WO2022221183A1 - Préparations de vésicules extracellulaires de fournierella - Google Patents

Préparations de vésicules extracellulaires de fournierella Download PDF

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Publication number
WO2022221183A1
WO2022221183A1 PCT/US2022/024243 US2022024243W WO2022221183A1 WO 2022221183 A1 WO2022221183 A1 WO 2022221183A1 US 2022024243 W US2022024243 W US 2022024243W WO 2022221183 A1 WO2022221183 A1 WO 2022221183A1
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WIPO (PCT)
Prior art keywords
evs
solution
fournierella massiliensis
excipient
fournierella
Prior art date
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PCT/US2022/024243
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English (en)
Inventor
Mark BODMER
Loise FRANCISCO-ANDERSON
Duncan MCHALE
Derek DORMAN
Collin MCKENNA
Bill Wang
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Evelo Biosciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Evelo Biosciences, Inc. filed Critical Evelo Biosciences, Inc.
Publication of WO2022221183A1 publication Critical patent/WO2022221183A1/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Extracellular vesicles (EVs) obtained from Fournierella massiliensis bacteria have therapeutic effects and are useful for the treatment and/or prevention of disease and/or health disorders.
  • EVs from Fournierella massiliensis bacteria can be prepared as a biomass (for example, isolated EVs can be resuspended in a buffer such as PBS).
  • EVs from Fournierella massiliensis bacteria can be prepared as solutions, dried forms and/or therapeutic compositions.
  • the solutions, dried forms and/or therapeutic compositions containing the Fournierella massiliensis EVs can also include an excipient that contains a bulking agent, and optionally one or more additional components, such as a lyoprotectant.
  • the Fournierella massiliensis EVs can be used in methods of treatment, e.g., as described herein.
  • a method of treating cancer in a human subject comprising administering (e.g., orally, intranasally or any other route) to the subject a dose (e.g., a therapeutically effective dose) of extracellular vesicles (EVs) from a Fournierella massiliensis strain and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles.
  • a dose e.g., a therapeutically effective dose
  • EVs extracellular vesicles
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a method of inducing pro-inflammatory cytokine release from dendritic cells in a human subject comprising administering (e.g., orally, intranasally or any other route) to the subject a dose (e.g. , a therapeutically effective dose) of extracellular vesicles (EVs) from a.
  • a dose e.g. , a therapeutically effective dose
  • EVs extracellular vesicles
  • Fournierella massiliensis strain and/or a composition e.g., a solution, dried form and/or therapeutic composition
  • the pro-inflammatory cytokine is TNFa (TNFa), IL (interleukin)- 12p70, and/or IFNy (IFNg).
  • a method of inducing pro- inflammatory cytokine levels in a human subject comprising administering (e.g., orally administering) to the subject a dose (e.g, a therapeutically effective dose) of extracellular vesicles (EVs) from a Fournierella massiliensis strain and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles.
  • a dose e.g., a therapeutically effective dose
  • EVs extracellular vesicles
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • the pro-inflammatory cytokine is TNFa(TNFa), IL (interleukin)- 12p70, IFNy(IFNg), IL-6, and/or ILi (IL1B). In some embodiments, the pro-inflammatory cytokine is TNFa(TNFa), IL- 12p70, and/or IFNy(IFNg).
  • the extracellular vesicles are from a Fournierella massiliensis strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Fournierella massiliensis Strain A (ATCC Deposit Number PTA- 126696).
  • the Fournierella massiliensis strain is the Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696).
  • the dose of EVs from Fournierella massiliensis bacteria is about 1 x 10 11 to about 1 x 10 14 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)).
  • the human subject has a cancer.
  • the cancer comprises a solid tumor.
  • the human subject has lung cancer. In some embodiments, the human subject has non-small cell lung cancer.
  • the human subject has breast cancer. In some embodiments, the human subject has triple negative breast cancer. In some embodiments, the human subject has metastatic triple negative breast cancer.
  • the Fournierella massiliensis EVs are administered orally.
  • administration of the extracellular vesicles (EVs) from a. Fournierella massiliensis strain and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles cause an improvement in Overall survival (OS) in the human subject.
  • OS Overall survival
  • administration of the extracellular vesicles (EVs) from a Fournierella massiliensis strain and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles cause an improvement in overall response rate (ORR) in the human subject.
  • administration of the extracellular vesicles (EVs) from a Fournierella massiliensis strain and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles cause an improvement in Clinical benefit rate (CBR) in the human subject.
  • CBR Clinical benefit rate
  • administration of the extracellular vesicles (EVs) from a Fournierella massiliensis strain and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles cause an improvement in Duration of response (DOR) in the human subject.
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • the extracellular vesicles (EVs) from a Fournierella massiliensis strain and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles reduce tumor growth in a CT26 preclinical model of cancer.
  • the dose is in the form of one or more enteric- coated capsules, optionally comprising an enteric-coating (e.g., enteric-coated capsules).
  • the dose is in the form of one or more tablets, optionally comprising an enteric-coating (e.g., enteric-coated tablets).
  • the dose is in the form of one or more mini-tablets.
  • the mini-tablets are enteric-coated mini-tablets.
  • the dose is in the form of a non enteric coated capsule comprising an enteric-coated mini-tablet.
  • Fournierella massiliensis extracellular vesicles have therapeutic effects and are useful for the treatment and/or prevention of disease and/or health disorders.
  • Therapeutic compositions of biomass, solutions and dried forms containing Fournierella massiliensis EVs can be prepared, e.g., as described herein.
  • Fournierella massiliensis EVs are prepared as solutions and as dried forms.
  • the solutions and dried forms are for use in preparing therapeutic compositions comprising Fournierella massiliensis EVs.
  • the dried forms comprising Fournierella 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 lyoprotectants can be used when preparing Fournierella massiliensis extracellular vesicles (EVs), e.g., for freeze drying.
  • Bulking agents including but not limited to sucrose, mannitol, and dextran (such as dextran 40k), can make dried forms (such as powders and/or lyophilates) easier to handle after drying.
  • Bulking agents can improve the powder and/or lyophilate properties, e.g., improve cake quality.
  • 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 decrease drying cycle time. In some embodiments, the excipient functions to maintain therapeutic efficacy of the EVs.
  • the disclosure provides extracellular vesicles (EVs) from Fournierella massiliensis bacteria.
  • the disclosure provides a lyophilate comprising Fournierella 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 disclosure provides a lyophilate comprising Fournierella massiliensis EVs, wherein the lyophilate has a particle numeration of about 6.24e9 to about 2.89el0 particles/mg lyophilate.
  • the disclosure provides a lyophilate comprising Fournierella 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.
  • the disclosure provides a lyophilate comprising Fournierella massiliensis EVs, wherein the particles have a hydrodynamic diameter (Z average, Zave) of about 132 nm to about 315.2 nm.
  • the disclosure provides a lyophilate comprising Fournierella 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.
  • the disclosure provides a lyophilate comprising Fournierella 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 Fournierella 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 Fournierella 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%.
