  • filtered supernatants are centrifuged to pelletF. massiliensis EVs (for example, at 100,000-150,000 x g for 1-3 hours at 4°C, at 200,000 x g for 1-3 hours at 4°C). In some embodiments, the F.
  • massiliensis EVs are further purified by resuspending the resulting EV pellets (for example, in PBS), and applying the resuspended EVs to an Optiprep (iodixanol) gradient or gradient (for example, a 30-60% discontinuous gradient, a 0-45% discontinuous gradient), followed by centrifugation (for example, at 200,000 x g for 4-20 hours at 4°C).
  • EV bands can be collected, diluted with PBS, and centrifuged to pellet the EVs (for example, at 150,000 x g for 3 hours at 4°C, at 200,000 x g for 1 hour at 4°C).
  • the purified F. massiliensis EVs can be stored, for example, at -80°C or -20°C until use.
  • the F. massiliensis EVs are further purified by treatment with DNase and/or proteinase K.
  • cultures of F. massiliensis bacteria can be centrifuged at 11,000 x g for 20-40 min at 4°C to pellet bacteria.
  • Culture supernatants may be passed through a 0.22 pm filter to exclude intact bacterial cells. Filtered supernatants may then be concentrated using methods that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration.
  • ammonium sulfate precipitation 1.5-3 M ammonium sulfate can be added to filtered supernatant slowly, while stirring at 4°C.
  • Precipitations can be incubated at 4°C for 8-48 hours and then centrifuged at 11,000 x g for 20-40 min at 4°C. The resulting pellets contain bacteria EVs and other debris.
  • filtered supernatants can be centrifuged at 100,000-200,000 x g for 1-16 hours at 4°C. The pellet of this centrifugation contains bacterial EVs and other debris such as large protein complexes.
  • a filtration technique such as through the use of an Amicon Ultra spin filter or by tangential flow filtration, supernatants can be filtered so as to retain species of molecular weight > 50 or 100 kDa.
  • F. massiliensis EVs can be obtained from bacteria cultures continuously during growth, or at selected time points during growth, for example, by connecting a bioreactor to an alternating tangential flow (ATF) system (for example, XCell ATF from Repligen).
  • ATF alternating tangential flow
  • the ATF system retains intact cells (>0.22 pm) in the bioreactor, and allows smaller components (for example, EVs, free proteins) to pass through a filter for collection.
  • the system may be configured so that the ⁇ 0.22 pm filtrate is then passed through a second filter of 100 kDa, allowing species such as EVs between 0.22 pm and 100 kDa to be collected, and species smaller than 100 kDa to be pumped back into the bioreactor.
  • the system may be configured to allow for medium in the bioreactor to be replenished and/or modified during growth of the culture.
  • EVs collected by this method may be further purified and/or concentrated by ultracentrifugation or filtration as described above for filtered supernatants.
  • F. massiliensis EVs obtained by methods provided herein may be further purified by size-based column chromatography, by affinity chromatography, by ion-exchange chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0.
  • the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in PBS and 3 volumes of 60% Optiprep are added to the sample.
  • the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep.
  • Samples are applied to a 0-45% discontinuous Optiprep gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C, for example, 4-24 hours at 4°C.
  • F. massiliensis EVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 pm filter to exclude intact cells. To further increase purity, isolated F. massiliensis EVs may be DNase or proteinase K treated.
  • purified EVs are processed as described previously (G. Norheim, et al. PLoS ONE. 10(9): e0134353 (2015)). Briefly, after sucrose gradient centrifugation, bands containing EVs are resuspended to a final concentration of 50 pg/mL in a solution containing 3% sucrose or other solution suitable for in vivo injection known to one skilled in the art. This solution may also contain adjuvant, for example aluminum hydroxide at a concentration of 0-0.5% (w/v). In some embodiments, for preparation of F.
  • EVs in PBS are sterile-filtered to ⁇ 0.22 pm.
  • samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 using filtration (for example, Amicon Ultra columns), dialysis, or ultracentrifugation (200,000 x g, > 3 hours, 4°C) and resuspension.
  • the sterility of the F. massiliensis EV preparations can be confirmed by plating a portion of the EVs onto agar medium used for standard culture of the bacteria used in the generation of the EVs and incubating using standard conditions.
  • select F. massiliensis EVs are isolated and enriched by chromatography and binding surface moieties on EVs.
  • select EVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.
  • F. massiliensis EVs are analyzed, for example, as described in Jeppesen, et al. Cell 177:428 (2019).
  • F. massiliensis EVs are lyophilized.
  • F. massiliensis EVs are gamma irradiated (for example, at 17.5 or 25 kGy).
  • E F. massiliensis Vs are UV irradiated.
  • F. massiliensis EVs are heat inactivated (for example, at 50°C for two hours or at 90°C for two hours).
  • F. massiliensis EVs are acid treated.
  • F. massiliensis EVs are oxygen sparged (for example, at 0.1 vvm for two hours).
  • the phase of growth can affect the amount or properties of bacteria and/or EVs produced by bacteria.
  • EVs can be isolated, for example, from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • the growth environment (for example, culture conditions) can affect the amount of EVs produced by bacteria.
  • the yield of EVs can be increased by an EV inducer, as provided in Table 1.
  • the methods can optionally include exposing a culture of bacteria to an EV inducer prior to isolating EVs from the bacterial culture.
  • the culture of bacteria can be exposed to an EV inducer at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • F. massiliensis EVs such as processed EVs (pmEVs) described herein are prepared (for example, artificially prepared) using any method known in the art.
  • the F. massiliensis pmEVs are prepared without a pmEV purification step.
  • bacteria from which the pmEVs described herein are released are killed using a method that leaves the bacterial pmEVs intact, and the resulting bacterial components, including the pmEVs, are used in the methods and compositions described herein.
  • the bacteria are killed using an antibiotic (for example, using an antibiotic described herein).
  • the bacteria are killed using UV irradiation.
  • the F. massiliensis pmEVs described herein are purified from one or more other bacterial components.
  • Methods for purifying pmEVs from bacteria (and optionally, other bacterial components) are known in the art.
  • pmEVs are prepared from bacterial cultures using methods described in Thein, et al. (J. Proteome Res. 9(12):6135-6147 (2010)) or Sandrini, et al. (Bio-protocol 4(21): el287 (2014)), each of which is hereby incorporated by reference in its entirety.
  • the bacteria are cultured to high optical density and then centrifuged to pellet bacteria (for example, at 10,000- 15,000 x g for 10- 15 min at room temperature or 4°C).
  • the supernatants are discarded and cell pellets are frozen at -80°C.
  • cell pellets are thawed on ice and resuspended in 100 mM Tris-HCl, pH 7.5 supplemented with 1 mg/mL DNase I.
  • cells are lysed using an Emulsiflex C-3 (Avestin, Inc.) under conditions recommended by the manufacturer.
  • debris and unlysed cells are pelleted by centrifugation at 10,000 x g for 15 min at 4°C. In some embodiments, supernatants are then centrifuged at 120,000 x g for 1 hour at 4°C. In some embodiments, pellets are resuspended in ice-cold 100 mM sodium carbonate, pH 11, incubated with agitation for 1 hour at 4°C, and then centrifuged at 120,000 x g for 1 hour at 4°C.
  • pellets are resuspended in 100 mM Tris-HCl, pH 7.5, re-centrifuged at 120,000 x g for 20 min at 4°C, and then resuspended in 0.1 M Tris-HCl, pH 7.5 or in PBS. In some embodiments, samples are stored at -20°C.
  • F. massiliensis pmEVs are obtained by methods adapted from Sandrini et al, 2014.
  • bacterial cultures are centrifuged at 10,000- 15,500 x g for 10-15 min at room temp or at 4°C.
  • cell pellets are frozen at -80°C and supernatants are discarded.
  • cell pellets are thawed on ice and resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA supplemented with 0.1 mg/mL lysozyme.
  • samples are incubated with mixing at room temp or at 37°C for 30 min.
  • samples are re-frozen at -80°C and thawed again on ice.
  • DNase I is added to a final concentration of 1.6 mg/mL and MgCh to a final concentration of 100 mM.
  • samples are sonicated using a QSonica Q500 sonicator with 7 cycles of 30 sec on and 30 sec off.
  • debris and unlysed cells are pelleted by centrifugation at 10,000 x g for 15 min. at 4°C. In some embodiments, supernatants are then centrifuged at 110,000 x g for 15 min at 4°C. In some embodiments, pellets are resuspended in 10 mM Tris-HCl, pH 8.0, 2% Triton X-100 and incubated 30-60 min with mixing at room temperature. In some embodiments, samples are centrifuged at 110,000 x g for 15 min at 4°C. In some embodiments, pellets are resuspended in PBS and stored at -20°C.
  • a method of forming (for example, preparing) isolated F. massiliensis pmEVs comprises the steps of: (a) centrifuging a bacterial culture, thereby forming a first pellet and a first supernatant, wherein the first pellet comprises cells; (b) discarding the first supernatant; (c) resuspending the first pellet in a solution; (d) lysing the cells; (e) centrifuging the lysed cells, thereby forming a second pellet and a second supernatant; (f) discarding the second pellet and centrifuging the second supernatant, thereby forming a third pellet and a third supernatant; (g) discarding the third supernatant and resuspending the third pellet in a second solution, thereby forming the isolated bacterial pmEVs.
  • the method further comprises the steps of: (h) centrifuging the solution of step (g), thereby forming a fourth pellet and a fourth supernatant; (i) discarding the fourth supernatant and resuspending the fourth pellet in a third solution. In some embodiments, the method further comprises the steps of: (j) centrifuging the solution of step (i), thereby forming a fifth pellet and a fifth supernatant; and (k) discarding the fifth supernatant and resuspending the fifth pellet in a fourth solution.
  • the centrifugation of step (a) is at 10,000 x g. In some embodiments the centrifugation of step (a) is for 10-15 minutes. In some embodiments, the centrifugation of step (a) is at 4°C or room temperature. In some embodiments, step (b) further comprises freezing the first pellet at -80 °C.
  • the solution in step (c) is 100 mM Tris-HCl, pH 7.5 supplemented with 1 mg/ml DNasel. In some embodiments, the solution in step (c) is 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, supplemented with 0.1 mg/ml lysozyme.
  • step (c) further comprises incubating for 30 minutes at 37°C or room temperature. In some embodiments, step (c) further comprises freezing the first pellet at -80°C. In some embodiments, step (c) further comprises adding DNase I to a final concentration of 1.6 mg/ml. In some embodiments, step (c) further comprises adding MgChto a final concentration of 100 mM.
  • the cells are lysed in step (d) via homogenization. In some embodiments, the cells are lysed in step (d) via emulsiflex C3. In some embodiments, the cells are lysed in step (d) via sonication.
  • the cells are sonicated in 7 cycles, wherein each cycle comprises 30 seconds of sonication and 30 seconds without sonication.
  • the centrifugation of step (e) is at 10,000 x g. In some embodiments, the centrifugation of step (e) is for 15 minutes. In some embodiments, the centrifugation of step (e) is at 4°C or room temperature.
  • the centrifugation of step (f) is at 120,000 x g. In some embodiments, the centrifugation of step (f) is at 110,000 x g. In some embodiments, the centrifugation of step (f) is for 1 hour. In some embodiments, the centrifugation of step (f) is for 15 minutes. In some embodiments, the centrifugation of step (f) is at 4°C or room temperature.
  • the second solution in step (g) is 100 mM sodium carbonate, pH 11. In some embodiments, the second solution in step (g) is 10 mM Tris- HCl pH 8.0, 2% triton X-100.
  • step (g) further comprises incubating the solution for 1 hour at 4°C. In some embodiments, step (g) further comprises incubating the solution for 30-60 minutes at room temperature. In some embodiments, the centrifugation of step (h) is at 120,000 x g. In some embodiments, the centrifugation of step (h) is at 110,000 x g. In some embodiments, the centrifugation of step (h) is for 1 hour. In some embodiments, the centrifugation of step (h) is for 15 minutes. In some embodiments, the centrifugation of step (h) is at 4°C or room temperature. In some embodiments, the third solution in step (i) is 100 mM Tris-HCl, pH 7.5.
  • the third solution in step (i) is PBS.
  • the centrifugation of step (j) is at 120,000 x g. In some embodiments, the centrifugation of step (j) is for 20 minutes. In some embodiments, the centrifugation of step (j) is at 4°C or room temperature.
  • the fourth solution in step (k) is 100 mM Tris- HC1, pH 7.5 or PBS.
  • F. massiliensis pmEVs obtained by methods provided herein may be further purified by size based column chromatography, by affinity chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column.
  • Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 35% Optiprep in PBS. In some embodiments, if filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3- 24 hours at 4°C.
  • pmEVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 pm filter to exclude intact cells. To further increase purity, isolated pmEVs may be DNase or proteinase K treated.
  • the sterility of the F. massiliensis pmEV preparations can be confirmed by plating a portion of the pmEVs onto agar medium used for standard culture of the bacteria used in the generation of the pmEVs and incubating using standard conditions.
  • select F. massiliensis pmEVs are isolated and enriched by chromatography and binding surface moi eties on pmEVs.
  • select pmEVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.
  • F. massiliensis pmEVs are analyzed, for example, as described in Jeppesen, et al. Cell 177:428 (2019).
  • F. massiliensis pmEVs are lyophilized.
  • F. massiliensis pmEVs are gamma irradiated (for example, at 17.5 or 25 kGy).
  • F. massiliensis pmEVs are UV irradiated.
  • F. massiliensis pmEVs are heat inactivated (for example, at 50°C for two hours or at 90°C for two hours).
  • F. massiliensis pmEVs are acid treated.
  • F. massiliensis pmEVs are oxygen sparged (for example, at 0.1 vvm for two hours).
  • pmEVs can be isolated , for example, from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • a solution includes F. massiliensis EVs and an excipient that comprises a bulking agent.
  • a solution includes F. massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • a solution includes F. massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the bulking agent comprises mannitol, sucrose, maltodextrin, dextran, Ficoll, or PVP-K30.
  • the excipient optionally includes an additional component such as trehalose, mannitol, sucrose, sorbitol, maltodextrin, dextran, poloxamer 188, maltodextrin, PVP-K30, Ficoll, citrate, arginine, and/or hydroxypropyl-B-cyclodextrin.
  • a solution contains a liquid preparation of EVs and an excipient that comprises a bulking agent, for example, an excipient from a stock of a formula provided in Tables A or F.
  • a solution includes a liquid preparation containing F. massiliensis (for example, obtained by isolating F. massiliensis EVs from aF. massiliensis bacterial culture (such as the supernatant) or a retentate) and an excipient that comprises a bulking agent, for example, a liquid preparation containing F. massiliensis EVs is combined with an excipient stock that comprises a bulking agent, for example, an excipient stock of a formula provided in Tables A or F, to prepare the solution.
  • F. massiliensis for example, obtained by isolating F. massiliensis EVs from aF. massiliensis bacterial culture (such as the supernatant) or a retentate
  • an excipient that comprises a bulking agent for
  • a “dried form” that contains F. massiliensis refers to the product resulting from drying a solution that contains F. massiliensis EVs. In some embodiments, the drying is performed by freeze-drying (lyophilization) or spray drying. In some embodiments, the dried form is a powder. As used herein, a powder refers to a type of dried form and includes a lyophilized powder, but includes powders, such as spray-dried powders, obtained by methods such as spray drying.
  • the resulting product is a lyophilate.
  • the dried form is a lyophilate.
  • a lyophilate refers to a type of dried form and includes a lyophilized powder and lyophilized cake.
  • the lyophilized cake is milled (for example, ground) to produce a lyophilized powder. Milling refers to mechanical size reduction of solids. Grinding is a type of milling, for example, that can be performed on dried forms. See, for example, Seibert et al., “MILLING OPERATIONS IN THE PHARMACEUTICAL INDUSTRY” in Chemical Engineering in the Pharmaceutical Industry: R&D to Manufacturing, Edited by David J. am Ende (2011).
  • the disclosure also provides dried forms, in some embodiments, such as lyophilates, that comprise F. massiliensis EVs (for example, EVs from a F. massiliensis strain and/or a combination of EVs from different F. massiliensis strains described herein), and an excipient.