  • 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 disclosure provides a powder comprising Fournierella massiliensis EVs, wherein the powder has a particle numeration of about 6.24e9 to about 2.89el0 particles/mg powder.
  • the disclosure provides a powder comprising Fournierella 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.
  • the disclosure provides a powder comprising Fournierella massiliensis EVs, wherein the particles have a hydrodynamic diameter (Z average, Zave) of about 132 nm to about 315.2 nm.
  • the disclosure provides a powder comprising Fournierella 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.
  • the disclosure provides a powder comprising Fournierella 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 Fournierella 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 Fournierella 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 disclosure provides a dried form comprising Fournierella massiliensis EVs, wherein the dried form has a particle numeration of about 6.24e9 to about 2.89el0 particles/mg dried form.
  • the disclosure provides a dried form comprising Fournierella 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.
  • the disclosure provides a dried form comprising Fournierella massiliensis EVs, wherein the particles have a hydrodynamic diameter (Z average, Zave) of about 132 nm to about 315.2 nm.
  • the disclosure provides a dried form comprising Fournierella 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.
  • the disclosure provides a dried form comprising Fournierella 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 Fournierella 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 therapeutic composition comprising the Fournierella massiliensis EVs, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the disclosure provides a solution comprising extracellular vesicles (EVs) from Fournierella massiliensis bacteria and an excipient that comprises a bulking agent.
  • EVs extracellular vesicles
  • the disclosure provides a solution consisting essentially of Fournierella massiliensis EVs and an excipient that comprises a bulking agent. [64] In some aspects, the disclosure provides a solution comprising Fournierella massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a solution consisting essentially of Fournierella massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a solution comprising Fournierella massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a solution consisting essentially of Fournierella 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 extracellular vesicles (EVs) from Fournierella massiliensis bacteria and an excipient that comprises a bulking agent.
  • EVs extracellular vesicles
  • the disclosure provides a dried form consisting essentially of Fournierella massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a dried form comprising Fournierella massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a dried form consisting essentially of Fournierella massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a dried form comprising Fournierella massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a dried form consisting essentially of Fournierella 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 Fournierella massiliensis EVs and an excipient that comprises a bulking agent. [77] In some aspects, the disclosure provides a powder consisting essentially of Fournierella massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a powder comprising Fournierella massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a powder consisting essentially of Fournierella massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a powder comprising Fournierella massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a powder consisting essentially of Fournierella 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 Fournierella massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a spray -dried powder consisting essentially of Fournierella massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a spray -dried powder comprising Fournierella massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a spray -dried powder consisting essentially of Fournierella massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a spray -dried powder comprising Fournierella massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a spray -dried powder consisting essentially of Fournierella 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 Fournierella massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a lyophilate consisting essentially of Fournierella massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a lyophilate comprising Fournierella massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a lyophilate consisting essentially of Fournierella massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a lyophilate comprising Fournierella massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a lyophilate consisting essentially of Fournierella 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 Fournierella massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a lyophilized powder consisting essentially of Fournierella massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a lyophilized powder comprising Fournierella massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a lyophilized powder consisting essentially of Fournierella massiliensis EVs and from an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a lyophilized powder comprising Fournierella massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a lyophilized powder consisting essentially of Fournierella 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 Fournierella massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a lyophilized cake consisting essentially of Fournierella massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a lyophilized cake comprising Fournierella massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a lyophilized cake consisting essentially of Fournierella massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a lyophilized cake comprising Fournierella massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a lyophilized cake consisting essentially of Fournierella massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a therapeutic composition comprising extracellular vesicles (EVs) from Fournierella massiliensis bacteria and an excipient that comprises a bulking agent.
  • EVs extracellular vesicles
  • the disclosure provides a therapeutic composition consisting essentially of Fournierella massiliensis EVs and an excipient that comprises a bulking agent.
  • the disclosure provides a therapeutic composition comprising Fournierella massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a therapeutic composition consisting essentially of Fournierella massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a therapeutic composition comprising Fournierella massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a therapeutic composition consisting essentially of Fournierella massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides a solution comprising Fournierella massiliensis extracellular vesicles (EVs) and an excipient stock of a formula provided in Table A, B, C, or D.
  • EVs Fournierella massiliensis extracellular vesicles
  • the disclosure provides a solution consisting essentially of Fournierella massiliensis extracellular vesicles (EVs) and an excipient stock of a formula provided in Table A, B, C, or D.
  • EVs Fournierella massiliensis extracellular vesicles
  • 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 Fournierella massiliensis extracellular vesicles (EVs) and an excipient stock of a formula provided in Table A, B, C, or D.
  • EVs Fournierella massiliensis extracellular vesicles
  • the disclosure provides a dried form consisting essentially of Fournierella massiliensis extracellular vesicles (EVs) and an excipient stock of a formula provided in Table A, B, C, or D.
  • EVs Fournierella massiliensis extracellular vesicles
  • 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 Fournierella massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D.
  • the disclosure provides a powder consisting essentially of Fournierella massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D.
  • 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 Fournierella massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D.
  • the disclosure provides a spray-dried powder consisting essentially of Fournierella massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D.
  • 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 Fournierella massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D.
  • the disclosure provides a lyophilate consisting essentially of Fournierella massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D.
  • 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 Fournierella massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D.
  • the disclosure provides a lyophilized powder consisting essentially of Fournierella massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D.
  • 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 Fournierella massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D.
  • the disclosure provides a lyophilized cake consisting essentially of Fournierella massiliensis EVs and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D.
  • 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 (e.g., human) (e.g., a subject in need of treatment), the method comprising: administering to the subject Fournierella massiliensis EVs or a solution, dried form, or therapeutic composition (e.g., a therapeutically effective amount thereof) described herein.
  • a subject e.g., human
  • therapeutic composition e.g., a therapeutically effective amount thereof
  • Fournierella massiliensis EVs or a solution, dried form, or therapeutic composition e.g., a therapeutically effective amount thereof
  • a subject e.g., human
  • therapeutic composition e.g., a therapeutically effective amount thereof
  • the disclosure provides use of Fournierella massiliensis EVs or a solution, dried form, or therapeutic composition (e.g., a therapeutically effective amount thereof) provided herein for the preparation of a medicament for treating a subject (e.g., human) (e.g., a subject in need of treatment).
  • a subject e.g., human
  • a subject in need of treatment e.g., a subject in need of treatment.
  • the Fournierella massiliensis EVs or solution/dried form/therapeutic composition is orally administered (e.g., 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)).
  • provided herein is 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.
  • the subject is in need of treatment (and/or prevention) of a cancer.
  • the subject is in need of induction of pro-inflammatory cytokine release from dendritic cells.
  • the subject is in need of induction of pro-inflammatory cytokine levels (e.g., in serum or PBMCS).
  • pro-inflammatory cytokine levels e.g., in serum or PBMCS.
  • the subject is in need of treatment (and/or prevention) of dysbiosis.