  • a dried form can include F. massiliensis EVs and an excipient that comprises a bulking agent.
  • a dried form can include F. massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • a dried form can include F. massiliensis EVs and an excipient that comprises a lyoprotectant.
  • F. massiliensis EVs are combined with an excipient that comprises a bulking agent and/or lyoprotectant, for example, to prepare a solution.
  • the solution is dried.
  • the resulting dried form (for example, lyophilate) contains F. massiliensis EVs and a component(s) of the excipient, for example, bulking agent and/or lyoprotectant (for example, in dried form).
  • the disclosure also provides dried forms of F. massiliensis EVs and an excipient.
  • the dried form is a lyophilate, for example, such as a lyophilized cake or lyophilized powder.
  • the dried form is a powder, for example, such as a spray-dried powder or lyophilized powder.
  • the bulking agent comprises mannitol, sucrose, maltodextrin, dextran, Ficoll, or PVP-K30.
  • the excipient includes an additional component such as trehalose, mannitol, sucrose, sorbitol, dextran, pol oxamer 188, maltodextrin, PVP-K30, Ficoll, citrate, arginine, and/or hydroxypropyl-B-cyclodextrin.
  • a dried form contains F. massiliensis EVs and an excipient, for example, that comprises a bulking agent, for example, an excipient from a stock of a formula provided in Tables A or F.
  • the dried form has a moisture content below about 6% (or below about 5%) (for example, as determined by Karl Fischer titration).
  • the dried form has about 10% to about 80% (by weight) of an excipient, for example, an excipient that comprises a bulking agent. In some embodiments, the dried form has about 10% to about 80% (by weight) of an excipient, for example, an excipient from a stock of a formula provided in Tables A or F. In some embodiments, the F. massiliensis EVs comprise about 1% to about 99% of the total solids by weight of the dried form. In some embodiments, the dried form has at least about lelO particles per mg of the dried form (for example, as determined by particles per mg, such as by NTA).
  • the particles of the dried form have a hydrodynamic diameter (Z average, Zave) of about 132 nm to about 315.2 nm after resuspension from the dried form (for example, resuspension in deionized water) (for example, as determined by dynamic light scattering).
  • the solutions and/or dried forms comprise F. massiliensis EVs substantially or entirely free of whole bacteria (for example, live bacteria, killed bacteria, and/or attenuated bacteria). In some embodiments, the solutions and/or dried forms comprise both F. massiliensis EVs and whole bacteria (for example, live bacteria, killed bacteria, and/or attenuated bacteria). In some embodiments, the solutions and/or dried forms comprise F. massiliensis EVs from one or more F. massiliensis strain. In some embodiments, the solutions and/or dried forms comprise gamma irradiated F. massiliensis EVs. In some embodiments, the F. massiliensis EVs are gamma irradiated after the EVs are isolated (for example, prepared).
  • NTA nanoparticle tracking analysis
  • DLS dynamic light scattering
  • Coulter counting reveals the numbers of bacteria and/or EVs from bacteria in a given sample.
  • Coulter counting reveals the numbers of particles with diameters of 0.7-10 pm.
  • the Coulter counter alone can reveal the number of bacteria and/or EVs in a sample.
  • NTA a Nanosight instrument can be obtained from Malvern Pananlytical.
  • the NS300 can visualize and measure particles in suspension in the size range 10-2000 nm.
  • NTA allows for counting of the numbers of particles that are, for example, 50-1000 nm in diameter.
  • DLS reveals the distribution of particles of different diameters within an approximate range of 1 nm - 3 pm.
  • F. massiliensis EVs are characterized by analytical methods known in the art (for example, Jeppesen, et al. Cell 177:428 (2019)).
  • the F. massiliensis EVs are quantified based on particle count.
  • particle count of an F. massiliensis EV preparation can be measured using NTA.
  • the F. massiliensis EVs are quantified based on the amount of protein, lipid, or carbohydrate.
  • total protein content of an F. massiliensis EV preparation is measured using the Bradford assay or BCA.
  • the F. massiliensis EVs are isolated away from one or more other bacterial components of the source bacteria.
  • the solution and/or dried form further comprises other bacterial components.
  • the F. massiliensis EV liquid preparation obtained from the source bacteria may be fractionated into subpopulations based on the physical properties (for example, size, density, protein content, and/or binding affinity) of the subpopulations.
  • One or more of the EV subpopulations (for example, as a liquid preparation) can then be incorporated into the solutions, powders and/or lyophilates of the invention.
  • solutions and/or dried forms (and therapeutic compositions thereof) comprising F. massiliensis EVs useful for the treatment and/or prevention of disease (for example, a cancer, an autoimmune disease, an inflammatory disease, dysbiosis, or a metabolic disease), as well as methods of making and/or identifying such EVs, and methods of using such solutions and/or dried forms (and therapeutic compositions thereof) (for example, for the treatment of a cancer, an autoimmune disease, an inflammatory disease, dysbiosis, or a metabolic disease, either alone or in combination with other therapeutics).
  • the therapeutic compositions comprise both F. massiliensis EVs, and whole F.
  • the therapeutic compositions comprise F. massiliensis EVs in the absence of F. massiliensis bacteria (for example, 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 compositions comprise F. massiliensis EVs and/or bacteria from one or more strain.
  • the solution and/or dried form is added to or incorporated into a food product (for example, a food or beverage) such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a probiotic, a food or beverage for patients, or an animal feed.
  • a food product for example, a food or beverage
  • a food or beverage for infants such as a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group
  • a functional food such as a beverage, a food or beverage for specified health use, a dietary supplement, a probiotic, a food or beverage for patients, or an animal feed.
  • the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products, including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, capsules, liquid
  • the solution and/or dried form is added to a food product or food supplement for animals, including humans.
  • the 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, cattle, horses, sheep, goats, chickens, ducks, ostriches, turkeys, dogs, cats, rabbits, hamsters, mice, rats, monkeys, and the like, but the animals are not limited thereto.
  • a solution and/or dried form provided herein is formulated into a therapeutic composition.
  • therapeutic compositions comprising a solution and/or dried form described herein.
  • the therapeutic composition comprises a solution and/or dried form provided herein and a pharmaceutically acceptable carrier.
  • the therapeutic composition comprises a pharmaceutically acceptable excipient, such as a glidant, lubricant, and/or diluent.
  • compositions comprising F. massiliensis EVs useful for the treatment and/or prevention of disease (for example, a cancer, an autoimmune disease, an inflammatory disease, dysbiosis, or a metabolic disease), as well as methods of making and/or identifying such EVs, and methods of using such therapeutic compositions (for example, for the treatment of a cancer, an autoimmune disease, an inflammatory disease, dysbiosis, or a metabolic disease, either alone or in combination with other therapeutics).
  • the therapeutic compositions comprise both F. massiliensis EVs and whole bacteria (for example, live bacteria, killed bacteria, attenuated bacteria).
  • the therapeutic compositions comprise F.
  • the therapeutic compositions comprise F. massiliensis EVs and/or bacteria from one or more strain.
  • therapeutic compositions for administration to a subject (for example, human subject).
  • the therapeutic compositions are combined with additional active and/or inactive materials to produce a final product, which may be in single dosage unit or in a multi-dose format.
  • the therapeutic composition is combined with an adjuvant such as an immuno-adjuvant (for example, a STING agonist, a TLR agonist, or a NOD agonist).
  • an adjuvant such as an immuno-adjuvant (for example, a STING agonist, a TLR agonist, or a NOD agonist).
  • the therapeutic composition comprises at least one carbohydrate.
  • the therapeutic composition comprises at least one lipid.
  • the lipid comprises at least one fatty acid selected from lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16: 1), margaric acid (17:0), heptadecenoic acid (17: 1), stearic acid (18:0), oleic acid (18: 1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20: 1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EP A), docosanoic acid (22:0), docosenoic acid (22: 1), docosapentaenoic acid (22:5), dococosanoic acid (22:0
  • the therapeutic composition comprises at least one supplemental mineral or mineral source.
  • supplemental mineral or mineral source examples include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium.
  • Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.
  • the therapeutic composition comprises at least one supplemental vitamin.
  • the at least one vitamin can be fat-soluble or water-soluble vitamins.
  • Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin.
  • Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.
  • the therapeutic composition comprises an excipient, such as a pharmaceutically acceptable excipient.
  • excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.
  • the excipient is a buffering agent.
  • suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.
  • the excipient comprises a preservative.
  • suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.
  • the therapeutic composition comprises a binder as an excipient.
  • suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof.
  • the therapeutic composition comprises a lubricant as an excipient.
  • suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil.
  • the therapeutic composition comprises a dispersion enhancer as an excipient.
  • suitable dispersants include starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose as high HLB emulsifier surfactants.
  • the therapeutic composition comprises a disintegrant as an excipient.
  • the disintegrant is a non-effervescent disintegrant.
  • suitable non-effervescent disintegrants include starches such as com starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pectin, and tragacanth.
  • the disintegrant is an effervescent disintegrant.
  • the therapeutic composition is a food product (for example, a food or beverage) such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.
  • a food product for example, a food or beverage
  • a food or beverage such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.
  • the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, and Chinese soups; soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products, including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, carb
  • the therapeutic composition is a food product for animals, including humans.
  • the 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, cattle, horses, sheep, goats, chickens, wild ducks, ostriches, domestic ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, and the like, but the animals are not limited thereto.
  • a therapeutic composition comprising a dried form is formulated as a solid dosage form, (also referred to as “solid dose form”) for example, for oral administration.
  • the solid dosage form comprises one or more excipients, for example, pharmaceutically acceptable excipients, in addition to the dried form.
  • the dried form in the solid dosage form contains isolated F. massiliensis EVs.
  • the F. massiliensis EVs in the solid dosage form are gamma irradiated.
  • the solid dosage form comprises a tablet, a minitablet, a capsule, or a powder; or a combination of these forms (for example, minitablets comprised in a capsule).
  • the solid dosage form described herein is a capsule. In some embodiments, the solid dosage form described herein is a tablet or a minitablet. Further, in some embodiments, a plurality of minitablets are in (for example, loaded into) a capsule.
  • the solid dosage form comprises a capsule.
  • the capsule is a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule or similar sizes, such as OOel (elongated size 00 capsule).
  • the capsule is a size 0 capsule.
  • the size of the capsule refers to the size of the tablet prior to application of an enteric coating.
  • the capsule is banded after loading (and prior to enterically coating the capsule).
  • the capsule is banded with an HPMC -based banding solution.
  • the solid dosage form comprises a tablet (> 4 mm) (for example, 5 mm- 17 mm).
  • the tablet is a 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, or 18 mm tablet.
  • the size refers to the diameter of the tablet, as is known in the art. As used herein, the size of the tablet refers to the size of the tablet prior to application of an enteric coating.
  • the solid dosage form comprises a minitablet.
  • the minitablet is in the size range of 1 mm-4 mm range.
  • the minitablet is a 1 mm minitablet, 1.5 mm minitablet, 2 mm minitablet, 3 mm minitablet, or 4 mm minitablet.
  • the size refers to the diameter of the minitablet, as is known in the art.
  • the size of the minitablet refers to the size of the minitablet prior to application of an enteric coating.
  • the minitablets are in a capsule.
  • the capsule is a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule or similar sizes, such as OOel (elongated size 00 capsule).
  • the capsule that contains the minitablets comprises HPMC (hydroxyl propyl methyl cellulose) or gelatin.
  • the minitablets are inside a capsule: the number of minitablets inside a capsule will depend on the size of the capsule and the size of the minitablets. As an example, a size 0 capsule can contain 31-35 (an average of 33) minitablets that are 3 mm minitablets.
  • the capsule is banded after loading. In some embodiments, the capsule is banded with an HPMC-based banding solution.
  • a therapeutic composition comprising a solution and/or dried is formulated as a suspension, for example a dried form is reconstituted or a solution is diluted), for example, for oral administration or for injection. Administration by injection includes intravenous (IV), intramuscular (IM), intratumoral (IT) and subcutaneous (SC) administration.
  • IV intravenous
  • IM intramuscular
  • IT intratumoral
  • SC subcutaneous
  • F. massiliensis EVs are in a buffer, for example, a pharmaceutically acceptable buffer, for example, saline or PBS.
  • a therapeutic composition comprising a solution and/or dried form (for example, that comprises F. massiliensis EVs and a bulking agent) is formulated as a suspension for example, a dried form is reconstituted; a solution is diluted), for example, for topical administration.
  • the suspension comprises one or more excipients, for example, pharmaceutically acceptable excipients.
  • the suspension comprises sucrose or glucose.
  • the F. massiliensis EVs in the solution or dried form are isolated F. massiliensis EVs.
  • the F. massiliensis EVs in the suspension are gamma irradiated.
  • a solid dosage form for example, capsule, tablet or minitablet described herein is enterically coated, for example, with one enteric coating layer or with two layers of enteric coating, for example, an inner enteric coating and an outer enteric coating.
  • the inner enteric coating and outer enteric coating are not identical (for example, the inner enteric coating and outer enteric coating do not contain the same components in the same amounts).
  • the enteric coating allows for release of the therapeutic agent (such as bacterial EVs, dried forms, and/or solid dosage forms thereof), for example, in the small intestine.
  • Release of the therapeutic agent in the small intestine allows the therapeutic agent to target and affect cells (for example, epithelial cells and/or immune cells) located at these specific locations, for example, which can cause a local effect in the gastrointestinal tract and/or cause a systemic effect (for example, an effect outside of the gastrointestinal tract).
  • cells for example, epithelial cells and/or immune cells located at these specific locations, for example, which can cause a local effect in the gastrointestinal tract and/or cause a systemic effect (for example, an effect outside of the gastrointestinal tract).
  • EUDRAGIT is the brand name for a diverse range of polymethacrylate-based copolymers. It includes anionic, cationic, and neutral copolymers based on methacrylic acid and methacrylic/acrylic esters or their derivatives.
  • Examples of other materials that can be used in the enteric coating include cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), poly(vinyl acetate phthalate) (PVAP), hydroxypropyl methylcellulose phthalate (HPMCP), fatty acids, waxes, shellac (esters of aleurtic acid), plastics, plant fibers, zein, Aqua-Zein® (an aqueous zein formulation containing no alcohol), amylose starch, starch derivatives, dextrins, methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetate succinate), methyl methacrylate-methacrylic acid copolymers, and/or sodium alginate.
  • CAP cellulose acetate phthalate
  • CAT cellulose acetate trimellitate
  • PVAP poly(vinyl acetate phthalate)
  • the enteric coating (for example, the one enteric coating or the inner enteric coating and/or the outer enteric coating) includes a methacrylic acid ethyl acrylate (MAE) copolymer (1 : 1).
  • MAE methacrylic acid ethyl acrylate
  • the one enteric coating includes methacrylic acid ethyl acrylate (MAE) copolymer (1 :1) (such as Kollicoat MAE 100P).
  • MAE methacrylic acid ethyl acrylate
  • the one enteric coating includes a Eudragit copolymer, for example, a Eudragit L (for example, Eudragit L 100-55; Eudragit L 30 D-55), a Eudragit S, a Eudragit RL, a Eudragit RS, a Eudragit E, or a Eudragit FS (for example, Eudragit FS 30 D).
  • a Eudragit L for example, Eudragit L 100-55; Eudragit L 30 D-55
  • Eudragit S for example, Eudragit L 100-55; Eudragit L 30 D-55
  • Eudragit S for example, Eudragit S
  • RL Eudragit RL
  • Eudragit RS Eudragit RS
  • Eudragit E Eudragit E
  • Eudragit FS for example, Eudragit FS 30 D
  • enteric coating for example, the one enteric coating or the inner enteric coating and/or the outer enteric coating
  • materials that can be used in the enteric coating include those described in, for example, U.S. 6312728; U.S. 6623759; U.S. 4775536; U.S. 5047258; U.S. 5292522; U.S. 6555124; U.S. 6638534; U.S. 2006/0210631; U.S. 2008/200482; U.S. 2005/0271778; U.S. 2004/0028737; WO 2005/044240.
  • methacrylic acid copolymers include: poly(methacrylic acid, methyl methacrylate) 1 : 1 sold, for example, under the Eudragit LI 00 trade name; poly(methacrylic acid, ethyl acrylate) 1 :1 sold, for example, under the Eudragit LI 00-55 trade name; partially-neutralized poly(methacrylic acid, ethyl acrylate) 1 : 1 sold, for example, under the Kollicoat MAE- 1 OOP trade name; and poly(methacrylic acid, methyl methacrylate) 1 :2 sold, for example, under the Eu
  • the disclosure also provides methods of preparing solutions of F. massiliensis EVs and an excipient that comprises a bulking agent.
  • the bulking agent comprises mannitol, sucrose, polyethylene glycol (PEG, such as PEG 6000), cyclodextrin, maltodextrin, dextran, Ficoll, or PVP-K30.
  • the excipient comprises a lyoprotectant.
  • the excipient optionally includes an additional component such as trehalose, mannitol, sucrose, sorbitol, dextran, poloxamer 188, maltodextrin, PVP-K30, Ficoll, citrate, arginine, and/or hydroxypropyl-B-cyclodextrin.
  • an additional component such as trehalose, mannitol, sucrose, sorbitol, dextran, poloxamer 188, maltodextrin, PVP-K30, Ficoll, citrate, arginine, and/or hydroxypropyl-B-cyclodextrin.
  • a liquid preparation of F. massiliensis EVs and an excipient that comprises a bulking agent are combined to prepare a solution.
  • a liquid preparation of F. massiliensis EVs for example, obtained by isolating F.
  • massiliensis EVs from a bacterial culture such as a supernatant or a retentate
  • an excipient that comprises a bulking agent for example, an excipient stock of a formula provided in Tables A or F
  • a liquid preparation containing F. massiliensis EVs for example, obtained by isolating F. massiliensis EVs from a bacterial culture (such as a supernatant or a retentate) and an excipient that comprises a bulking agent are combined, for example, a liquid preparation containing F. massiliensis EVs (for example, obtained by isolating F.
  • massiliensis EVs from a bacterial culture (such as a supernatant or a retentate) or a retentate) are combined with an excipient that comprises a bulking agent, for example, such as mannitol or an excipient of an excipient stock of a formula provided in Tables A or F, to prepare the solution.
  • a bulking agent for example, such as mannitol or an excipient of an excipient stock of a formula provided in Tables A or F
  • the disclosure also provides methods of preparing dried forms of F. massiliensis EVs.
  • the method is used to prepare a lyophilate such as a lyophilized powder and/or a lyophilized cake.
  • the method is used to prepare a powder such as a lyophilized powder and/or a spray-dried powder.
  • the excipient comprises a bulking agent.
  • the bulking agent comprises mannitol, sucrose, polyethylene glycol (PEG, such as PEG 6000), cyclodextrin, maltodextrin, dextran, Ficoll, or PVP-K30.
  • the excipient comprises a lyoprotectant.
  • the excipient optionally includes an additional component such as trehalose, mannitol, sucrose, sorbitol, dextran, poloxamer 188, maltodextrin, PVP-K30, Ficoll, citrate, arginine, and/or hydroxypropyl-B-cyclodextrin.
  • an additional component such as trehalose, mannitol, sucrose, sorbitol, dextran, poloxamer 188, maltodextrin, PVP-K30, Ficoll, citrate, arginine, and/or hydroxypropyl-B-cyclodextrin.
  • a liquid preparation containing F. massiliensis EVs for example, obtained by isolating F.
  • massiliensis EVs from a bacterial culture(such as a supernatant or a retentate) is be combined with an excipient that comprises a bulking agent, such as mannitol or an excipient of an excipient stock of a formula provided in Tables A or F; and dried (for example, by lyophilization or spray drying) to thereby prepare a dried form.
  • the dried form has a moisture content below about 6%, below about 5%, below about 4%, between about 0.5% to about 5%, between about 1% to about 5%, between about 1% to about 4%, between about 1.5% to about 4%, or between about 2% to about 3%, (for example, as determined by Karl Fischer titration).
  • the dried form has about 10% to about 80% (by weight) of an excipient, for example, an excipient that comprises a bulking agent. In some embodiments, the dried form has about 10% to about 80% (by weight) of an excipient, for example, an excipient from a stock of a formula provided in Tables A or F. In some embodiments, the F. massiliensis EVs comprise about 1% to about 99% of the total solids by weight of the dried form. In some embodiments, the dried form has at least about lelO particles per mg of the dried form (for example, as determined by particles per mg, such as by NTA).
  • the particles in the dried form have a hydrodynamic diameter (Z average, Zave) of about 130 nm to about 300 nm after resuspension from the dried form (for example, resuspension in deionized water) (for example, as determined by dynamic light scattering).
  • the dried form is a lyophilate.
  • the lyophilate is a lyophilized powder or a lyophilized cake.
  • the dried form is a powder.
  • the powder is a lyophilized powder or a spray- dried powder.
  • a method of preparing a solution that comprises F. massiliensis EVs includes: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises a bulking agent, thereby preparing the solution.
  • a method of preparing a solution that comprises F. massiliensis EVs includes: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises a bulking agent and a lyoprotectant, thereby preparing the solution.
  • a method of preparing a solution that comprises F. massiliensis EVs includes: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises a lyoprotectant, thereby preparing the solution.
  • a method of preparing a solution that comprises F. massiliensis EVs includes: combining a liquid preparation that comprises F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution.
  • the F. massiliensis EVs are from Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696).
  • the disclosure provides a solution prepared by a method described herein.
  • the disclosure provides a method of preparing a dried form that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; and drying the solution, thereby preparing the dried form.
  • the disclosure provides a method of preparing a dried form that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; drying the solution to prepare a cake, and milling (for example, grinding the cake, thereby preparing the dried form.
  • the disclosure provides a method of preparing a dried form that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; and drying the solution, thereby preparing the dried form.
  • the disclosure provides a method of preparing a dried form that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; drying the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the dried form.
  • the disclosure provides a method of preparing a dried form that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; and drying the solution, thereby preparing the dried form.
  • the disclosure provides a method of preparing a dried form that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; drying the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the dried form.
  • the drying comprises lyophilization.
  • the drying comprises spray drying.
  • the method further comprises combining the dried form with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a dried form prepared by a method described herein.
  • the disclosure provides a method of preparing a powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; and drying the solution, thereby preparing the powder.
  • the disclosure provides a method of preparing a powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; drying the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the powder.
  • the disclosure provides a method of preparing a powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; and drying the solution, thereby preparing the powder.
  • the disclosure provides a method of preparing a powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; drying the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the powder.
  • the disclosure provides a method of preparing a powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; and drying the solution, thereby preparing the powder.
  • the disclosure provides a method of preparing a powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; drying the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the powder.
  • the drying comprises lyophilization.
  • the drying comprises spray drying.
  • the method further comprises combining the powder with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a powder prepared by a method described herein.
  • the disclosure provides a method of preparing a spray-dried powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; and spray drying the solution, thereby preparing the spray-dried powder.
  • the disclosure provides a method of preparing a spray-dried powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; and spray drying the solution, thereby preparing the spray-dried powder.
  • the disclosure provides a method of preparing a spray-dried powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; and spray drying the solution, thereby preparing the spray-dried powder.
  • the method further comprises combining the spray-dried powder with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a spray-dried powder prepared by a method described herein.
  • the disclosure provides a method of preparing a lyophilate that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilate.
  • the disclosure provides a method of preparing a lyophilate that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; freeze drying (lyophilizing) the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the lyophilate.
  • the disclosure provides a method of preparing a lyophilate that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilate.
  • the disclosure provides a method of preparing a lyophilate that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; freeze drying (lyophilizing) the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the lyophilate.
  • the disclosure provides a method of preparing a lyophilate that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilate.
  • the disclosure provides a method of preparing a lyophilate that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; freeze drying (lyophilizing) the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the lyophilate.
  • the method further comprises combining the lyophilate with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilate prepared by a method described herein.
  • the disclosure provides a method of preparing a lyophilized powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilized powder.
  • the disclosure provides a method of preparing a lyophilized powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; freeze drying (lyophilizing) the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the lyophilized powder.
  • the disclosure provides a method of preparing a lyophilized powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilized powder.
  • the disclosure provides a method of preparing a lyophilized powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; freeze drying (lyophilizing) the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the lyophilized powder.
  • the disclosure provides a method of preparing a lyophilized powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilized powder.
  • the disclosure provides a method of preparing a lyophilized powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; freeze drying (lyophilizing) the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the lyophilized powder.
  • the method further comprises combining the lyophilized powder with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilized powder prepared by a method described herein.
  • the disclosure provides a method of preparing a lyophilized cake that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent to prepare a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilized cake.
  • the disclosure provides a method of preparing a lyophilized cake that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a bulking agent and a lyoprotectant to prepare a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilized cake.
  • the disclosure provides a method of preparing a lyophilized cake that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with an excipient that comprises (or consists essentially of) a lyoprotectant to prepare a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilized cake.
  • the disclosure provides a lyophilized cake prepared by a method described herein.
  • the disclosure provides a method of preparing a solution that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution.
  • the F. massiliensis EVs are from Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696).
  • the disclosure provides a solution prepared by a method described herein.
  • the disclosure provides a method of preparing a dried form that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; and drying the solution, thereby preparing the dried form.
  • the disclosure provides a method of preparing a dried form that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; drying the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the dried form.
  • the F. massiliensis EVs are from Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696).
  • the drying comprises lyophilization.
  • the drying comprises spray drying.
  • the method further comprises combining the dried form with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a dried form prepared by a method described herein.
  • the disclosure provides a method of preparing a powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; and drying the solution, thereby preparing the powder.
  • the disclosure provides a method of preparing a powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; drying the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the powder.
  • the F. massiliensis EVs are from Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696).
  • the drying comprises lyophilization.
  • the drying comprises spray drying.
  • the method further comprises combining the powder with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a powder prepared by a method described herein.
  • the disclosure provides a method of preparing a spray-dried powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; and spray drying the solution, thereby preparing the spray-dried powder.
  • the F. massiliensis EVs are from Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696).
  • the method further comprises combining the spray-dried powder with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a spray-dried powder prepared by a method described herein.
  • the disclosure provides a method of preparing a lyophilate that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilate.
  • the disclosure provides a method of preparing a lyophilate that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; freeze drying (lyophilizing) the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the lyophilate.
  • the F. massiliensis EVs are from Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696).
  • the method further comprises combining the lyophilate with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilate prepared by a method described herein.
  • the disclosure provides a method of preparing a lyophilized powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; and freeze drying (lyophilizing) the solution, thereby preparing the lyophilized powder.
  • the disclosure provides a method of preparing a lyophilized powder that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; freeze drying (lyophilizing) the solution to prepare a cake, and milling (for example, grinding) the cake, thereby preparing the lyophilized powder.
  • the F. massiliensis EVs are from Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696).
  • the method further comprises combining the lyophilized powder with an additional ingredient.
  • the additional ingredient comprises an excipient, for example, a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilized powder prepared by a method described herein.
  • the disclosure provides a method of preparing a lyophilized cake that comprises F. massiliensis EVs, the method comprising: combining a liquid preparation that comprises F. massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A or F, thereby preparing a solution; and freeze drying (lyophilizing) the solution, thereby preparing a lyophilized cake.
  • the disclosure provides a lyophilized cake prepared by a method described herein.
  • the disclosure also provides methods of preparing therapeutic compositions.
  • the method includes combining a solution or dried form described herein with a pharmaceutically acceptable excipient, such as a glidant, lubricant, and/or diluent, thereby preparing a therapeutic composition.
  • a pharmaceutically acceptable excipient such as a glidant, lubricant, and/or diluent
  • the disclosure also provides methods of preparing therapeutic compositions, such as solid dosage forms, that contain a dried form described herein.
  • the solid dosage form is a capsule, tablet, or minitablet.
  • the disclosure also provides methods of making a solid dosage form (for example, for oral administration) (for example, for pharmaceutical use) that comprises a dried form.
  • the dried form comprises F. massiliensis EVs and an excipient that comprises a bulking agent.
  • the dried form comprises F. massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the dried form comprises F. massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the dried form also contains one or more additional components.
  • the dried form is combined with one or more pharmaceutically acceptable excipients.
  • the solid dosage form is enterically coated, for example, with a coating described herein.
  • a method of making the solid dosage form includes: loading a dried form into a capsule, thereby preparing a capsule, and thereby preparing the solid dosage form; optionally combining the dried form with a pharmaceutically acceptable excipient prior to loading into the capsule; and/or optionally banding the capsule after loading the capsule (for example, optionally banding the capsule after loading the capsule).
  • a method of making the solid dosage form includes: compressing a dried form described herein into a minitablet, thereby preparing a minitablet and thereby preparing the solid dosage form; optionally combining the dried form with a pharmaceutically acceptable excipient prior to compressing; optionally filling a capsule with a plurality of enterically coated minitablets.
  • a method of making the solid dosage form includes: compressing a powder described herein into a tablet, thereby preparing a tablet, and thereby preparing the solid dosage form; optionally combining the dried form with a pharmaceutically acceptable excipient prior to compressing.
  • the method comprises performing wet granulation on a powder prior to combining the powder and one or more (for example, one, two or three) excipients into a therapeutic composition, such as a solid dosage form.
  • the wet granulation comprises (i) mixing the powder with a granulating fluid (for example, water, ethanol, or isopropanol, alone or in combination).
  • the wet granulation comprises mixing the powder with water.
  • the wet granulation comprises (ii) drying mixed powder and granulating fluid (for example, drying on a fluid bed dryer).
  • the wet granulation comprises (iii) milling (for example, grinding) the dried powder and granulating fluid.
  • the milled (for example, ground) powder and granulating fluid are then combined with the one or more (for example, one, two or three) excipients to prepare a therapeutic composition, such as a solid dosage form.
  • the powder is a lyophilized powder.
  • the powder is a spray-dried powder.
  • a dried form described herein is reconstituted in a liquid (for example, a buffer, juice, or water) to prepare a therapeutic composition.
  • a liquid for example, a buffer, juice, or water
  • a solution is resuspended (for example, diluted) in a liquid (for example, a buffer, juice, or water) to prepare a therapeutic composition.
  • a liquid for example, a buffer, juice, or water
  • a therapeutic composition comprising a dried form described herein is reconstituted in a liquid (for example, a buffer, juice, or water) to prepare a suspension.
  • a liquid for example, a buffer, juice, or water
  • a therapeutic composition comprising a solution is resuspended (for example, diluted) in a liquid (for example, a buffer, juice, or water) to prepare a suspension.
  • a liquid for example, a buffer, juice, or water
  • Powders and frozen biomass can be gammairradiated.
  • powders for example, of F. massiliensis EVs
  • frozen biomasses for example, of F. massiliensis EVs
  • frozen biomasses are gamma-irradiated at 25 kGy radiation unit in the presence of dry ice.
  • the methods provided herein include the administration to a subject of a therapeutic composition described herein either alone or in combination with an additional therapeutic agent.
  • the additional therapeutic agent is an immunosuppressant, an anti-inflammatory agent, a steroid, and/or a cancer therapeutic.
  • the therapeutic composition comprising F. massiliensis EVs is administered to the subject before the additional therapeutic agent is administered (for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days before).
  • the therapeutic composition comprising F. massiliensis EVs is administered to the subject after the additional therapeutic agent is administered (for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours after or at least 1,
  • the therapeutic composition comprising F. massiliensis EVs and the additional therapeutic agent are administered to the subject simultaneously or nearly simultaneously (for example, administrations occur within an hour of each other).
  • an antibiotic is administered to the subject before the therapeutic composition comprising F. massiliensis EVs is administered to the subject (for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days before).
  • an antibiotic is administered to the subject after therapeutic composition comprising F. massiliensis EVs is administered to the subject (for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2,
  • the therapeutic composition comprising F. massiliensis EVs and the antibiotic are administered to the subject simultaneously or nearly simultaneously (for example, administrations occur within an hour of each other).