  • the solution/dried form/therapeutic composition is administered in combination with an additional therapeutic agent. In some embodiments of the method/solution/dried form/therapeutic composition/use provided herein, the solution/dried form/therapeutic composition is administered in combination with a direct cytotoxic agent, an immune activator, or a checkpoint inhibitor.
  • 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparationthat comprises Fournierella massiliensis EVs with an excipient that comprises (or consists essentially of) lyoprotectant to prepare a solution; drying the solution to prepare a cake, and
  • 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 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 powder that comprises Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises composition comprising Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D, 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises composition comprising Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D, 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises composition comprising Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D, 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D, thereby preparing a solution; and drying the solution, thereby preparing the powder.
  • the disclosure provides a method of preparing a powder that comprises Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D, 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D, 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 Fournierella 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 Table A, B, C, or D, 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D, 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D, 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D, 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D, 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 drying comprises lyophilization.
  • the method further comprises combining the dried form with an excipient, e.g., a glidant, lubricant, and/or diluent.
  • an excipient e.g., a glidant, lubricant, and/or diluent.
  • the disclosure provides a dried form 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, polyethylene glycol (PEG, such as PEG 6000), cyclodextrin, maltodextrin, sucrose, dextran, Ficoll, or PVP- K30.
  • PEG polyethylene glycol
  • the bulking agent comprises mannitol.
  • the excipient comprises an additional component.
  • the additional component 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 an excipient stock of a formula provided in provided in Table A, B, C, or D.
  • 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 solution, dried form, or therapeutic composition provided herein about 30% to about 60% (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.
  • 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.
  • 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.
  • 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 at least 6.24e9 particles per mg of the dried form (for example, as determined by particles per mg, such as by NTA).
  • 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).
  • 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).
  • the dried form comprises about 6.7e8 to about 2.55el0 particles/mg dried form.
  • particle numeration is determined on 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
  • the particles have a mean size of the most dominant DLS integrated peak of between about 43.72 nm to about 79.18 nm.
  • 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.
  • 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
  • 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 strain. 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 can contain EVs from one or more bacterial strain in addition to EVs from Fournierella massiliensis.
  • a solution, dried form, or therapeutic composition provided herein can contain EVs from one bacterial strain in addition to EVs from Fournierella massiliensis .
  • the bacterial strain used as a source of EVs may be selected based on the properties of the bacteria (e.g., growth characteristics, yield, ability to modulate an immune response in an assay or a subject).
  • a solution and/or dried form comprising Fournierella massiliensis EVs can comprise an excipient that comprises a bulking agent.
  • Fournierella massiliensis EVs or a solution, dried form, or therapeutic composition provided herein comprising EVs from Fournierella massiliensis bacteria can be used for the treatment or prevention of a disease and/or a health disorder, e.g., in a subject (e.g., human).
  • a dried form (or a therapeutic composition thereof) provided herein comprising EVs from Fournierella massiliensis bacteria can be prepared as a solid dose form, such as a tablet, a minitablet, a capsule, or a dried form; or a combination of these forms (e.g., minitablets comprised in a capsule).
  • the solid dose form can comprise a coating (e.g., enteric coating).
  • a dried form (or a therapeutic composition thereof) provided herein comprising EVs from Fournierella massiliensis bacteria can be reconstituted.
  • a solution (or a therapeutic composition thereof) provided herein comprising EVs from Fournierella massiliensis bacteria can be used as suspension, e.g., diluted to a suspension or used in undiluted form.
  • a therapeutic composition comprising Fournierella massiliensis EVs or a solution and/or dried form comprising EVs from Fournierella massiliensis bacteria can be prepared as provided herein.
  • the therapeutic composition comprising a dried form can be formulated into a solid dose form, such as a tablet, a minitablet, a capsule, or a dried form; or can be reconstituted in a suspension.
  • Fournierella massiliensis EVs or a solution, dried form, or therapeutic composition provided herein can comprise gamma irradiated EVs from Fournierella massiliensis bacteria.
  • the gamma irradiated EVs from Fournierella massiliensis bacteria can be formulated into therapeutic composition.
  • the gamma irradiated EVs from Fournierella massiliensis bacteria can be formulated into a solid dose form, such as a tablet, a minitablet, a capsule, or a dried form; or can be 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.
  • the gamma irradiated F. massiliensis EVs are formulated into therapeutic composition.
  • 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.
  • Fournierella massiliensis EVs or a solution, dried form, or therapeutic composition provided herein comprising EVs from Fournierella massiliensis bacteria can be orally administered.
  • Fournierella massiliensis EVs or a solution, dried form, or therapeutic composition provided herein comprising EVs from Fournierella massiliensis bacteria can be administered intranasally.
  • a solution, dried form, or therapeutic composition provided herein comprising F. massiliensis EVs is administered by inhalation.
  • Fournierella massiliensis EVs or a solution, dried form, or therapeutic composition provided herein comprising EVs from Fournierella massiliensis bacteria can be administered intravenously.
  • Fournierella massiliensis EVs or a solution, dried form, or therapeutic composition provided herein comprising EVs from Fournierella massiliensis bacteria can be administered by injection.
  • a solution, dried form, or therapeutic composition provided herein comprising F. massiliensis EVs is administered by injection, for example, intratumorally or subtumorally, 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 Fournierella massiliensis EVs and/or solutions and/or dried forms comprising EVs from Fournierella massiliensis bacteria useful for the treatment and/or prevention of a disease or a health disorder (e.g., adverse health disorders) (e.g., a cancer or dysbiosis), as well as methods of making and/or identifying such Fournierella massiliensis EVs and/or solutions and/or dried forms and therapeutic compositions, and methods of using such Fournierella massiliensis EVs and/or solutions and/or dried forms, and therapeutic compositions thereof (e.g., for the treatment of a cancer or dysbiosis, either alone or in combination with other therapeutics).
  • a health disorder e.g., adverse health disorders
  • therapeutic compositions e.g., for the treatment of a cancer or dysbiosis, either alone or in combination with other therapeutics.
  • the therapeutic compositions can comprise both EVs from Fournierella massiliensis bacteria and whole bacteria, e.g., Fournierella massiliensis bacteria from which the EVs were obtained, such as live bacteria, killed bacteria, attenuated bacteria.
  • the therapeutic compositions comprise EVs from Fournierella massiliensis bacteria in the absence of the 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 comprises Fournierella massiliensis EVs.
  • the Fournierella massiliensis EVs can be isolated EVs, e.g., 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 Fournierella massiliensis EVs or solution, dried form, or therapeutic composition comprises isolated Fournierella massiliensis EVs (e.g., from one or more strains of bacteria (e.g., a therapeutically effective amount thereof).
  • the Fournier ella massiliensis EVs or solution, dried form, or therapeutic composition comprises isolated Fournierella massiliensis EVs (e.g., from one strain of bacteria (e.g., bacteria of interest) (e.g., a therapeutically effective amount thereof).
  • the Fournierella massiliensis EVs or solution, dried form, or therapeutic composition comprises EVs from Fournierella massiliensis bacteria.
  • the solution, dried form, or therapeutic composition comprises EVs from more than one strain of bacteria (e.g., EVs from a strain in addition to the Fournierella massiliensis EVs).