  • the additional therapeutic agent is a cancer therapeutic.
  • the cancer therapeutic is a chemotherapeutic agent.
  • chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophy cin 8); dolast
  • the cancer therapeutic is a cancer immunotherapy agent.
  • Immunotherapy refers to a treatment that uses a subject’s immune system to treat cancer, for example, checkpoint inhibitors, cancer vaccines, cytokines, cell therapy, CAR-T cells, and dendritic cell therapy.
  • checkpoint inhibitors include Nivolumab (BMS, anti-PD-1), Pembrolizumab (Merck, anti-PD-1), Ipilimumab (BMS, anti-CTLA-4), MEDI4736 (AstraZeneca, anti-PD-Ll), and MPDL3280A (Roche, anti-PD-Ll).
  • Other immunotherapies may be tumor vaccines, such as Gardail, Cervarix, BCG, sipulencel-T, Gpl00:209-217, AGS-003, DCVax-L, Algenpantucel-L, Tergenpantucel-L, TG4010, ProstAtak, Prostvac-V/R-TRICOM, Rindopepimul, E75 peptide acetate, IMA901, POL-103A, Belagenpumatucel-L, GSK1572932A, MDX-1279, GV1001, and Tecemotide.
  • tumor vaccines such as Gardail, Cervarix, BCG, sipulencel-T, Gpl00:209-217, AGS-003, DCVax-L, Algenpantucel-L, Tergenpantucel-L, TG4010, ProstAtak, Prostvac-V/R-TRICOM, Rindopepimul, E75
  • the immunotherapy agent may be administered via injection (for example, intravenously, intratumorally, subcutaneously, or into lymph nodes), but may also be administered orally, topically, or via aerosol.
  • Immunotherapies may comprise adjuvants such as cytokines.
  • the immunotherapy agent is an immune checkpoint inhibitor.
  • Immune checkpoint inhibition broadly refers to inhibiting the checkpoints that cancer cells can produce to prevent or downregulate an immune response.
  • immune checkpoint proteins include, but are not limited to, CTLA4, PD-1, PD-L1, PD- L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3 or VISTA.
  • Immune checkpoint inhibitors can be antibodies or antigen binding fragments thereof that bind to and inhibit an immune checkpoint protein.
  • immune checkpoint inhibitors include, but are not limited to, nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI- Al l 10, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0010718C (avelumab), AUR-012 and STI-A1010.
  • the methods provided herein include the administration of a therapeutic composition described herein in combination with one or more additional therapeutic agents.
  • the methods disclosed herein include the administration of two immunotherapy agents (for example, immune checkpoint inhibitor).
  • the methods provided herein include the administration of a pharmaceutical composition described herein in combination with a PD-1 inhibitor (such as pemrolizumab or nivolumab or pidilizumab) or a CLTA-4 inhibitor (such as ipilimumab) or a PD-L1 inhibitor (such as avelumab).
  • a PD-1 inhibitor such as pemrolizumab or nivolumab or pidilizumab
  • CLTA-4 inhibitor such as ipilimumab
  • PD-L1 inhibitor such as avelumab
  • the immunotherapy agent is an antibody or antigen binding fragment thereof that, for example, binds to a cancer-associated antigen.
  • cancer-associated antigens include, but are not limited to, adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTCI, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin Dl, Cyclin-Al, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM
  • the immunotherapy agent is a cancer vaccine and/or a component of a cancer vaccine (for example, an antigenic peptide and/or protein).
  • the cancer vaccine can be a protein vaccine, a nucleic acid vaccine or a combination thereof.
  • the cancer vaccine comprises a polypeptide comprising an epitope of a cancer-associated antigen.
  • the cancer vaccine comprises a nucleic acid (for example, DNA or RNA, such as mRNA) that encodes an epitope of a cancer-associated antigen.
  • cancer-associated antigens include, but are not limited to, adipophilin, AIM-2, ALDH1 Al, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTCI, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin DI, Cyclin-Al, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen (“ETA”), ETV6-AML1 fusion protein, EZH2, FGF5, FLT3-ITD
  • the antigen is a neo-antigen.
  • the cancer vaccine is administered with an adjuvant.
  • adjuvants include, but are not limited to, an immune modulatory protein, Adjuvant 65, a-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, P-Glucan Peptide, CpG ODN DNA, GPI-0100, lipid A, lipopolysaccharide, Lipovant, Montanide, N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A , cholera toxin (CT) and heat-labile toxin from enterotoxigenic Escherichia coli (LT) including derivatives of these (CTB, mmCT, CTA1-DD, LTB, LTK63, LTR72, dmLT) and trehalose dimycolate.
  • CTB cholera toxin
  • LT heat-labile toxin from entero
  • the immunotherapy agent is an immune modulating protein to the subject.
  • the immune modulatory protein is a cytokine or chemokine.
  • immune modulating proteins include, but are not limited to, B lymphocyte chemoattractant ("BLC"), C-C motif chemokine 11 (“Eotaxin-1”), Eosinophil chemotactic protein 2 (“Eotaxin-2”), Granulocyte colony-stimulating factor (“G-CSF”), Granulocyte macrophage colony-stimulating factor (“GM-CSF”), 1-309, Intercellular Adhesion Molecule 1 ("ICAM-1"), Interferon alpha (“IFN-alpha”), Interferon beta (“IFN- beta”) Interferon gamma (“IFN-gamma”), Interlukin-1 alpha (“IL-1 alpha”), Interlukin-1 beta (“IL-1 beta”), Interleukin 1 receptor antagonist (“IL-1 ra”), Interleukin-2 (“IL
  • the cancer therapeutic is an anti-cancer compound.
  • anti-cancer compounds include, but are not limited to, Alemtuzumab (Campath®), Alitretinoin (Panretin®), Anastrozole (Arimidex®), Bevacizumab (Avastin®), Bexarotene (Targretin®), Bortezomib (Velcade®), Bosutinib (Bosulif®), Brentuximab vedotin (Adcetris®), Cabozantinib (CometriqTM), Carfilzomib (KyprolisTM), Cetuximab (Erbitux®), Crizotinib (Xalkori®), Dasatinib (Sprycel®), Denileukin diftitox (Ontak®), Erlotinib hydrochloride (Tarceva®), Everolimus (Afinitor®), Exemestane
  • Exemplary anti-cancer compounds that modify the function of proteins that regulate gene expression and other cellular functions are Vorinostat (Zolinza®), Bexarotene (Targretin®) and Romidepsin (Istodax®), Alitretinoin (Panretin®), and Tretinoin (Vesanoid®).
  • Exemplary anti-cancer compounds that induce apoptosis are Bortezomib (Velcade®), Carfilzomib (KyprolisTM), and Pralatrexate (Folotyn®).
  • exemplary anti-cancer compounds are small molecule inhibitors and conjugates thereof of, for example, Janus kinase, ALK, Bcl-2, PARP, PI3K, VEGF receptor, Braf, MEK, CDK, and HSP90.
  • exemplary platinum-based anti-cancer compounds include, for example, cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, Nedaplatin, Triplatin, and Lipoplatin.
  • Other metal-based drugs suitable for treatment include, but are not limited to ruthenium- based compounds, ferrocene derivatives, titanium-based compounds, and gallium-based compounds.
  • the cancer therapeutic is a radioactive moiety that comprises a radionuclide.
  • radionuclides include, but are not limited to Cr-51, Cs-131, Ce-134, Se-75, Ru-97, 1-125, Eu-149, Os-189m, Sb-119, 1-123, Ho-161, Sb-117, Ce-139, In-111, Rh-103m, Ga-67, Tl-201, Pd-103, Au-195, Hg-197, Sr-87m, Pt-191, P- 33, Er-169, Ru-103, Yb-169, Au-199, Sn-121, Tm-167, Yb-175, In-113m, Sn-113, Lu- 177, Rh-105, Sn-117m, Cu-67, Sc-47, Pt-195m, Ce-141, 1-131, Tb-161, As-77, Pt-197, Sm-153, Gd-159, Tm-173, Pr-143, Au-
  • the cancer therapeutic is an antibiotic.
  • antibiotics broadly refers to compounds capable of inhibiting or preventing a bacterial infection. Antibiotics can be classified in a number of ways, including their use for specific infections, their mechanism of action, their bioavailability, or their spectrum of target microbe (for example, Gram-negative vs. Gram-positive bacteria, aerobic vs. anaerobic bacteria, etc.) and these may be used to kill specific bacteria in specific areas of the host (“niches”) (Leekha, et al 2011.
  • antibiotics can be used to selectively target bacteria of a specific niche.
  • antibiotics known to treat a particular infection that includes a cancer niche may be used to target cancer-associated bacteria, including cancer-associated bacteria in that niche.
  • antibiotics are administered after the therapeutic composition comprising F. massiliensis EVs. In some embodiments, antibiotics are administered before therapeutic composition comprising F. massiliensis EVs.
  • antibiotics can be selected based on their bactericidal or bacteriostatic properties.
  • Bactericidal antibiotics include mechanisms of action that disrupt the cell wall (for example, P-lactams), the cell membrane (for example, daptomycin), or bacterial DNA (for example, fluoroquinolones).
  • Bacteriostatic agents inhibit bacterial replication and include sulfonamides, tetracyclines, and macrolides, and act by inhibiting protein synthesis.
  • some drugs can be bactericidal in certain organisms and bacteriostatic in others, knowing the target organism allows one skilled in the art to select an antibiotic with the appropriate properties.
  • bacteriostatic antibiotics inhibit the activity of bactericidal antibiotics.
  • bactericidal and bacteriostatic antibiotics are not combined.
  • Antibiotics include, but are not limited to aminoglycosides, ansamycins, carbacephems, carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptide antibiotics, quinolones, fluoroquinolone, sulfonamides, tetracyclines, and anti- mycobacterial compounds, and combinations thereof.
  • Aminoglycosides include, but are not limited to Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, and Spectinomycin. Aminoglycosides are effective, for example, against Gram-negative bacteria, such as Escherichia coH, Klebsiella, Pseudomonas aeruginosa, and Francisella tularensis, and against certain aerobic bacteria but less effective against obligate/facultative anaerobes. Aminoglycosides are believed to bind to the bacterial 30S or 50S ribosomal subunit thereby inhibiting bacterial protein synthesis.
  • Ansamycins include, but are not limited to, Geldanamycin, Herbimycin, Rifamycin, and Streptovaricin.
  • Geldanamycin and Herbimycin are believed to inhibit or alter the function of Heat Shock Protein 90.
  • Carbacephems include, but are not limited to, Loracarbef. Carbacephems are believed to inhibit bacterial cell wall synthesis.
  • Carbapenems include, but are not limited to, Ertapenem, Doripenem, Imipenem/Cilastatin, and Meropenem. Carbapenems are bactericidal for both Grampositive 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, Cefazolin, Cefalotin, Cefalothin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil,and Ceftobiprole.
  • Selected Cephalosporins are effective, for example, against Gram-negative bacteria and against Gram-positive bacteria, including Pseudomonas, certain Cephalosporins are effective against methicillin-resistant Staphylococcus aureus (MRSA). Cephalosporins are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Glycopeptides include, but are not limited to, Teicoplanin, Vancomycin, and Telavancin. Glycopeptides are effective, for example, against aerobic and anaerobic Gram-positive bacteria including MRSA and Clostridium difficile. Glycopeptides are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Lincosamides include, but are not limited to, Clindamycin and Lincomycin. Lincosamides are effective, for example, against anaerobic bacteria, as well as Staphylococcus and Streptococcus. Lincosamides are believed to bind to the bacterial 50S ribosomal subunit thereby inhibiting bacterial protein synthesis.
  • Lipopeptides include, but are not limited to, Daptomycin. Lipopeptides are effective, for example, against Gram-positive bacteria. Lipopeptides are believed to bind to the bacterial membrane and cause rapid depolarization.
  • Macrolides include, but are not limited to, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, and Spiramycin. Macrolides are effective, for example, against Streptococcus and Mycoplasma. Macrolides are believed to bind to the bacterial or 50S ribosomal subunit, thereby inhibiting bacterial protein synthesis.
  • Monobactams include, but are not limited to, Aztreonam. Monobactams are effective, for example, against Gram-negative bacteria. Monobactams are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Nitrofurans include, but are not limited to, Furazolidone and Nitrofurantoin.
  • Oxazolidonones include, but are not limited to, Linezolid, Posizolid, Radezolid, and Torezolid. Oxazolidonones are believed to be protein synthesis inhibitors.
  • Penicillins include, but are not limited to, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cioxacillin, Dicloxacillin, Flucioxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Temocillin and Ticarcillin.
  • Penicillins are effective, for example, against Gram-positive bacteria, facultative anaerobes, for example, Streptococcus, Borrelia, and Treponema. Penicillins are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Penicillin combinations include, but are not limited to, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam, and Ticarcillin/clavulanate.
  • Polypeptide antibiotics include, but are not limited to, Bacitracin, Colistin, and Polymyxin B and E.
  • Polypeptide Antibiotics are effective, for example, against Gramnegative bacteria. Certain polypeptide antibiotics are believed to inhibit isoprenyl pyrophosphate involved in synthesis of the peptidoglycan layer of bacterial cell walls, while others destabilize the bacterial outer membrane by displacing bacterial counterions.
  • Quinolones and Fluoroquinolone include, but are not limited to, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, and Temafloxacin.
  • Quinol ones/Fluoroquinol one are effective, for example, against Streptococcus and Neisseria.
  • Quinolones/Fluoroquinolone are believed to inhibit the bacterial DNA gyrase or topoisomerase IV, thereby inhibiting DNA replication and transcription.
  • Sulfonamides include, but are not limited to, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole (Co- trimoxazole), and Sulfonamidochrysoidine.
  • Sulfonamides are believed to inhibit folate synthesis by competitive inhibition of dihydropteroate synthetase, thereby inhibiting nucleic acid synthesis.
  • Tetracyclines include, but are not limited to, Demeclocy cline, Doxycycline, Minocycline, Oxytetracycline, and Tetracycline. Tetracyclines are effective, for example, against Gram-negative bacteria. Tetracyclines are believed to bind to the bacterial 30S ribosomal subunit thereby inhibiting bacterial protein synthesis.
  • Anti-mycobacterial compounds include, but are not limited to, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, and Streptomycin.
  • Suitable antibiotics also include arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin/dalfopristin, tigecycline, tinidazole, trimethoprim amoxicillin/clavulanate, ampicillin/sulbactam, amphomycin ristocetin, azithromycin, bacitracin, buforin II, carbomycin, cecropin Pl, clarithromycin, erythromycins, furazolidone, fusidic acid, Na fusidate, gramicidin, imipenem, indolicidin, josamycin, magainan II, metronidazole, nitroimidazoles, mikamycin, mutacin B-Ny266, mutacin B-JH1 140, mutacin J-T8, nisin, nisin A, novobiocin, oleando
  • the additional therapeutic agent is an immunosuppressive agent, a DMARD, a pain-control drug, a steroid, a non-steroidal antiinflammatory drug (NSAID), or a cytokine antagonist, and combinations thereof.
  • Representative agents include, but are not limited to, cyclosporin, retinoids, corticosteroids, propionic acid derivative, acetic acid derivative, enolic acid derivatives, fenamic acid derivatives, Cox-2 inhibitors, lumiracoxib, ibuprophen, cholin magnesium salicylate, fenoprofen, salsalate, difunisal, tolmetin, ketoprofen, flurbiprofen, oxaprozin, indomethacin, sulindac, etodolac, ketorolac, nabumetone, naproxen, valdecoxib, etoricoxib, MK0966; rofecoxib, acetominophen, Celecoxib, Diclofenac, tramadol, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefanamic acid, meclofenamic acid
  • the additional therapeutic agent is an immunosuppressive agent.