  • the Fournierella massiliensis EVs are lyophilized.
  • the Fournierella massiliensis EVs are gamma irradiated.
  • the Fournierella massiliensis EVs are UV irradiated.
  • the Fournierella massiliensis EVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).
  • the Fournierella massiliensis EVs are acid treated.
  • the Fournierella massiliensis EVs are oxygen sparged (e.g., at 0.1 vvm for two hours).
  • the Fournierella massiliensis EVs are from a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Fournierella massiliensis Strain A (ATCC Deposit Number PTA- 126696). In some embodiments, the Fournierella massiliensis EVs are from a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Fournierella massiliensis Strain A (ATCC Deposit Number PTA- 126696). In some embodiments, the Fournierella massiliensis bacteria are from Fournierella massiliensis Strain A (ATCC Deposit Number PTA- 126696).
  • the Fournierella massiliensis EVs obtained from bacteria that have been selected based on certain desirable properties, such as reduced toxicity and adverse effects (e.g., by removing or deleting lipopolysaccharide (LPS)), enhanced oral delivery (e.g., by improving acid resistance, muco-adherence and/or penetration and/or resistance to bile acids, resistance to anti-bacterial peptides and/or antibody neutralization), target desired cell types (e.g., M-cells, goblet cells, enterocytes, dendritic cells, macrophages), improved bioavailability systemically or in an appropriate niche (e.g., mesenteric lymph nodes, Peyer’s patches, lamina intestinal, lymph nodes, and/or blood), enhanced immunomodulatory and/or therapeutic effect (e.g., either alone or in combination with another therapeutic agent), enhanced immune activation , and/or manufacturing attributes (e.g., growth characteristics, yield, greater stability, improved freeze-thaw tolerance, shorter generation
  • LPS lipopol
  • the Fournierella 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 (e.g., by removing or deleting lipopolysaccharide (LPS)), enhanced oral delivery (e.g., 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 (e.g., M-cells, goblet cells, enterocytes, dendritic cells, macrophages), improved bioavailability systemically or in an appropriate niche (e.g., mesenteric lymph nodes, Peyer’s patches, lamina intestinal, lymph nodes, and/or blood), enhanced immunomodulatory and/or therapeutic effect (e.g., either alone or in combination with another therapeutic agent), enhanced immune activation, and/or improved manufacturing attributes (e.g.,
  • Fournierella massiliensis EVs and/or solutions and/or dried forms (or therapeutic compositions thereof) comprising EVs from Fournierella massiliensis bacteria useful for the treatment and/or prevention of a disease or a health disorder (e.g., a cancer or dysbiosis), 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 (e.g., for the treatment of a cancer or dysbiosis), either alone or in combination with one or more other therapeutics.
  • a disease or a health disorder e.g., a cancer or dysbiosis
  • Therapeutic compositions containing Fournierella massiliensis EVs and/or a solution and/or dried form can provide potency comparable to or greater than therapeutic compositions that contain the whole Fournierella massiliensis bacteria from which the EVs were obtained.
  • a therapeutic composition containing solutions and/or dried forms can provide potency comparable to or greater than a comparable therapeutic composition that contains whole bacteria of the same Fournierella massiliensis bacterial strain from which the EVs were obtained.
  • Such EV- and/or solution- and/or dried form- containing therapeutic compositions can allow the administration of higher doses and elicit a comparable or greater (e.g., more effective) response than observed with a comparable therapeutic composition that contains whole bacteria of the same Fournierella massiliensis bacterial strain from which the EVs were obtained.
  • a therapeutic composition containing Fournierella massiliensis EVs and/or a solution and/or dried form can contain less microbially-derived material (based on particle count or protein content), as compared to a therapeutic composition that contains the whole Fournierella massiliensis bacteria of the same bacterial strain from which the EVs were obtained, while providing an equivalent or greater therapeutic benefit to the subject receiving such therapeutic composition.
  • EVs from Fournierella massiliensis bacteria can be administered at doses e.g., of about lxlO 7 to about lxlO 15 particles, e.g., as measured by NTA.
  • the dose of EVs is about 1 x 10 5 to about 7 x 10 13 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)).
  • the dose of EVs from Fournierella massiliensis bacteria is about 1 x 10 10 to about 7 x 10 13 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)).
  • EVs from Fournierella massiliensis bacteria can be administered at doses e.g., of about 5 mg to about 900 mg total protein, e.g., as measured by Bradford assay.
  • EVs from Fournierella massiliensis bacteria can be administered at doses e.g., of about 5 mg to about 900 mg total protein, e.g., as measured by BCA assay.
  • provided herein are methods of treating a subject who has a cancer comprising administering to the subject a therapeutic composition Fournierella massiliensis EVs and/or or a solution and/or dried form described herein.
  • methods of inducing pro-inflammatory cytokine release from dendritic cells in a subject comprising administering to the subject a therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form described herein.
  • provided herein are methods of inducing pro-inflammatory cytokine levels in a subject comprising administering to the subject a therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form described herein.
  • methods of treating a subject who has a dysbiosis comprising administering to the subject a therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form described herein.
  • the method further comprises administering to the subject an antibiotic. In some embodiments, the method further comprises the administration of an additional treatment for cancer. In some embodiments, the therapeutic composition or Fournierella massiliensis EVs and/or a solution, dried form, and/or lyophilate can be for use in combination with one or more cancer therapies.
  • 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 a solution, and/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 Fournierella massiliensis EVs and/or a solution and/or dried form for use in the treatment and/or prevention of a disease (e.g., a cancer or a dysbiosis) or a health disorder, either alone or in combination with one or more other (e.g.., additional) therapeutic agent.
  • a disease e.g., a cancer or a dysbiosis
  • a health disorder either alone or in combination with one or more other (e.g.., additional) therapeutic agent.
  • a therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form for use in treating and/or preventing a cancer in a subject (e.g., human).
  • the therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form can be used either alone or in combination with one or more other therapeutic agent for the treatment of the cancer.
  • a therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form for use in treating and/or preventing a dysbiosis in a subject (e.g., human).
  • the therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form can be used either alone or in combination with therapeutic agent for the treatment of the dysbiosis.
  • the therapeutic composition or a solution and/or dried form is for use in combination with an antibiotic.
  • the therapeutic composition or a solution and/or dried 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
  • the therapeutic composition or a solution and/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 a solution and/or dried form is for use in combination with one or more immune suppressant(s) and/or an anti-inflammatory agent(s).
  • the therapeutic composition or a solution and/or dried form is for use in combination with one or more other metabolic disease therapeutic agents.
  • the therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form can be for use in combination with an antibiotic.
  • the therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form can be for use in combination with another therapeutic bacterium and/or EVs obtained from one or more other bacterial strains (e.g., therapeutic bacterium).
  • the therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form can be for use in combination with one or more cancer therapy agents.
  • a therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form for the preparation of a medicament for the treatment and/or prevention of a disease (e.g., a cancer or a dysbiosis), either alone or in combination with another therapeutic agent.
  • a disease e.g., a cancer or a dysbiosis
  • the use is in combination with another therapeutic bacterium and/or EVs obtained from one or more other bacterial strains (e.g., therapeutic bacterium).
  • a therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form for the preparation of a medicament for treating and/or preventing an immune disorder (e.g., a cancer) in a subject (e.g., human).
  • the therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form can be for use either alone or in combination with another therapeutic agent for the cancer.
  • a therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form for the preparation of a medicament for treating and/or preventing a dysbiosis in a subject (e.g., human).
  • the therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form can be for use either alone or in combination with another therapeutic agent for the dysbiosis.
  • the therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form can be for use in combination with an antibiotic.
  • the therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form can be for use in combination with another therapeutic bacterium and/or EVs obtained from one or more other bacterial strains (e.g., therapeutic bacterium).
  • the therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form can be for use in combination with one or more other cancer therapy agent(s).
  • the therapeutic composition or a solution and/or dried form is for use in combination with an antibiotic.
  • the therapeutic composition or a solution and/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)
  • the therapeutic composition or a solution and/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 a solution and/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 a solution and/or dried form is for use in combination with one or more other metabolic disease therapeutic agent(s).
  • a therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form, e.g., as described herein, comprising EVs from Fournierella massiliensis bacteria can provide a therapeutically effective amount of Fournierella massiliensis EVs to a subject, e.g., a human.
  • a therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form, e.g., as described herein, comprising EVs from Fournierella massiliensis bacteria can provide a non-natural amount of the therapeutically effective components (e.g., present in the Fournierella massiliensis EVs to a subject, e.g., a human.
  • a therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form, e.g., as described herein, comprising EVs from Fournierella massiliensis bacteria can provide unnatural quantity of the therapeutically effective components (e.g., present in the EVs to a subject, e.g., a human.
  • a therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form, e.g., as described herein, comprising EVs from Fournierella massiliensis bacteria can bring about one or more changes to a subject, e.g., human, e.g., to treat or prevent a disease or a health disorder.
  • a therapeutic composition or Fournierella massiliensis EVs and/or a solution and/or dried form, e.g., as described herein, comprising EVs from Fournierella massiliensis bacteria has potential for significant utility, e.g., to affect a subject, e.g., a human, e.g., to treat or prevent a disease or a health disorder.
  • 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-D.
  • a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A-D, 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.
  • F. massiliensis EVs for example, a liquid preparation thereof
  • 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-D.
  • 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 Table A 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 three graphs showing levels (pg/ml) of TNFa, IL-12p70, and IFNg from dendritic cells following 24 hr Fournierella massiliensis Strain A EV treatment (“F. massiliensis EVs”) vs untreated (media control) wells.
  • Figure 2 A and 2B are graphs showing tumor growth over time (days)as mean tumor volume +SEM post implant ( Figure 2A) and as median tumor volume at day 20 post implantation ( Figure 2B).
  • Tumor-bearing mice were treated orally with vehicle, Fournierella massiliensis Strain A EVs (“F. massiliensis EVs”) (2 xlO 11 particles, b.i.d.) or anti-PD-1 (200ug, q.4d.). Data analyzed using ANOVA with Tukey’s post-test.
  • Figure 3 is a graph showing a comparison of orally-administered Fournierella massiliensis Strain A EVs (“F. massiliensis EVs”) to its parent microbe (“F. massiliensis microbe”) in CT26 tumor-bearing mice.
  • Fournierella massiliensis Strain A EVs were administered orally at either 2x10 11 or 2x10 9 particles beginning at day 10 for 12 days vs anti-PD-1.
  • the parental strain Fournierella massiliensis was administered at its highest deliverable concentration of 2xl0 9 cells. Vehicle controls for both the EVs and microbe were included. Data are median and range.
  • Figure 4A-4C is a series of graphs showing Fournierella massiliensis Strain A EVs (“F. massiliensis EVs”) activate and sustain IFNy+ cytolytic and helper lymphocytes, DCs and IP- 10 in the TME.
  • F. massiliensis EVs Fournierella massiliensis Strain A EVs
  • Anti-PD-1 anti-PD-1 antibody
  • FIG. 5 is a series of bar graphs showing the cytokine profde of Fournierella massiliensis Strain A EVs (“F. massiliensis EVs”) in U937 macrophages.
  • the cytokines measured were: TNF (TNFa), IP-10, IL-Ib, IL-6 and IL-10. Cytokine values are presented as the percentage of the LPS control.
  • Figure 6 is a graph showing median tumor volume ⁇ range at day 21 post implantation after treatment with an anti-PD- 1 antibody or serial dilutions of Fournierella massiliensis Strain A EVs (“F. massiliensis EVs”). Data analyzed using one-way ANOVA and Tukey’s post-hoc analysis.
  • Figure 7 is a graph showing moisture content of lyophilized Fournierella massiliensis EV powders.
  • Figure 8 is a graph showing particle count of lyophilized Fournierella massiliensis EV powders.
  • Figure 9 is a graph showing average particle size by DLS of lyophilized Fournierella massiliensis EV powders.
  • Figure 10 is a graph showing electrokinetic potential of the dominant subpopulation of lyophilized Fournierella massiliensis EV powder by DLS.
  • Figure 11 is a graph showing particle size of the dominant subpopulation of lyophilized Fournierella massiliensis EV powders.
  • Figure 12A-C shows frozen and freeze-dried (lyophyilized) Fournierella massiliensis EVs (“Am.”) at 2xlO n , 2xl0 9 , and 2xl0 7 particles per dose compared to the vehicles (PBS and Formulation 7 (“Form 7”)) and an anti-PD 1.
  • the disclosure provides Fournierella massiliensis EVs, and solutions, dried forms and therapeutic compositions that contain extracellular vesicles (EVs) from Fournierella massiliensis bacteria, and methods for preparing and using the same.
  • 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, 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.
  • adjuvant or “Adjuvant therapy” broadly refers to an agent that affects an immunological or physiological response in a patient or subject (e.g., 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 (e.g., a therapeutic 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), and subcutaneous (SC) administration.
  • a therapeutic composition described herein can be administered in any form by any effective route, including but not limited to oral, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal (e.g., using any standard patch), intradermal, ophthalmic, (intra)nasally, local, non-oral, such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, (trans)rectal, vaginal, intra-arterial, and intrathecal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), implanted, intravesical, intrapulmonary, intraduodenal, intragastrical, and intrabronchial.
  • transdermal e.g., using any standard patch
  • intradermal e.g., using any standard patch
  • intradermal e.g., using any standard patch
  • intradermal e.
  • a therapeutic composition described herein is administered orally, rectally, topically, intravesically, by injection into or adjacent to a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously.
  • a therapeutic composition described herein is administered orally or intravenously.
  • a therapeutic composition described herein is administered orally.
  • antibody may refer to both an intact antibody and an antigen binding fragment thereof.
  • Intact antibodies are glycoproteins that include at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the term “antibody” includes, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies and antigen-binding antibody fragments.
  • antigen binding fragment and “antigen-binding portion” of an antibody, as used herein, refer to one or more fragments of an antibody that retain the ability to bind to an antigen.