  • immunosuppressive agents include, but are not limited to, corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine, theophylline, cromolyn sodium, anti -leukotrienes, anti-cholinergic drugs for rhinitis, TLR antagonists, inflammasome inhibitors, anti-cholinergic decongestants, mast-cell stabilizers, monoclonal anti-IgE antibodies, vaccines (for example, vaccines used for vaccination where the amount of an allergen is gradually increased), cytokine inhibitors, such as anti- IL-6 antibodies, TNF
  • a method of delivering a therapeutic composition described herein for example, a therapeutic composition comprising a solution or dried form described herein
  • the therapeutic composition is administered in conjunction with the administration of an additional therapeutic agent.
  • the therapeutic composition comprising a solution or dried form described herein is coformulated with the additional therapeutic agent.
  • the therapeutic composition comprising a solution or dried form described herein is co-administered with the additional therapeutic agent.
  • the additional therapeutic agent is administered to the subject before administration of the therapeutic composition comprising a solution or dried form described herein (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes before, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours before, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days before).
  • the additional therapeutic agent is administered to the subject after administration of the therapeutic composition comprising a solution or dried form described herein (for example, 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,
  • the same mode of delivery is used to deliver both the therapeutic composition comprising a solution or dried form described herein and the additional therapeutic agent.
  • different modes of delivery are used to administer the therapeutic composition comprising a solution or dried form described herein and the additional therapeutic agent.
  • the therapeutic composition comprising a solution or dried form described herein is administered orally while the additional therapeutic agent is administered via injection (for example, an intravenous, intramuscular and/or intratumoral injection).
  • the therapeutic composition described herein is administered once a day. In some embodiments, the therapeutic composition described herein is administered twice a day. In some embodiments, the therapeutic composition described herein is formulated for a daily dose. In some embodiments, the therapeutic composition described herein is formulated for twice a day dose, wherein each dose is half of the daily dose.
  • the therapeutic compositions described herein are administered in conjunction with any other conventional anti-cancer treatment, such as, for example, radiation therapy and surgical resection of the tumor. These treatments may be applied as necessary and/or as indicated and may occur before, concurrent with or after administration of the therapeutic composition comprising a solution or dried form described herein.
  • the dosage regimen can be any of a variety of methods and amounts, and can be determined by one skilled in the art according to known clinical factors. As is known in the medical arts, dosages for any one patient can depend on many factors, including the subject's species, size, body surface area, age, sex, immunocompetence, and general health, the particular microorganism to be administered, duration and route of administration, the kind and stage of the disease, for example, tumor size, and other compounds such as drugs being administered concurrently or near-concurrently. In addition to the above factors, such levels can be affected by the infectivity of the microorganism, and the nature of the microorganism, as can be determined by one skilled in the art.
  • appropriate minimum dosage levels of microorganisms can be levels sufficient for the microorganism to survive, grow and replicate.
  • the dose of a therapeutic composition comprising a solution or dried form described herein may be appropriately set or adjusted in accordance with the dosage form, the route of administration, the degree or stage of a target disease, and the like.
  • the general effective dose of the agents may range between 0.01 mg/kg body weight/day and 1000 mg/kg body weight/day, between 0.1 mg/kg body weight/day and 1000 mg/kg body weight/day, 0.5 mg/kg body weight/day and 500 mg/kg body weight/day, 1 mg/kg body weight/day and 100 mg/kg body weight/day, or between 5 mg/kg body weight/day and 50 mg/kg body weight/day.
  • the effective dose may be 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, or 1000 mg/kg body weight/day or more, but the dose is not limited thereto.
  • the dose administered to a subject is sufficient to prevent disease (for example, autoimmune disease, inflammatory disease, metabolic disease, or cancer), delay its onset, or slow or stop its progression, or relieve one or more symptoms of the disease.
  • disease for example, autoimmune disease, inflammatory disease, metabolic disease, or cancer
  • dosage will depend upon a variety of factors including the strength of the particular agent (for example, therapeutic agent) employed, as well as the age, species, condition, and body weight of the subject.
  • the size of the dose will also be determined by the route, timing, and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular therapeutic agent and the desired physiological effect.
  • Suitable doses and dosage regimens can be determined by conventional rangefinding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached.
  • An effective dosage and treatment protocol can be determined by routine and conventional means, starting for example, with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Animal studies are commonly used to determine the maximal tolerable dose ("MTD”) of bioactive agent per kilogram weight. Those skilled in the art regularly extrapolate doses for efficacy, while avoiding toxicity, in other species, including humans.
  • MTD maximal tolerable dose
  • the dosages of the therapeutic agents used in accordance with the invention vary depending on the active agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage.
  • the dose should be sufficient to result in slowing, and preferably regressing, the growth of a tumor and most preferably causing complete regression of the cancer, or reduction in the size or number of metastases
  • the dose should be sufficient to result in slowing of progression of the disease for which the subject is being treated, and preferably amelioration of one or more symptoms of the disease for which the subject is being treated.
  • Separate administrations can include any number of two or more administrations, including two, three, four, five or six administrations.
  • One skilled in the art can readily determine the number of administrations to perform or the desirability of performing one or more additional administrations according to methods known in the art for monitoring therapeutic methods and other monitoring methods provided herein.
  • the methods provided herein include methods of providing to the subject one or more administrations of a pharmaceutical composition, where the number of administrations can be determined by monitoring the subject, and, based on the results of the monitoring, determining whether or not to provide one or more additional administrations. Deciding on whether or not to provide one or more additional administrations can be based on a variety of monitoring results.
  • the time period between administrations can be any of a variety of time periods.
  • the time period between administrations can be a function of any of a variety of factors, including monitoring steps, as described in relation to the number of administrations, the time period for a subject to mount an immune response.
  • the time period can be a function of the time period for a subject to mount an immune response; for example, the time period can be more than the time period for a subject to mount an immune response, such as more than about one week, more than about ten days, more than about two weeks, or more than about a month; in another example, the time period can be less than the time period for a subject to mount an immune response, such as less than about one week, less than about ten days, less than about two weeks, or less than about a month.
  • the delivery of an additional therapeutic agent in combination with the therapeutic composition described herein reduces the adverse effects and/or improves the efficacy of the additional therapeutic agent.
  • the effective dose of an additional therapeutic agent described herein is the amount of the additional therapeutic agent that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, with the least toxicity to the subject.
  • the effective dosage level can be identified using the methods described herein and will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions or agents administered, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts.
  • an effective dose of an additional therapeutic agent will be the amount of the additional therapeutic agent which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the toxicity of an additional therapeutic agent is the level of adverse effects experienced by the subject during and following treatment.
  • Adverse events associated with additional therapy toxicity can include, but are not limited to, abdominal pain, acid indigestion, acid reflux, allergic reactions, alopecia, anaphylaxis, anemia, anxiety, lack of appetite, arthralgias, asthenia, ataxia, azotemia, loss of balance, bone pain, bleeding, blood clots, low blood pressure, elevated blood pressure, difficulty breathing, bronchitis, bruising, low white blood cell count, low red blood cell count, low platelet count, cardiotoxicity, cystitis, hemorrhagic cystitis, arrhythmias, heart valve disease, cardiomyopathy, coronary artery disease, cataracts, central neurotoxicity, cognitive impairment, confusion, conjunctivitis, constipation, coughing, cramping, cystitis, deep vein thrombosis, dehydration, depression, diarrhea, dizziness, dry mouth, dry skin, dyspepsi
  • the methods and therapeutic compositions described herein relate to the treatment or prevention of a disease or disorder associated a pathological immune response, such as an autoimmune disease, an allergic reaction and/or an inflammatory disease.
  • a disease or disorder is an inflammatory bowel disease (for example, Crohn’s disease or ulcerative colitis).
  • the disease or disorder is psoriasis.
  • the disease or disorder is atopic dermatitis.
  • a “subject in need thereof’ includes any subject that has a disease or disorder associated with a pathological immune response (for example, an inflammatory bowel disease), as well as any subject with an increased likelihood of acquiring a such a disease or disorder.
  • a pathological immune response for example, an inflammatory bowel disease
  • the therapeutic compositions described herein can be used, for example, as a pharmaceutical composition for preventing or treating (reducing, partially or completely, the adverse effects of) an autoimmune disease, such as chronic inflammatory bowel disease, systemic lupus erythematosus, psoriasis, muckle-wells syndrome, rheumatoid arthritis, multiple sclerosis, or Hashimoto's disease; an allergic disease, such as a food allergy, pollenosis, or asthma; an infectious disease, such as an infection with Clostridium difficile; an inflammatory disease such as a TNF-mediated inflammatory disease (for example, an inflammatory disease of the gastrointestinal tract, such as pouchitis, a cardiovascular inflammatory condition, such as atherosclerosis, or an inflammatory lung disease, such as chronic obstructive pulmonary disease); a pharmaceutical composition for suppressing rejection in organ transplantation or other situations in which tissue rejection might occur; a supplement, food, or beverage for improving immune functions; or a reagent for suppressing the proliferation or
  • the methods provided herein are useful for the treatment of inflammation.
  • the inflammation of any tissue and organs of the body including musculoskeletal inflammation, vascular inflammation, neural inflammation, digestive system inflammation, ocular inflammation, inflammation of the reproductive system, and other inflammation, as discussed below.
  • Immune disorders of the musculoskeletal system include, but are not limited, to those conditions affecting skeletal joints, including joints of the hand, wrist, elbow, shoulder, jaw, spine, neck, hip, knew, ankle, and foot, and conditions affecting tissues connecting muscles to bones such as tendons.
  • immune disorders which may 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), tendonitis, synovitis, tenosynovitis, bursitis, fibrositis (fibromyalgia), epicondylitis, myositis, and osteitis (including, for example, Paget's disease, osteitis pubis, and osteitis fibrosa cystic).
  • 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
  • tendonitis synovitis, ten
  • Ocular immune disorders refers to a immune disorder that affects any structure of the eye, including the eye lids.
  • ocular immune disorders which may be treated with the methods and compositions described herein include, but are not limited to, blepharitis, blepharochalasis, conjunctivitis, dacryoadenitis, keratitis, keratoconjunctivitis sicca (dry eye), scleritis, trichiasis, and uveitis
  • Examples of nervous system immune disorders which may be treated with the methods and compositions described herein include, but are not limited to, encephalitis, Guillain-Barre syndrome, meningitis, neuromyotonia, narcolepsy, multiple sclerosis, myelitis and schizophrenia.
  • Examples of inflammation of the vasculature or lymphatic system which may be treated with the methods and compositions described herein include, but are not limited to, arthrosclerosis, arthritis, phlebitis, vasculitis, and lymphangitis.
  • Examples of digestive system immune disorders which may be treated with the methods and pharmaceutical compositions described herein include, but are not limited to, cholangitis, cholecystitis, enteritis, enterocolitis, gastritis, gastroenteritis, inflammatory bowel disease, ileitis, and proctitis.
  • Inflammatory bowel diseases include, for example, certain art-recognized forms of a group of related conditions.
  • Crohn's disease regional bowel disease, for example, inactive and active forms
  • ulcerative colitis for example, inactive and active forms
  • the inflammatory bowel disease encompasses irritable bowel syndrome, microscopic colitis, lymphocytic- plasmocytic enteritis, coeliac disease, collagenous colitis, lymphocytic colitis and eosinophilic enterocolitis.
  • Other less common forms of IBD include indeterminate colitis, pseudomembranous colitis (necrotizing colitis), ischemic inflammatory bowel disease, Behcet’s disease, sarcoidosis, scleroderma, IBD-associated dysplasia, dysplasia associated masses or lesions, and primary sclerosing cholangitis.
  • reproductive system immune disorders which may be treated with the methods and pharmaceutical compositions described herein include, but are not limited to, cervicitis, chorioamnionitis, endometritis, epididymitis, omphalitis, oophoritis, orchitis, salpingitis, tubo-ovarian abscess, urethritis, vaginitis, vulvitis, and vulvodynia.
  • the methods and therapeutic compositions described herein may be used to treat autoimmune conditions having an inflammatory component.
  • Such conditions include, but are not limited to, acute disseminated alopecia universalise, Behcet's disease, Chagas' disease, chronic fatigue syndrome, dysautonomia, encephalomyelitis, ankylosing spondylitis, aplastic anemia, hidradenitis suppurativa, autoimmune hepatitis, autoimmune oophoritis, celiac disease, Crohn's disease, diabetes mellitus type 1, giant cell arteritis, Goodpasture's syndrome, Grave's disease, Guillain-Barre syndrome, Hashimoto's disease, Henoch-Schonlein purpura, Kawasaki's disease, lupus erythematosus, microscopic colitis, microscopic polyarteritis, mixed connective tissue disease, Muckle-Wells syndrome, multiple sclerosis, myasthenia gravis, opso
  • T-cell mediated hypersensitivity diseases having an inflammatory component.
  • Such conditions include, but are not limited to, contact hypersensitivity, contact dermatitis (including that due to poison ivy), uticaria, skin allergies, respiratory allergies (hay fever, allergic rhinitis, house dust mite allergy) and gluten-sensitive enteropathy (Celiac disease).
  • immune disorders which may be treated with the methods and therapeutic compositions include, for example, appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, hepatitis, hidradenitis suppurativa, ulceris, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis, pleuritis, pneumonitis, prostatitis, pyelonephritis, and stomatitis, transplant rejection (involving organs such as kidney, liver, heart, lung, pancreas (for example, islet cells), bone marrow, cornea, small bowel, skin allografts, skin homografts, and heart valve xenografts, serum sickness, and graft vs host
  • Preferred treatments include treatment of transplant rejection, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Type 1 diabetes, asthma, inflammatory bowel disease, systemic lupus erythematosus, psoriasis, chronic obstructive pulmonary disease, and inflammation accompanying infectious conditions (for example, sepsis).
  • the methods and therapeutic compositions described herein relate to the treatment or prevention of a metabolic disease or disorder a, such as type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, non-alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglylceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) or a related disease.
  • a metabolic disease or disorder a such as type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, non-alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglylceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic
  • the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema.
  • the methods and pharmaceutical compositions described herein relate to the treatment of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH).
  • NAFLD nonalcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • a “subject in need thereof’ includes any subject that has a metabolic disease or disorder, as well as any subject with an increased likelihood of acquiring a such a disease or disorder.
  • the therapeutic compositions described herein can be used, for example, for preventing or treating (reducing, partially or completely, the adverse effects of) a metabolic disease, such as type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, non-alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglylceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), or a related disease.
  • the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema.
  • the methods and therapeutic compositions described herein relate to the treatment of cancer.
  • any cancer can be treated using the methods described herein.
  • cancers that may treated by methods and pharmaceutical compositions described herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • Hodgkin's disease Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
  • the methods and pharmaceutical compositions provided herein relate to the treatment of a leukemia.
  • leukemia diseases include, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia
  • carcinomas include, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant
  • the methods and therapeutic compositions provided herein relate to the treatment of a sarcoma.
  • Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing' s sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma,
  • Additional exemplary neoplasias that can be treated using the methods and therapeutic compositions described herein include Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small- cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, plasmacytoma, colorectal cancer, rectal cancer, and adrenal cortical cancer.
  • the cancer treated is a melanoma.
  • melanomas are Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.
  • the cancer comprises breast cancer (for example, triple negative breast cancer).
  • the cancer comprises colorectal cancer (for example, microsatellite stable (MSS) colorectal cancer).
  • MSS microsatellite stable
  • the cancer comprises renal cell carcinoma.
  • the cancer comprises lung cancer (for example, non-small cell lung cancer).
  • the cancer comprises bladder cancer.
  • the cancer comprises gastroesophageal cancer.
  • tumors that can be treated using methods and therapeutic compositions described herein include lymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, cancer of the thyroid, head and neck cancer, cancer of the central nervous system, cancer of the peripheral nervous system, skin cancer, kidney cancer, as well as metastases of all the above.
  • tumors include hepatocellular carcinoma, hepatoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, invasive ductal carcinoma, papillary adenocarcinoma, melanoma, pulmonary squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (well differentiated, moderately differentiated, poorly differentiated or undifferentiated), bronchioloalveolar carcinoma, renal cell carcinoma, hypernephroma, hypernephroid adenocarcinoma, bile duct carcinoma,
  • Cancers treated in certain embodiments also include precancerous lesions, for example, actinic keratosis (solar keratosis), moles (dysplastic nevi), acitinic chelitis (farmer's lip), cutaneous horns, Barrett's esophagus, atrophic gastritis, dyskeratosis congenita, sideropenic dysphagia, lichen planus, oral submucous fibrosis, actinic (solar) elastosis and cervical dysplasia.
  • precancerous lesions for example, actinic keratosis (solar keratosis), moles (dysplastic nevi), acitinic chelitis (farmer's lip), cutaneous horns, Barrett's esophagus, atrophic gastritis, dyskeratosis congenita, sideropenic dysphagia, lichen planus, oral subm
  • Cancers treated in some embodiments include non-cancerous or benign tumors, for example, of endodermal, ectodermal or mesenchymal origin, including, but not limited to cholangioma, colonic polyp, adenoma, papilloma, cystadenoma, liver cell adenoma, hydatidiform mole, renal tubular adenoma, squamous cell papilloma, gastric polyp, hemangioma, osteoma, chondroma, lipoma, fibroma, lymphangioma, leiomyoma, rhabdomyoma, astrocytoma, nevus, meningioma, and ganglioneuroma.
  • cholangioma colonic polyp
  • adenoma papilloma
  • cystadenoma cystadenoma
  • liver cell adenoma hydatidi
  • the methods and therapeutic compositions described herein relate to the treatment of liver diseases.
  • diseases include, but are not limited to, Alagille Syndrome, Alcohol-Related Liver Disease, Alpha-1 Antitrypsin Deficiency, Autoimmune Hepatitis, Benign Liver Tumors, Biliary Atresia, Cirrhosis, Galactosemia, Gilbert Syndrome, Hemochromatosis, Hepatitis A, Hepatitis B, Hepatitis C, Hepatic Encephalopathy, Intrahepatic Cholestasis of Pregnancy (ICP), Lysosomal Acid Lipase Deficiency (LAL-D), Liver Cysts, Liver Cancer, Newborn Jaundice, Primary Biliary Cholangitis (PBC), Primary Sclerosing Cholangitis (PSC), Reye Syndrome, Type I Glycogen Storage Disease, and Wilson Disease.
  • ICP Pregnancy
  • LAL-D Lysosomal Acid Lipase Deficiency
  • the methods and therapeutic compositions described herein may be used to treat neurodegenerative and neurological diseases.
  • the neurodegenerative and/or neurological disease is Parkinson’s disease, Alzheimer’s disease, prion disease, Huntington’s disease, motor neuron diseases (MND), spinocerebellar ataxia, spinal muscular atrophy, dystonia, idiopathicintracranial hypertension, epilepsy, nervous system disease, central nervous system disease, movement disorders, multiple sclerosis, encephalopathy, peripheral neuropathy or postoperative cognitive dysfunction.
  • the gut microbiome also called the “gut microbiota” can have a significant impact on an individual’s health through microbial activity and influence (local and/or distal) on immune and other cells of the host (Walker, W.A., Dysbiosis. The Microbiota in Gastrointestinal Pathophysiology. Chapter 25. 2017; Weiss and Thierry, Mechanisms and consequences of intestinal dysbiosis. Cellular and Molecular Life Sciences. (2017) 74(16):2959-2977. Zurich Open Repository and Archive, doi.org/10.1007/s00018-017- 2509-x)).
  • a healthy host-gut microbiome homeostasis is sometimes referred to as a “eubiosis” or “normobiosis,” whereas a detrimental change in the host microbiome composition and/or its diversity can lead to an unhealthy imbalance in the microbiome, or a “dysbiosis” (Hooks and O’Malley. Dysbiosis and its discontents. American Society for Microbiology. Oct 2017. Vol. 8. Issue 5. mBio 8:e01492-17. https://doi.org/10.1128/mBio.01492-17).
  • Dysbiosis, and associated local or distal host inflammatory or immune effects may occur where microbiome homeostasis is lost or diminished, resulting in: increased susceptibility to pathogens; altered host bacterial metabolic activity; induction of host proinflammatory activity and/or reduction of host anti-inflammatory activity.
  • Such effects are mediated in part by interactions between host immune cells (for example, T cells, dendritic cells, mast cells, NK cells, intestinal epithelial lymphocytes (IEC), macrophages and phagocytes) and cytokines, and other substances released by such cells and other host cells.
  • a dysbiosis may occur within the gastrointestinal tract (a “gastrointestinal dysbiosis” or “gut dysbiosis”) or may occur outside the lumen of the gastrointestinal tract (a “distal dysbiosis”).
  • Gastrointestinal dysbiosis is often associated with a reduction in integrity of the intestinal epithelial barrier, reduced tight junction integrity and increased intestinal permeability.
  • Citi, S. Intestinal Barriers protect against disease, Science 359: 1098-99 (2016); Srinivasan et al., TEER measurement techniques for in vitro barrier model systems. J. Lab. Autom. 20: 107-126 (2015).
  • a gastrointestinal dysbiosis can have physiological and immune effects within and outside the gastrointestinal tract.
  • the presence of a dysbiosis can be associated with a wide variety of diseases and conditions including: infection, cancer, autoimmune disorders (for example, systemic lupus erythematosus (SLE)) or inflammatory disorders (for example, functional gastrointestinal disorders such as inflammatory bowel disease (IBD), ulcerative colitis, and Crohn’s disease), neuroinflammatory diseases (for example, multiple sclerosis), transplant disorders (for example, graft-versus-host disease), fatty liver disease, type I diabetes, rheumatoid arthritis, Sjogren’s syndrome, celiac disease, cystic fibrosis, chronic obstructive pulmonary disorder (COPD), and other diseases and conditions associated with immune dysfunction.
  • autoimmune disorders for example, systemic lupus erythematosus (SLE)
  • inflammatory disorders for example, functional gastrointestinal disorders such as inflammatory bowel disease (IBD), ulcerative colitis, and Crohn’s disease
  • neuroinflammatory diseases for example, multiple sclerosis
  • transplant disorders for example,
  • exemplary therapeutic compositions disclosed herein can treat a dysbiosis and its effects by modifying the immune activity present at the site of dysbiosis.
  • such compositions can modify a dysbiosis via effects on host immune cells, resulting in, for example, an effect on secretion of cytokines , reducing inflammation in the subject recipient or via changes in metabolite production.
  • compositions disclosed herein that are useful for treatment of disorders associated with a dysbiosis contain one or more types of EVs derived from immunomodulatory bacteria. Such compositions are capable of affecting the recipient host’s immune function, in the gastrointestinal tract, and/or a systemic effect at distal sites outside the subject’s gastrointestinal tract.
  • compositions disclosed herein that are useful for treatment of disorders associated with a dysbiosis contain a population of immunomodulatory bacteria of a single bacterial species (for example, a single strain) and/or a population of EVs derived from immunomodulatory bacteria of a single bacterial species (for example, a single strain). Such compositions are capable of affecting the recipient host’s immune function, in the gastrointestinal tract, and /or a systemic effect at distal sites outside the subject’s gastrointestinal tract.
  • compositions containing an isolated population of EVs derived from immunomodulatory bacteria are administered (for example, orally) to a mammalian recipient in an amount effective to treat a dysbiosis and one or more of its effects in the recipient.
  • the dysbiosis may be a gastrointestinal tract dysbiosis or a distal dysbiosis.
  • compositions of the instant invention can treat a gastrointestinal dysbiosis and one or more of its effects on host immune cells, resulting in an effect on cytokines secretion, reducing inflammation in the subject recipient.
  • the therapeutic compositions can treat a gastrointestinal dysbiosis and one or more of its effects by modulating the recipient immune response via cellular and cytokine modulation to reduce gut permeability by increasing the integrity of the intestinal epithelial barrier.
  • the therapeutic compositions can treat a distal dysbiosis and one or more of its effects by modulating the recipient immune response at the site of dysbiosis via modulation of host immune cells.
  • compositions are useful for treatment of disorders associated with a dysbiosis, which compositions contain one or more types of bacteria and/or EVs capable of altering the relative proportions of host immune cell subpopulations, for example, subpopulations of T cells, immune lymphoid cells, dendritic cells, NK cells and other immune cells, or the function thereof, in the recipient.
  • compositions are useful for treatment of disorders associated with a dysbiosis, which compositions contain a population of EVs of a single immunomodulatory bacterial species (for example, a single strain) capable of altering the relative proportions of immune cell subpopulations, for example, T cell subpopulations, immune lymphoid cells, NK cells and other immune cells, or the function thereof, in the recipient subject.
  • a single immunomodulatory bacterial species for example, a single strain
  • immune cell subpopulations for example, T cell subpopulations, immune lymphoid cells, NK cells and other immune cells, or the function thereof, in the recipient subject.
  • the invention provides methods of treating a gastrointestinal dysbiosis and one or more of its effects by orally administering to a subject in need thereof a therapeutic composition which alters the microbiome population existing at the site of the dysbiosis.
  • the therapeutic composition can contain one or more types of EVs from immunomodulatory bacteria or a population of EVs of a single immunomodulatory bacterial species (for example, a single strain).
  • the invention provides methods of treating a distal dysbiosis and one or more of its effects by orally administering to a subject in need thereof a therapeutic composition which alters the subject’s immune response outside the gastrointestinal tract.
  • the therapeutic composition can contain one or more types of EVs from immunomodulatory bacteria or a population of EVs of a single immunomodulatory bacterial species (for example, a single strain).
  • therapeutic compositions useful for treatment of disorders associated with a dysbiosis stimulate secretion of one or more antiinflammatory cytokines by host immune cells.
  • Anti-inflammatory cytokines include, but are not limited to, IL-10, IL-13, IL-9, IL-4, IL-5, TGFP, and combinations thereof.
  • pharmaceutical compositions useful for treatment of disorders associated with a dysbiosis that decrease (for example, inhibit) secretion of one or more pro-inflammatory cytokines by host immune cells.
  • Pro-inflammatory cytokines include, but are not limited to, TFNy, IL-12p70, IL- la, IL-6, IL-8, MCP1, MIPla, MIPlp, TNFa, and combinations thereof.
  • Other exemplary cytokines are known in the art and are described herein.
  • the invention provides a method of treating or preventing a disorder associated with a dysbiosis in a subject in need thereof, comprising administering (for example, orally administering) to the subject a therapeutic composition in the form of a probiotic or medical food comprising F. massiliensis bacteria or EVs in an amount sufficient to alter the microbiome at a site of the dysbiosis, such that the disorder associated with the dysbiosis is treated.
  • a therapeutic composition of the instant invention in the form of a probiotic or medical food may be used to prevent or delay the onset of a dysbiosis in a subject at risk for developing a dysbiosis.
  • engineered bacteria for the production of the F. massiliensis EVs described herein.
  • the engineered bacteria are modified to enhance certain desirable properties.
  • the engineered bacteria are modified to enhance the immunomodulatory and/or therapeutic effect of the F. massiliensis EVs (for example, either alone or in combination with another therapeutic agent), to reduce toxicity and/or to improve bacterial and/or EV manufacturing (for example, higher oxygen tolerance, improved freeze-thaw tolerance, shorter generation times).
  • the engineered bacteria may be produced using any technique known in the art, including but not limited to site- directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, CRISPR/Cas9, or any combination thereof.
  • the bacterium is modified by directed evolution.
  • the directed evolution comprises exposure of the bacterium to an environmental condition and selection of bacterium with improved survival and/or growth under the environmental condition.
  • the method comprises a screen of mutagenized bacteria using an assay that identifies enhanced bacterium.
  • the method further comprises mutagenizing the bacteria (for example, by exposure to chemical mutagens and/or UV radiation) or exposing them to a therapeutic agent (for example, antibiotic) followed by an assay to detect bacteria having the desired phenotype (for example, an in vivo assay, an ex vivo assay, or an in vitro assay).
  • Example 1 Purification and preparation of extracellular vesicles (EVs) from bacteria
  • Extracellular vesicles (such as smEVs) are purified and prepared from bacterial cultures using methods known to those skilled in the art (S. Bin Park, et al. PLoS ONE. 6(3):el7629 (2011)).
  • bacterial cultures are centrifuged at 10,000-15,500 x g for 10-40 min at 4°C or room temperature to pellet bacteria.
  • Culture supernatants are then filtered to include material ⁇ 0.22 pm (for example, via a 0.22 pm or 0.45 pm filter) and to exclude intact bacterial cells.
  • Filtered supernatants are concentrated using methods that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration. Briefly, for ammonium sulfate precipitation, 1.5-3 M ammonium sulfate is added to filtered supernatant slowly, while stirring at 4°C.
  • Precipitations are incubated at 4°C for 8-48 hours and then centrifuged at 11,000 x g for 20-40 min at 4°C.
  • the pellets contain EVs and other debris.
  • using ultracentrifugation filtered supernatants are centrifuged at 100,000-200,000 x g for 1-16 hours at 4°C.
  • the pellet of this centrifugation contains EVs and other debris.
  • using a filtration technique using an Amicon Ultra spin filter or by tangential flow filtration, supernatants are filtered so as to retain species of molecular weight > 50, 100, 300, or 500 kDa.
  • EVs are obtained from bacterial cultures continuously during growth, or at selected time points during growth, by connecting a bioreactor to an alternating tangential flow (ATF) system (for example, XCell ATF from Repligen) according to manufacturer’s instructions.
  • ATF alternating tangential flow
  • the ATF system retains intact cells (> 0.22 pm) in the bioreactor, and allows smaller components (for example, EVs, free proteins) to pass through a filter for collection.
  • the system may be configured so that the ⁇ 0.22 pm filtrate is then passed through a second filter of 100 kDa, allowing species such as EVs between 0.22 pm and 100 kDa to be collected, and species smaller than 100 kDa to be pumped back into the bioreactor.
  • the system may be configured to allow for medium in the bioreactor to be replenished and/or modified during growth of the culture.
  • EVs collected by this method may be further purified and/or concentrated by ultracentrifugation or filtration as described above for filtered supernatants.
  • EVs obtained by methods described above may be further purified by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column.
  • Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 45% Optiprep in PBS. If filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 45% Optiprep. Samples are applied to a 0-45% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Alternatively, high resolution density gradient fractionation could be used to separate EVs based on density. Preparation
  • EVs are serially diluted onto agar medium used for routine culture of the bacteria being tested and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 pm filter to exclude intact cells. To further increase purity, isolated EVs may be DNase or proteinase K treated.
  • samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 using filtration (for example, Amicon Ultra columns), dialysis, or ultracentrifugation (following 15-fold or greater dilution in PBS, 200,000 x g, 1-3 hours, 4°C) and resuspension in PBS.
  • filtration for example, Amicon Ultra columns
  • dialysis for example, dialysis
  • ultracentrifugation followeding 15-fold or greater dilution in PBS, 200,000 x g, 1-3 hours, 4°C
  • EVs may be heated, irradiated, and/or lyophilized prior to administration (as described herein).
  • Example 2 Manipulating bacteria through stress to produce various amounts of EVs and/or to vary content of EVs
  • Bacteria may be subjected to single stressors or stressors in combination. The effects of different stressors on different bacteria is determined empirically by varying the stress condition and determining the IC50 value (the conditions required to inhibit cell growth by 50%).
  • EV purification, quantification, and characterization occurs. EV production is quantified (1) in complex samples of bacteria and EVs by nanoparticle tracking analysis (NTA) or transmission electron microscopy (TEM); or (2) following EV purification by NTA, lipid quantification, or protein quantification. EV content is assessed following purification by methods described above.
  • NTA nanoparticle tracking analysis
  • TEM transmission electron microscopy
  • Bacteria are cultivated under standard growth conditions with the addition of sublethal concentrations of antibiotics. This may include 0.1-1 pg/mL chloramphenicol, or 0.1-0.3 pg/mL gentamicin, or similar concentrations of other antibiotics (for example, ampicillin, polymyxin B). Host antimicrobial products such as lysozyme, defensins, and Reg proteins may be used in place of antibiotics. Bacterially-produced antimicrobial peptides, including bacteriocins and mi crocins may also be used.
  • Bacteria are cultivated under standard growth conditions, but at higher or lower temperatures than are typical for their growth. Alternatively, bacteria are grown under standard conditions, and then subjected to cold shock or heat shock by incubation for a short period of time at low or high temperatures respectively. For example, bacteria grown at 37°C are incubated for 1 hour at 4°C-18°C for cold shock or 42°C-50°C for heat shock.