  • binding fragments encompassed within the term "antigen-binding fragment” of an antibody include Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chain antibodies, NANOBODIES®, isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of an intact antibody.
  • These antibody fragments can be obtained using conventional recombinant and/or enzymatic techniques and can be screened for antigen binding in the same manner as intact antibodies.
  • carcinomas which are cancers of the epithelial tissue (e.g., skin, squamous cells); sarcomas which are cancers of the connective tissue (e.g., bone, cartilage, fat, muscle, blood vessels, etc.); leukemias which are cancers of blood forming tissue (e.g., 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 (e.g., skin, squamous cells)
  • sarcomas which are cancers of the connective tissue (e.g., bone, cartilage, fat, muscle, blood vessels, etc.)
  • leukemias which are cancers of blood forming tissue (e.g., bone marrow tissue)
  • lymphomas and myelomas which are cancers of immune cells
  • central nervous system cancers which include cancers from brain and spinal tissue.
  • cancer(s) and” “neoplasm(s)” are used herein interchangeably.
  • 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.
  • the cancer comprises a solid tumor.
  • the cancer comprises a metastasis.
  • a “carbohydrate” refers to a sugar or polymer of sugars.
  • saccharide polysaccharide
  • carbohydrate oligosaccharide
  • Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one on each carbon atom of the molecule.
  • Carbohydrates generally have the molecular formula CnHmOn.
  • a carbohydrate may be a monosaccharide, a disaccharide, trisaccharide, 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. Typically, an oligosaccharide includes between three and six monosaccharide units (e.g., raffmose, 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, orN- acetylglucosamine, a nitrogen-containing form of glucose (e.g., 2’-fluororibose, deoxyribose, and hexose).
  • Carbohydrates may exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.
  • 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.
  • 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, e.g., another EV (e.g., from another strain), with bacteria (e.g., 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-administration 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, e.g., mucosal or skin surfaces (or any other microbiome niche) in which the normal diversity and/or function of the host gut microbiome ecological networks ( ’’microbiome”) are disrupted.
  • a state of dysbiosis may result in a diseased state, or it may be unhealthy under only certain conditions or only if present for a prolonged period.
  • Dysbiosis may be due to a variety of factors, including, environmental factors, infectious agents , host genotype, host diet and/or stress.
  • a dysbiosis may result in: a change (e.g., increase or decrease) in the prevalence of one or more bacteria types (e.g., anaerobic), species and/or strains, change (e.g., increase or decrease) in diversity of the host microbiome population composition; a change (e.g., increase or reduction) of one or more populations of symbiont organisms resulting in a reduction or loss of one or more beneficial effects; overgrowth of one or more populations of pathogens (e.g., pathogenic bacteria); and/or the presence of, and/or overgrowth of, symbiotic organisms that cause disease only when certain conditions are present.
  • 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 (e.g., in a DTH animal model) or tumor size (e.g., in an animal tumor model)).
  • the term “effective dose” is the amount of 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 (e.g., increased) or decreased through manipulation of the environment in which the bacterial cells are being cultured (e.g., by media or temperature alterations).
  • 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 (e.g., lymph node), absorption (e.g., gastrointestinal), and/or yield (e.g., 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 (e.g., 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 (e.g., by lysozyme and/or lysostaphin) and/or physically disrupting (e.g., 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
  • chemically disrupting e.g., by lysozyme and/or lysostaphin
  • physically disrupting e.g., by mechanical force
  • 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.
  • 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 etal. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, T, et al, Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., etal, J Molec Biol 215:403 (1990); Guide to Huge Computers, Mrtin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al.
  • Immunotherapy is treatment that uses a subject’s immune system to treat disease (e.g., 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 e.g., immune disease, inflammatory disease, metabolic disease, cancer
  • checkpoint inhibitors e.g., 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 L 3 fold, 10 L 4 fold, 10 L 5 fold, 10 L 6 fold, and/or 10 L 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 (e.g., in a DTH animal model) or tumor size (e.g., 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- ghitamyl-meso-diaminopimelic acid (iE-DAP), and desmuramylpeptides (DMP).
  • MDP N-acetylmuramyl-L-alanyl-D-isoglutamine
  • iE-DAP gamma-D- ghitamyl-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 28S regions with ITS4 and 5 between the 8S and 5.8S and 5.8S and 28S 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, e.g., substantially free of other components.
  • leukemia includes broadly progressive, malignant diseases of the hematopoietic organs/systems 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.
  • Metal refers to any and all molecular compounds, compositions, molecules, ions, co-factors, catalysts or nutrients used as substrates in any cellular or bacterial metabolic reaction or resulting as product compounds, compositions, molecules, ions, co-factors, catalysts or nutrients from any cellular or bacterial metabolic reaction.
  • Microbiome broadly refers to the microbes residing on or in body site of a subject or patient.
  • Microbes in a microbiome may include bacteria, viruses, eukaryotic microorganisms, and/or viruses.
  • Individual microbes in a microbiome may be metabolically active, dormant, latent, or exist as spores, may exist planktonically or in biofdms, 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 or treatment conditions (e.g., antibiotic treatment, exposure to different microbes).
  • the microbiome occurs at a mucosal surface.
  • the microbiome is a gut 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.
  • “Modified” in reference to a bacteria broadly refers to a bacteria that has undergone a change from its wild-type form.
  • Bacterial modification can result from engineering bacteria. Examples of bacterial modifications include genetic modification, gene expression modification, phenotype modification, formulation modification, chemical modification, and dose or concentration. Examples of improved properties are described throughout this specification and include, e.g., attenuation, auxotrophy, homing, or antigenicity.
  • Phenotype modification might include, by way of example, bacteria growth in media that modify the phenotype of a bacterium such that it increases or decreases virulence.
  • 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, e.g., 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 e.g., Claesson MJ, Wang Q, O’Sullivan O, Greene-Diniz R, Cole JR, Ross RP, and O’Toole PW. 2010. Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Res 38: e200. Konstantinidis KT, Ramette A, and Tiedje JM. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940.
  • OTUs For complete genomes, MLSTs, specific genes, other than 16S, or sets of genes OTUs that share > 95% average nucleotide identity are considered the same OTU. See e.g., Achtman M, and Wagner M. 2008. Microbial diversity and the genetic nature of microbial species. Nat. Rev. Microbiol. 6: 431-440. Konstantinidis KT, Ramette A, and Tiedje JM. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Uond B Biol Sci 361: 1929-1940. 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 (e.g., “house-keeping” genes), or a combination thereof.
  • Operational Taxonomic Units (OTUs) with taxonomic assignments made to, e.g., 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 (e.g., 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, e.g., 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 (e.g., 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, e.g., other microbe strains or forms (e.g., bacteria, virus, my coplasm, 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. In one embodiment, substantially free of residual habitat products may also mean that the microbial composition contains no detectable viral (including bacteria, viruses (e.g., phage)), fungal, mycoplasmal contaminants.
  • it means that fewer than lxl0 2 %, lxl0 3 %, lxl0 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.