  • bacteria are cultivated under conditions where one or more nutrients are limited. Bacteria may be subjected to nutritional stress throughout growth or shifted from a rich medium to a poor medium.
  • Some examples of media components that are limited are carbon, nitrogen, iron, and sulfur.
  • An example medium is M9 minimal medium (Sigma-Aldrich), which contains low glucose as the sole carbon source.
  • M9 minimal medium Sigma-Aldrich
  • iron availability is varied by altering the concentration of hemin in media and/or by varying the type of porphyrin or other iron carrier present in the media, as cells grown in low hemin conditions were found to produce greater numbers of EVs (S. Stubbs et al. Letters in Applied Microbiology. 29:31- 36 (1999).
  • Media components are also manipulated by the addition of chelators such as EDTA and deferoxamine.
  • Bacteria are grown to saturation and incubated past the saturation point for various periods of time.
  • conditioned media is used to mimic saturating environments during exponential growth.
  • Conditioned media is prepared by removing intact cells from saturated cultures by centrifugation and filtration, and conditioned media may be further treated to concentrate or remove specific components.
  • Bacteria are cultivated in or exposed for brief periods to medium containing NaCl, bile salts, or other salts.
  • UV stress is achieved by cultivating bacteria under a UV lamp or by exposing bacteria to UV using an instrument such as a Stratalinker (Agilent). UV may be administered throughout the entire cultivation period, in short bursts, or for a single defined period following growth.
  • Stratalinker Stratalinker
  • Bacteria are cultivated in the presence of sublethal concentrations of hydrogen peroxide (250-1,000 pM) to induce stress in the form of reactive oxygen species. Anaerobic bacteria are cultivated in or exposed to concentrations of oxygen that are toxic to them.
  • Bacteria are cultivated in or exposed to detergent, such as sodium dodecyl sulfate (SDS) or deoxy cholate. pH stress
  • Bacteria are cultivated in or exposed for limited times to media of different pH.
  • Example 3 Profiling EV composition and content
  • EVs may be characterized by any one of various methods including, but not limited to, NanoSight characterization, SDS-PAGE gel electrophoresis, Western blot, ELISA, liquid chromatography-mass spectrometry and mass spectrometry, dynamic light scattering, lipid levels, total protein, lipid to protein ratios, nucleic acid analysis and/or zeta potential.
  • Nanoparticle tracking analysis is used to characterize the size distribution of purified EVs. Purified EV preparations are run on a NanoSight machine (Malvern Instruments) to assess EV size and concentration. SDS-PAGE Gel Electrophoresis
  • samples are run on a gel, for example a Bolt Bis-Tris Plus 4-12% gel (Thermo-Fisher Scientific), using standard techniques. Samples are boiled in lx SDS sample buffer for 10 minutes, cooled to 4°C, and then centrifuged at 16,000 x g for 1 min. Samples are then run on a SDS-PAGE gel and stained using one of several standard techniques (for example, Silver staining, Coomassie Blue, Gel Code Blue) for visualization of bands.
  • a gel for example a Bolt Bis-Tris Plus 4-12% gel (Thermo-Fisher Scientific)
  • Samples are boiled in lx SDS sample buffer for 10 minutes, cooled to 4°C, and then centrifuged at 16,000 x g for 1 min. Samples are then run on a SDS-PAGE gel and stained using one of several standard techniques (for example, Silver staining, Coomassie Blue, Gel Code Blue) for visualization of bands.
  • EV proteins are separated by SDS-PAGE as described above and subjected to Western blot analysis (Cvjetkovic et al., Sci. Rep. 6, 36338 (2016)) and are quantified via ELISA.
  • EV proteins are identified and quantified by Mass Spectrometry techniques.
  • EV proteins may be prepared for LC-MS/MS using standard techniques including protein reduction using dithiothreitol solution (DTT) and protein digestion using enzymes such as LysC and trypsin as described in Erickson et al, 2017 (Molecular Cell, VOLUME 65, ISSUE 2, P361-370, JANUARY 19, 2017).
  • DTT dithiothreitol solution
  • peptides are prepared as described by Liu et al. 2010 (JOURNAL OF BACTERIOLOGY, June 2010, p. 2852-2860 Vol. 192, No. 11), Kieselbach and Oscarsson 2017 (Data Brief.
  • peptide preparations are run directly on liquid chromatography and mass spectrometry devices for protein identification within a single sample.
  • peptide digests from different samples are labeled with isobaric tags using the iTRAQ Reagent-8plex Multiplex Kit (Applied Biosystems, Foster City, CA) or TMT lOplex and 1 Iplex Label Reagents (Thermo Fischer Scientific, San Jose, CA, USA).
  • iTRAQ Reagent-8plex Multiplex Kit Applied Biosystems, Foster City, CA
  • TMT lOplex and 1 Iplex Label Reagents Thermo Fischer Scientific, San Jose, CA, USA.
  • Each peptide digest is labeled with a different isobaric tag and then the labeled digests are combined into one sample mixture.
  • the combined peptide mixture is analyzed by LC-MS/MS for both identification and quantification.
  • a database search is performed using the LC-MS/MS data to identify the labeled peptides and the corresponding proteins.
  • the fragmentation of the attached tag generates a low molecular mass reporter ion that is used to obtain a relative quantitation of the peptides and proteins present in each EV.
  • metabolic content is ascertained using liquid chromatography techniques combined with mass spectrometry.
  • a LC-MS system includes a 4000 QTRAP triple quadrupole mass spectrometer (AB SCIEX) combined with 1100 Series pump (Agilent) and an HTS PAL autosampler (Leap Technologies).
  • the column is eluted by flowing a 5% mobile phase [lOmM ammonium formate, 0.1% formic acid in water] for 1 minute at a rate of 250 pL/minute followed by a linear gradient over 10 minutes to a solution of 40% mobile phase [acetonitrile with 0.1% formic acid].
  • the ion spray voltage is set to 4.5 kV and the source temperature is 450°C.
  • DLS measurements including the distribution of particles of different sizes in different EV preparations are taken using instruments such as the DynaPro NanoStar (Wyatt Technology) and the Zetasizer Nano ZS (Malvern Instruments). Lipid Levels
  • Lipid levels are quantified using FM4-64 (Life Technologies), by methods similar to those described by A. J. McBroom et al. J Bacterial 188:5385-5392. and A. Frias, et al. Microb Ecol. 59:476-486 (2010). Samples are incubated with FM4-64 (3.3 pg/mL in PBS for 10 minutes at 37°C in the dark). After excitation at 515 nm, emission at 635 nm is measured using a Spectramax M5 plate reader (Molecular Devices). Absolute concentrations are determined by comparison of unknown samples to standards (such as palmitoyloleoylphosphatidylglycerol (POPG) vesicles) of known concentrations.
  • POPG palmitoyloleoylphosphatidylglycerol
  • Lipidomics can be used to identify the lipids present in the EVs.
  • Protein levels are quantified by standard assays such as the Bradford and BCA assays.
  • the Bradford assays are run using Quick Start Bradford lx Dye Reagent (BioRad), according to manufacturer’s protocols.
  • BCA assays are run using the Pierce BCA Protein Assay Kit (Thermo-Fisher Scientific). Absolute concentrations are determined by comparison to a standard curve generated from BSA of known concentrations.
  • protein concentration can be calculated using the Beer-Lambert equation using the sample absorbance at 280 nm (A280) as measured on a Nanodrop spectrophotometer (Thermo-Fisher Scientific).
  • proteomics may be used to identify proteins in the sample.
  • Lipid:protein ratios are generated by dividing lipid concentrations by protein concentrations. These provide a measure of the purity of vesicles as compared to free protein in each preparation.
  • Nucleic acids are extracted from EVs and quantified using a Qubit fluorimeter. Size distribution is assessed using a BioAnalyzer and the material is sequenced.
  • Enriched media is used to grow and prepare the bacteria for in vitro and in vivo use and, ultimately, for EV preparations.
  • media may contain sugar, yeast extracts, plant-based peptones, buffers, salts, trace elements, surfactants, anti-foaming agents, and vitamins.
  • Composition of complex components such as yeast extracts and peptones may be undefined or partially defined (including approximate concentrations of amino acids, sugars etc.).
  • Microbial metabolism may be dependent on the availability of resources such as carbon and nitrogen. Various sugars or other carbon sources may be tested.
  • media may be prepared and the selected bacterium grown as shown by Saarela et al., J. Applied Microbiology. 2005. 99: 1330-1339, which is hereby incorporated by reference. Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze-drying survival, storage stability, and acid and bile exposure of the selected bacterium produced without milk-based ingredients.
  • the media is sterilized. Sterilization may be accomplished by Ultra High Temperature (UHT) processing.
  • UHT Ultra High Temperature
  • the UHT processing is performed at very high temperature for short periods of time.
  • the UHT range may be from 135-180°C.
  • the medium may be sterilized from between 10 to 30 seconds at 135°C.
  • Inoculum can be prepared in flasks or in smaller bioreactors and growth is monitored.
  • the inoculum size may be between approximately 0.5 and 3% of the total bioreactor volume.
  • bioreactor volume can be at least 2 L, 10 L, 80 L, 100 L, 250 L, 1000 L, 2500 L, 5000 L, 10,000 L.
  • the bioreactor Before the inoculation, the bioreactor is prepared with medium at desired pH, temperature, and oxygen concentration.
  • the initial pH of the culture medium may be different that the process set-point. pH stress may be detrimental at low cell centration; the initial pH could be between pH 7.5 and the process set-point. For example, pH may be set between 4.5 and 8.0.
  • the pH can be controlled through the use of sodium hydroxide, potassium hydroxide, or ammonium hydroxide.
  • the temperature may be controlled from 25°C to 45°C, for example at 37°C. Anaerobic conditions are created by reducing the level of oxygen in the culture broth from around 8 mg/L to 0 mg/L.
  • N2, CO2, and H2 nitrogen or gas mixtures
  • no gases are used and anaerobic conditions are established by cells consuming remaining oxygen from the medium.
  • the bioreactor fermentation time can vary. For example, fermentation time can vary from approximately 5 hours to 48 hours.
  • Reviving bacteria from a frozen state may require special considerations.
  • Production medium may stress cells after a thaw; a specific thaw medium may be required to consistently start a seed train from thawed material.
  • the kinetics of transfer or passage of seed material to fresh medium may be influenced by the current state of the bacteria (ex. exponential growth, stationary growth, unstressed, stressed).
  • Inoculation of the production fermenter(s) can impact growth kinetics and cellular activity.
  • the initial state of the bioreactor system must be optimized to facilitate successful and consistent production.
  • the fraction of seed culture to total medium (for example, a percentage) has a dramatic impact on growth kinetics.
  • the range may be 1-5% of the fermenter’s working volume.
  • the initial pH of the culture medium may be different from the process set-point. pH stress may be detrimental at low cell concentration; the initial pH may be between pH 7.5 and the process set-point. Agitation and gas flow into the system during inoculation may be different from the process set-points. Physical and chemical stresses due to both conditions may be detrimental at low cell concentration.
  • Process conditions and control settings may influence the kinetics of microbial growth and cellular activity. Shifts in process conditions may change membrane composition, production of metabolites, growth rate, cellular stress, etc.
  • Optimal temperature range for growth may vary with strain. The range may be 20-40°C.
  • Optimal pH for cell growth and performance of downstream activity may vary with strain. The range may be pH 5-8. Gasses dissolved in the medium may be used by cells for metabolism. Adjusting concentrations of O2, CO2, and N2 throughout the process may be required. Availability of nutrients may shift cellular growth. Bacteria may have alternate kinetics when excess nutrients are available.
  • Bacteria may be preconditioned shortly before harvest to better prepare them for the physical and chemical stresses involved in separation and downstream processing. A change in temperature (often reducing to 20-5°C) may reduce cellular metabolism, slowing growth (and/or death) and physiological change when removed from the fermenter. Effectiveness of centrifugal concentration may be influenced by culture pH. Raising pH by 1-2 points can improve effectiveness of concentration but can also be detrimental to cells. Bacteria may be stressed shortly before harvest by increasing the concentration of salts and/or sugars in the medium. Cells stressed in this way may better survive freezing and lyophilization during downstream.
  • Separation methods and technology may impact how efficiently bacteria are separated from the culture medium.
  • Solids may be removed using centrifugation techniques. Effectiveness of centrifugal concentration can be influenced by culture pH or by the use of flocculating agents. Raising pH by 1-2 points may improve effectiveness of concentration but can also be detrimental to cells.
  • Bacteria may be stressed shortly before harvest by increasing the concentration of salts and/or sugars in the medium. Cells stressed in this way may better survive freezing and lyophilization during downstream. Additionally, Bacteria may also be separated via filtration. Filtration is superior to centrifugation techniques for purification if the cells require excessive g-minutes to successfully centrifuge. Excipients can be added before after separation.
  • Excipients can be added for cryo protection or for protection during lyophilization.
  • Excipients can include, but are not limited to, sucrose, trehalose, or lactose, and these may be alternatively mixed with buffer and anti-oxidants.
  • droplets of cell pellets mixed with excipients are submerged in liquid nitrogen.
  • Harvesting can be performed by continuous centrifugation.
  • Product may be resuspended with various excipients to a desired final concentration.
  • Excipients can be added for cryo protection or for protection during lyophilization.
  • Excipients can include, but are not limited to, sucrose, trehalose, or lactose, and these may be alternatively mixed with buffer and anti-oxidants.
  • droplets of cell pellets mixed with excipients are submerged in liquid nitrogen.
  • Lyophilization of material including live bacteria, vesicles, or other bacterial derivative includes a freezing, primary drying, and secondary drying phase.
  • Lyophilization begins with freezing.
  • the product material may or may not be mixed with a lyoprotectant or stabilizer prior to the freezing stage.
  • a product may be frozen prior to the loading of the lyophilizer, or under controlled conditions on the shelf of the lyophilizer.
  • the primary drying phase ice is removed via sublimation. Here, a vacuum is generated and an appropriate amount of heat is supplied to the material. The ice will sublime while keeping the product temperature below freezing, and below the material’s critical temperature (T c ). The temperature of the shelf on which the material is loaded and the chamber vacuum can be manipulated to achieve the desired product temperature.
  • product-bound water molecules are removed.
  • the temperature is generally raised higher than in the primary drying phase to break any physico-chemical interactions that have formed between the water molecules and the product material.
  • the chamber may be filled with an inert gas, such as nitrogen.
  • the product may be sealed within the freeze dryer under dry conditions, in a glass vial or other similar container, preventing exposure to atmospheric water and contaminates.
  • the equipment used in EV isolation includes a Sorvall RC-5C centrifuge with SLA-3000 rotor; an Optima XE-90 Ultracentrifuge by Beckman-Coulter 45Ti rotor; a Sorvall wX+ Ultra Series Centrifuge by Thermo Scientific; and a Fiberlite F37L-8xl00 rotor.
  • Bacteria must be pelleted and filtered away from supernatant in order to recover EVs and not bacteria.
  • Pellet bacterial culture is generated by using a Sorvall RC-5C centrifuge with the SLA-3000 rotor and centrifuge culture for a minimum of 15 min at a minimum of 7,000 rpm. And then decanting the supernatant into new and sterile container.
  • the supernatant is filtered through a 0.2 pm filter.
  • a 0.45 pm capsule filter is attached ahead of the 0.2 pm vacuum filter.
  • the filtered supernatant is stored at 4°C.
  • the filtered supernatant can then be concentrated using TFF.
  • Density gradients are used for EV purification. During ultracentrifugation, particles in the sample will move, and separate, within the graded density medium based on their ‘buoyant’ densities. In this way EVs are separated from other particles, such as sugars, lipids, or other proteins, in the sample.
  • EV purification For EV purification, four different percentages of the density medium (60% Optiprep) are used, a 45% layer, a 35% layer, a 25%, and a 15% layer. This will create the graded layers. A 0% layer is added at the top consisting of sterile lx PBS. The 45% gradient layer should contain the crude EV sample. 5 ml of sample is added to 15 ml of Optiprep. If crude EV sample is less than 5 ml, bring up to volume using sterile lx PBS.
  • the 45% gradient mixture is pipetted up and down to mix.
  • the sample is then pipetted into a labeled clean and sterile ultracentrifuge tube.
  • a 10 ml serological pipette is used to slowly add 13 ml of 35% gradient mixture.
  • 13 ml of the 25% gradient mixture is added, followed by 13 ml of the 15% mixture and finally 6 ml of sterile lx PBS.
  • the ultracentrifuge tubes are balanced with sterile lx PBS.
  • the gradients are carefully placed in a rotor and the ultracentrifuge is set for 200,000 x g and 4°C. The gradients are centrifuged for a minimum of 16 hours.
  • a clean pipette is used to remove fraction(s) of interest, which are added to 15 ml conical tube. These ‘purified’ EV samples are kept at 4°C.
  • lOx volume of PBS are added to purified EVs.
  • the ultracentrifuge is set for 200,000 x g and 4°C. Centrifuge and spun for 1 hour. The tubes are carefully removed from ultracentrifuge and the supernatant decanted.
  • the purified EVs are washed until all sample has been pelleted, lx PBS is added to the purified pellets, which are placed in a container. The container is placed on a shaker set at speed 70 at 4°C overnight or longer.
  • the ‘purified’ EV pellets are resuspended with additional sterile IxPBS.
  • the resuspended purified EV samples are stored at 4°C or at -80°C.
  • Example 6 Veillonella parvula and Fournierella massiliensis smEVs
  • Clarified supernatant was then concentrated approximately 25-fold by Tangential Flow Filtration (TFF) using a 300 kd mPES hollow fiber filter. Concentrates were further purified via a 5-volume continuous diafiltration to remove residual medium components and waste products.
  • Example 7 Veillonella parvula and Fournierella massiliensis smEVs powders
  • the Veillonella parvula strain used as a source of EVs was the Veillonella bacteria deposited as ATCC Deposit number PTA-125691. See also WO 2019/157003.
  • the Fournierella massiliensis strain used as a source of EVs was the Fournierella massiliensis bacteria deposited as ATCC Deposit number PTA-126696. See also PCT/US21/36927 (WO 2021/252838).
  • Each excipient stock (see Table A below) was prepared as a 15% (w/w) stock solution for the purpose of mixing with purified EV concentrate. Stocks were filter sterilized with a 0.2 pm bottle top filter and stored under ambient conditions until use. Each stabilizer solution was mixed by mass with purified concentrate in a ratio of 0.5875:1 to create an EV-stabilizer “slurry”.
  • each powder was analyzed for moisture content (by Karl Fischer titration (KF)), particle size distribution and zeta-potential (by dynamic light scattering (DLS)), and particles (quantification by nanoparticle tracking analysis (NTA)). Results are reported in Tables C, D, and E below.
  • the DLS method measures particle size and electrokinetic potential (charge) of nanoparticles based on the diffraction of monochromatic light from a laser source. DLS measurements (either size or charge) may be analyzed as an average of the entire sample distribution or broken down into up to three distinct subpopulations per sample. Results are reported as either the average of the entire distribution (e.g., z-average), or as the average of the most dominant subpopulation (e.g., peak size, peak zeta-potential).
  • Table A Excipient stock formulations by relative concentration (%w:w).
  • Table B General conservative lyophilization cycle for EVs.
  • Table C Particle count by NTA and moisture content by KF.
  • Table D Particle size distribution determined by DLS, including average size of the distribution and the size of the dominant subpopulation (peak size).
  • Table E Electrokinetic potential of the dominant subpopulation determined by DLS.
  • Figure 2 is a graph showing particle count of lyophilized EV powders, the data in Table C.
  • Figure 3 is a graph showing average particle size by DLS of lyophilized EV powders, the data in Table D.
  • Figure 4 is a graph showing electrokinetic potential of the dominant subpopulation of lyophilized EV powder by DLS, the data shown in table E.
  • Figure 5 is a graph showing particle size of the dominant subpopulation of lyophilized EV powders, the data shown in Table D.
  • Example 8 Additional Stocks for Drying
  • F. massiliensis EVs are dried, such as by freeze drying or spray drying, using one of the stockes provided in Table F.
  • Table F Stocks comprising excipients by relative concentration (%w:w)

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Abstract

L'invention concerne des solutions et des formes séchées de vésicules extracellulaires de Fournierella massiliensis (VE de F. massiliensis) qui sont utiles en tant qu'agents thérapeutiques, ainsi que des compositions thérapeutiques à base de celles-ci.
PCT/US2022/052846 2021-12-14 2022-12-14 Préparations de vésicules extracellulaires de bactéries fournierella massiliensis WO2023114300A1 (fr)

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Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4775536A (en) 1986-02-24 1988-10-04 Bristol-Myers Company Enteric coated tablet and process for making
US5047258A (en) 1989-07-14 1991-09-10 Sterling Drug Inc. Aqueous spray-coating process
US5292522A (en) 1989-06-20 1994-03-08 Rohm Gmbh Aqueous film coating agent for solid medicaments
US6312728B1 (en) 1998-07-07 2001-11-06 Cascade Development, Inc. Sustained release pharmaceutical preparation
US6555124B1 (en) 1996-08-01 2003-04-29 Basf Aktiengesellschaft Use of (meth)acrylic acid copolymers to increase the permeability of mucous membranes
US6623759B2 (en) 1996-06-28 2003-09-23 Astrazeneca Ab Stable drug form for oral administration with benzimidazole derivatives as active ingredient and process for the preparation thereof
US6638534B1 (en) 1998-07-28 2003-10-28 Tanabe Seiyaku Co., Ltd. Preparation capable of releasing drug at target site in intestine
US20040028737A1 (en) 2002-08-12 2004-02-12 Kopran Research Laboratories Limited Enteric coated stable oral pharmaceutical composition of acid unstable drug and process for preparing the same
WO2005044240A2 (fr) 2003-10-31 2005-05-19 Dexcel, Ltd. Formulation stable contenant du lansoprazole
US20050271778A1 (en) 2002-12-20 2005-12-08 Roehm Gmbh & Co., Kg Method for producing coated pharmaceuticals and food supplements with concentration gradients in the coating thereof
US20060210631A1 (en) 2005-03-21 2006-09-21 Patel Ashish A Multi-particulate, modified-release composition
US20080200482A1 (en) 2005-07-12 2008-08-21 Evonik Roehm Gmbh Use of a Partially Neutralized, Anionic (Meth)Acrylate Copolymer as a Coating for the Production of a Medicament Releasing Active Substance at Reduced Ph Values
US9233074B2 (en) 2013-03-01 2016-01-12 Bpsi Holdings, Llc Delayed release film coatings containing calcium silicate and substrates coated therewith
WO2019051380A1 (fr) * 2017-09-08 2019-03-14 Evelo Biosciences, Inc. Vésicules extracellulaires (ev) bactériennes
WO2019157003A1 (fr) 2018-02-06 2019-08-15 Evelo Biosciences, Inc. Compositions et méthodes pour traiter le cancer et des troubles immunitaires au moyen de bacteries veillonelles
WO2020252149A1 (fr) * 2019-06-11 2020-12-17 Evelo Biosciences, Inc. Vésicules extracellulaires microbiennes traitées
WO2021252838A1 (fr) 2020-06-11 2021-12-16 Evelo Biosciences, Inc. Compositions et méthodes de traitement de maladies et de troubles à l'aide de fournierella massiliensis

Patent Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4775536A (en) 1986-02-24 1988-10-04 Bristol-Myers Company Enteric coated tablet and process for making
US5292522A (en) 1989-06-20 1994-03-08 Rohm Gmbh Aqueous film coating agent for solid medicaments
US5047258A (en) 1989-07-14 1991-09-10 Sterling Drug Inc. Aqueous spray-coating process
US6623759B2 (en) 1996-06-28 2003-09-23 Astrazeneca Ab Stable drug form for oral administration with benzimidazole derivatives as active ingredient and process for the preparation thereof
US6555124B1 (en) 1996-08-01 2003-04-29 Basf Aktiengesellschaft Use of (meth)acrylic acid copolymers to increase the permeability of mucous membranes
US6312728B1 (en) 1998-07-07 2001-11-06 Cascade Development, Inc. Sustained release pharmaceutical preparation
US6638534B1 (en) 1998-07-28 2003-10-28 Tanabe Seiyaku Co., Ltd. Preparation capable of releasing drug at target site in intestine
US20040028737A1 (en) 2002-08-12 2004-02-12 Kopran Research Laboratories Limited Enteric coated stable oral pharmaceutical composition of acid unstable drug and process for preparing the same
US20050271778A1 (en) 2002-12-20 2005-12-08 Roehm Gmbh & Co., Kg Method for producing coated pharmaceuticals and food supplements with concentration gradients in the coating thereof
WO2005044240A2 (fr) 2003-10-31 2005-05-19 Dexcel, Ltd. Formulation stable contenant du lansoprazole
US20060210631A1 (en) 2005-03-21 2006-09-21 Patel Ashish A Multi-particulate, modified-release composition
US20080200482A1 (en) 2005-07-12 2008-08-21 Evonik Roehm Gmbh Use of a Partially Neutralized, Anionic (Meth)Acrylate Copolymer as a Coating for the Production of a Medicament Releasing Active Substance at Reduced Ph Values
US9233074B2 (en) 2013-03-01 2016-01-12 Bpsi Holdings, Llc Delayed release film coatings containing calcium silicate and substrates coated therewith
WO2019051380A1 (fr) * 2017-09-08 2019-03-14 Evelo Biosciences, Inc. Vésicules extracellulaires (ev) bactériennes
WO2019157003A1 (fr) 2018-02-06 2019-08-15 Evelo Biosciences, Inc. Compositions et méthodes pour traiter le cancer et des troubles immunitaires au moyen de bacteries veillonelles
WO2020252149A1 (fr) * 2019-06-11 2020-12-17 Evelo Biosciences, Inc. Vésicules extracellulaires microbiennes traitées
WO2021252838A1 (fr) 2020-06-11 2021-12-16 Evelo Biosciences, Inc. Compositions et méthodes de traitement de maladies et de troubles à l'aide de fournierella massiliensis

Non-Patent Citations (36)

* Cited by examiner, † Cited by third party
Title
"Guide to Huge Computers", 1994, ACADEMIC PRESS
A. FRIAS ET AL., MICROB ECOL, vol. 59, 2010, pages 476 - 486
ACHTMAN MWAGNER M: "Microbial diversity and the genetic nature of microbial species", NAT. REV. MICROBIOL., vol. 6, 2008, pages 431 - 440, XP037115178, DOI: 10.1038/nrmicro1872
ATSCHUL, S. F. ET AL., J MOLEC BIOL, vol. 215, 1990, pages 403
CARDING ET AL.: "Dysbiosis of the gut microbiota in disease", MICROB. ECOL. HEALTH DIS., vol. 26, 2015, pages 10
CARILLO ET AL., SIAM J APPLIED MATH, vol. 48, 1988, pages 1073
CITI, S: "Intestinal Barriers protect against disease", SCIENCE, vol. 359, 2018, pages 1098 - 99
CLAESSON MJWANG QO'SULLIVAN OGREENE-DINIZ RCOLE JRROSS RPO'TOOLE PW: "Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions", NUCLEIC ACIDS RES, vol. 38, 2010, pages e200, XP055250083, DOI: 10.1093/nar/gkq873
CVJETKOVIC ET AL., SCI. REP., vol. 6, 2016, pages 36338
DETTMER ET AL.: "Mass spectrometry-based metabolomics", MASS SPECTROM REV, vol. 26, no. 1, 2007, pages 51 - 78, XP002485404, DOI: 10.1002/mas.20108
DEVEREUX, J. ET AL., NUCLEIC ACIDS RESEARCH, vol. 12, no. I, 1984, pages 387
ERICKSON ET AL., MOLECULAR CELL, vol. 65, 19 January 2017 (2017-01-19), pages 361 - 370
G. NORHEIM ET AL., PLOS ONE, vol. 10, no. 9, 2015, pages e0134353
HOOKSO'MALLEY: "mBio", vol. 8, October 2017, AMERICAN SOCIETY FOR MICROBIOLOGY, article "Dysbiosis and its discontents", pages: e01492 - 17
I. MACDONALDM. KUEHN, J BACTERIAL, vol. 188, no. 13, pages 5385 - 5392
JEPPESEN ET AL., CELL, vol. 177, 2019, pages 428
KIESELBACHOSCARSSON, DATA BRIEF, vol. 10, February 2017 (2017-02-01), pages 426 - 431
KONSTANTINIDIS KTRAMETTE ATIEDJE JM: "The bacterial species definition in the genomic era", PHILOS TRANS R SOC LOND B BIOL SCI, vol. 361, 2006, pages 1929 - 1940
LEEKHA ET AL.: "General Principles of Antimicrobial Therapy", MAYO CLIN PROC, vol. 86, no. 2, 2011, pages 156 - 167, XP055372896, DOI: 10.4065/mcp.2010.0639
LEVY ET AL.: "Dysbiosis and the Immune System", NATURE REVIEWS IMMUNOLOGY, vol. 17, April 2017 (2017-04-01), pages 219, XP055953368, DOI: 10.1038/nri.2017.7
LIU ET AL., JOURNAL OF BACTERIOLOGY, vol. 192, no. 11, June 2010 (2010-06-01), pages 2852 - 2860
LYNCH ET AL.: "The Human Microbiome in Health and Disease", N. ENGL. J. MED ., vol. 375, 2016, pages 2369 - 79
PEARSON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 2444
ROBERTS ET AL.: "Targeted Metabolomics", CURR PROTOC MOL BIOL., vol. 30, 2012, pages 1 - 24
S. BIN PARK ET AL., PLOS ONE, vol. 6, no. 3, 2011, pages e 17629
S. STUBBS ET AL., LETTERS IN APPLIED MICROBIOLOGY, vol. 29, 1999, pages 31 - 36
SAARELA ET AL., J. APPLIED MICROBIOLOGY., vol. 99, 2005, pages 1330 - 1339
SANDRINI ET AL., BIO-PROTOCOL, vol. 4, no. 21, 2014, pages e1287
SCHOCH ET AL.: "Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi", PNAS, vol. 109, 2012, pages 6241 - 6246, XP055194048, DOI: 10.1073/pnas.1117018109
SEIBERT ET AL.: "Chemical Engineering in the Pharmaceutical Industry: R&D to Manufacturing", 2011, article "MILLING OPERATIONS IN THE PHARMACEUTICAL INDUSTRY"
SRINIVASAN ET AL.: "TEER measurement techniques for in vitro barrier model systems", J. LAB. AUTOM., vol. 20, 2015, pages 107 - 126, XP055560328, DOI: 10.1177/2211068214561025
THEIN ET AL., J. PROTEOME RES., vol. 9, no. 12, 2010, pages 6135 - 6147
TOGO AMADOU HAMIDOU ET AL: "Fournierella massiliensis gen. nov., sp. nov., a new human-associated member of the family Ruminococcaceae", vol. 67, no. 5, 1 May 2017 (2017-05-01), GB, pages 1393 - 1399, XP055842193, ISSN: 1466-5026, Retrieved from the Internet <URL:https://www.microbiologyresearch.org/docserver/fulltext/ijsem/67/5/1393_ijsem001826.pdf?expires=1631880630&id=id&accname=guest&checksum=7C62B8D3399C4FC118801346CAB1931F> [retrieved on 20170101], DOI: 10.1099/ijsem.0.001826 *
VILDHEDE ET AL., DRUG METABOLISM AND DISPOSITION, 8 February 2018 (2018-02-08)
WALKER, W.A.: "The Microbiota in Gastrointestinal Pathophysiology", 2017, article "Dysbiosis"
WEISSTHIERRY: "Mechanisms and consequences of intestinal dysbiosis", CELLULAR AND MOLECULAR LIFE SCIENCES, vol. 74, no. 16, 2017, pages 2959 - 2977, XP036272294, DOI: 10.1007/s00018-017-2509-x

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