  • 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 (e.g., a dilution of 10 8 or 10 9 ), such as through multiple 10-fold serial dilutions. This can further be confirmed by showing that multiple isolated colonies have similar cell shapes and Gram staining behavior.
  • Other methods for confirming adequate purity include genetic analysis (e.g., PCR, DNA sequencing), serology and antigen analysis, enzymatic and metabolic analysis, and methods using instrumentation such as flow cytometry with reagents that distinguish desired constituents from contaminants.
  • 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 (e.g., 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 (e.g.
  • 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 (e.g., 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. In the case in which one strain (compared with another of the same species) has gained or lost antibiotic resistance or gained or lost a biosynthetic capability (such as an auxotrophic strain), strains may be differentiated by selection or counter-selection using an antibiotic or nutrient/metabolite, respectively.
  • the terms “subject” or “patient” refers to any mammal. A subject or a patient described as “in need thereof’ refers to one in need of a treatment (or prevention) for a disease.
  • Mammals include humans, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows, sheep, goats, pigs), and household pets (e.g., dogs, cats, rodents).
  • the subject may be a human.
  • the subject may be a non-human mammal including but not limited to of a dog, a cat, a cow, a horse, a pig, a donkey, a goat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, a gorilla or a chimpanzee.
  • the subject may be healthy, or may be suffering from a disease or disorder at any developmental stage.
  • 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, e.g. , the administration of one or more agents, such that onset of at least one symptom of the disease is delayed or prevented.
  • EVs Fournierella massiliensis extracellular vesicles
  • solutions and/or dried forms, and therapeutic compositions that comprise Fournierella massiliensis extracellular vesicles (EVs).
  • solutions and/or dried forms, and therapeutic compositions that comprise EVs obtained from Fournierella massiliensis bacteria.
  • the Fournierella massiliensis bacteria are high yield strains of Fournierella massiliensis bacteria.
  • the high yield strain produces at least 3x10 13 mEVs per liter from a bioreactor-grown culture.
  • solutions and/or dried forms, and therapeutic compositions that comprise extracellular vesicles (EVs) obtained from Fournierella massiliensis bacteria.
  • EVs extracellular vesicles
  • 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, hereby incorporated by reference in its entirety.
  • Fournierella massiliensis bacteria from which EVs are obtained are lyophilized.
  • Fournierella massiliensis bacteria from which EVs are obtained are gamma irradiated (e.g., at 17.5 or 25 kGy).
  • Fournierella massiliensis bacteria from which EVs are obtained are UV irradiated.
  • Fournierella massiliensis bacteria from which EVs are obtained are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).
  • Fournierella massiliensis bacteria from which EVs are obtained are acid treated.
  • Fournierella massiliensis bacteria from which EVs are obtained are oxygen sparged (e.g., at 0.1 vvm for two hours).
  • the Fournierella massiliensis EVs are lyophilized.
  • the Fournier ella massiliensis EVs are gamma irradiated (e.g., at 17.5 or 25 kGy).
  • the Fournier ella massiliensis EVs are UV irradiated.
  • the Fournier ella massiliensis EVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).
  • the Fournierella massiliensis EVs are acid treated.
  • the Fournierella massiliensis EVs are oxygen sparged (e.g., at 0.1 vvm for two hours).
  • the phase of growth can affect the amount or properties of bacteria and/or EVs produced by Fournierella massiliensis bacteria.
  • EVs can be isolated, e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • the Fournierella massiliensis EVs are from one strain of bacteria, e.g., a strain provided herein.
  • the Fournierella massiliensis EVs are from one strain of bacteria (e.g., a strain provided herein) or from more than one strain.
  • the EVs are from Fournierella massiliensis bacteria, e.g., from a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696). In some embodiments, the EVs are from Fournierella massiliensis bacteria, e.g., from Fournierella massiliensis Strain A (ATCC Deposit Number PTA-126696).
  • the Fournierella massiliensis bacterial strain is a bacterial strain having a genome that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to a strain listed in Table 2.
  • EVs are obtained from a strain of Fournierella massiliensis bacteria comprising a genomic sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the genomic sequence of the strain of bacteria deposited with the ATCC Deposit number as provided in Table 2.
  • 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
  • the EVs described herein are obtained from a strain of Fournierella massiliensis bacteria comprising a 16S sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the 16S sequence as provided in Table 2.
  • 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
  • the Fournierella massiliensis EVs of the therapeutic compositions described herein are lyophilized.
  • the Fournierella massiliensis EVs of the therapeutic compositions described herein are gamma irradiated (e.g., at 17.5 or 25 kGy).
  • the Fournierella massiliensis EVs of the therapeutic compositions described herein are UV irradiated.
  • the Fournierella massiliensis EVs of the therapeutic compositions described herein are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).
  • the Fournierella massiliensis EVs of the therapeutic compositions described herein are acid treated.
  • the Fournierella massiliensis EVs of the therapeutic compositions described herein are oxygen sparged (e.g., at 0.1 vvm for two hours).
  • Table 2 Exemplary Fournierella massiliensis Bacterial Strain
  • ATCC is a depository affording permanence of the deposit and ready accessibility thereto by the public if a patent is granted. All restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent. The material will be available during the pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122.
  • the deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited plasmid, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of the patent, whichever period is longer. Applicant acknowledges its duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit.
  • the Fournierella massiliensis bacteria from which the EVs are obtained are modified (e.g., engineered) to reduce toxicity or other adverse effects, to enhance delivery) (e.g., oral delivery) of the EVs (e.g., by improving acid resistance, muco-adherence and/or penetration and/or resistance to bile acids, digestive enzymes, resistance to anti-microbial peptides and/or antibody neutralization), to target desired cell types (e.g., M-cells, goblet cells, enterocytes, dendritic cells, macrophages), to enhance their immunomodulatory and/or therapeutic effect of the EVs (e.g., either alone or in combination with another therapeutic agent), and/or to enhance immune activation or suppression by the EVs (e.g., through modified production of polysaccharides, pili, fimbriae, adhesins).
  • target desired cell types e.g., M-cells, goblet cells, enterocytes, dendritic cells, macrophag
  • the engineered Fournierella massiliensis bacteria described herein are modified to improve Fournierella massiliensis EV manufacturing (e.g., higher oxygen tolerance, stability, improved freeze- thaw tolerance, shorter generation times).
  • the engineered Fournierella massiliensis 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 Fournierella massiliensis bacteria may be produced using any technique known in the art, including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, or any combination thereof.
  • Modified EVs include site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, or any combination thereof.
  • the Fournierella 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 Fournierella massiliensis EVs described herein are engineered such that they comprise, are linked to, and/or are bound by a magnetic and/or paramagnetic moiety (e.g., 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 (e.g., 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 Fournierella massiliensis 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.
  • the Fournierella massiliensis EVs (such as secreted EVs (smEVs)) described herein are prepared using any method known in the art.
  • the Fournierella massiliensis EVs (such as secreted EVs (smEVs)) are prepared without an EV purification step.
  • Fournierella 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 Fournierella massiliensis bacteria are killed using an antibiotic (e.g., using an antibiotic described herein).
  • the bacteria are killed using UV irradiation.
  • the Fournierella massiliensis bacteria are heat-killed.
  • the Fournierella massiliensis EVs described herein are purified from one or more other bacterial components. Methods for purifying Fournierella massiliensis EVs from bacteria are known in the art. In some embodiments, Fournierella massiliensis EVs are prepared from bacterial cultures using methods described in S. Bin Park, et al. PLoS ONE. 6(3):el7629 (2011) or G. Norheim, et al.
  • the Fournierella massiliensis bacteria are cultured to high optical density and then centrifuged to pellet bacteria (e.g., at 10,000 x g for 30 min at 4°C, at 15,500 x g for 15 min at 4°C).
  • the culture supernatants are then passed through filters to exclude intact bacterial cells (e.g., a 0.22 pm filter).
  • the supernatants are then subjected to tangential flow filtration, during which the supernatant is concentrated, species smaller than 100 kDa are removed, and the media is partially exchanged with PBS.
  • filtered supernatants are centrifuged to pellet Fournierella massiliensis EVs (e.g., at 100,000-150,000 x g for 1-3 hours at 4°C, at 200,000 x g for 1-3 hours at 4°C).
  • the Fournierella massiliensis EVs are further purified by resuspending the resulting EV pellets (e.g., in PBS), and applying the resuspended EVs to an Optiprep (iodixanol) gradient or gradient (e.g., a 30- 60% discontinuous gradient, a 0-45% discontinuous gradient), followed by centrifugation (e.g., at 200,000 x g for 4-20 hours at 4°C).
  • EV bands can be collected, diluted with PBS, and centrifuged to pellet the EVs (e.g., at 150,000 x g for 3 hours at 4°C, at 200,000 x g for 1 hour at 4°C).
  • the purified Fournierella massiliensis EVs can be stored, for example, at -80°C or -20°C until use.
  • the EVs are further purified by treatment with DNase and/or proteinase K.
  • cultures of Fournierella massiliensis bacteria are 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 Fournierella massiliensis 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.
  • Fournierella 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 (e.g., XCell ATF from Repligen).
  • ATF alternating tangential flow
  • the ATF system retains intact cells (>0.22 pm) in the bioreactor, and allows smaller components (e.g., 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 mih and 100 kDato be collected, and species smaller than 100 kDato 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.
  • Fournierella massiliensis EVs collected by this method may be further purified and/or concentrated by ultracentrifugation or filtration as described above for filtered supernatants.
  • Fournierella 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 ultracentrifiigation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient.
  • 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.
  • Optiprep gradient method if ammonium sulfate precipitation or ultracentrifiigation were used to concentrate the filtered supernatants, pellets are resuspended in PBS and 3 volumes of 60% Optiprep are added to the sample. In some embodiments, if filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep. Samples are applied to a 0-45% discontinuous Optiprep gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C, e.g., 4-24 hours at 4°C.
  • 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 (e.g., Amicon Ultra columns), dialysis, or ultracentrifugation (200,000 x g, > 3 hours, 4°C) and resuspension.
  • filtration e.g., Amicon Ultra columns
  • dialysis e.g., dialysis
  • ultracentrifugation 200,000 x g, > 3 hours, 4°C
  • the sterility of the Fournierella 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 Fournierella massiliensis EVs are isolated and enriched by chromatography and binding surface moieties on EVs.
  • select Fournierella massiliensis 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.
  • Fournierella massiliensis EVs are lyophilized.
  • Fournierella massiliensis EVs are gamma irradiated (e.g., at 17.5 or 25 kGy).
  • Fournierella massiliensis EVs are UV irradiated.
  • Fournierella massiliensis EVs are heat inactivated
  • Fournierella massiliensis EVs are acid treated.
  • Fournierella massiliensis EVs are oxygen sparged
  • the phase of growth can affect the amount or properties of bacteria and/or EVs produced by Fournierella massiliensis bacteria.
  • EVs can be isolated, e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • the growth environment e.g., culture conditions
  • the growth environment can affect the amount of
  • the yield of EVs can be increased by an EV inducer, as provided in Table 4.
  • the method can optionally include exposing a culture of bacteria to an EV inducer prior to isolating EVs from the bacterial culture.
  • the culture of bacteria can be exposed to an EV inducer at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • the Fournierella massiliensis EVs (such as processed EVs (pmEVs)) described herein are prepared (for example, artificially prepared) using any method known in the art.
  • the Fournierella 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 Fournierella 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, etal. ( J. Proteome Res. 9(12):6135-6147 (2010)) or Sandrini, etal. ⁇ 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.
  • supernatants are then centrifuged at 120,000 x g for 1 hour at 4°C.
  • 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.
  • samples are stored at -20°C.
  • Fournierella 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
  • 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 Fournierella 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 supematant;(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 MgCkto a final concentration of 100 mM. In some embodiments, the cells are lysed in step (d) via homogenization. In some embodiments, the cells are lysed in step (d) via emulsiflex C3.
  • the cells are lysed in step (d) via sonication. In some embodiments, the cells are sonicated in 7 cycles, wherein each cycle comprises 30 seconds of sonication and 30 seconds without sonication. In some embodiments, the centrifugation of step (e) is at 10,000 x g. In some embodiments, the centrifugation of step (e) is for 15 minutes. In some embodiments, the centrifugation of step (e) is at 4°C or room temperature.
  • 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- HCl, pH 7.5 or PBS.
  • Fournierella 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 Fournierella 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 Fournierella massiliensis pmEVs are isolated and enriched by chromatography and binding surface moieties 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.
  • Fournierella massiliensis pmEVs are analyzed, for example, as described in Jeppesen, et al. Cell 177:428 (2019).
  • Fournierella massiliensis pmEVs are lyophilized.
  • Fournierella massiliensis pmEVs are gamma irradiated (for example, at 17.5 or 25 kGy).
  • Fournierella massiliensis pmEVs are UV irradiated.
  • Fournierella massiliensis pmEVs are heat inactivated (for example, at 50°C for two hours or at 90°C for two hours).
  • Fournierella massiliensis pmEVs are acid treated.
  • Fournierella 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.
  • the solutions and dried forms that contain Fournierella 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 Fournierella 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 Fournierella massiliensis EVs (for example, obtained by isolating the EVs from a bacterial culture) to prepare a dried form such as a lyophilate, making it easier to handle (and optionally, further formulate, for example, into a therapeutic composition) after drying.
  • PEG polyethylene glycol
  • dextran such as dextran 40k
  • 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 Fournierella massiliensis EVs (for example, obtained by isolating the EVs from a bacterial culture) to protect the Fournierella massiliensis EVs while lyophilizing or spray drying.
  • a bulking agent and/or lyoprotectant is included from an excipient stock that is added to Fournierella massiliensis EVs (for example, purified and/or concentrated Fournierella massiliensis EVs) to produce a solution, and/or to produce a dried form upon subsequent drying, for example, of the solution.
  • a dried form such as a lyophilate contains between about 5% and about 100% Fournierella massiliensis EVs solids by weight. In some embodiments, prior to drying (such as by lyophilization), the total solids, including Fournierella 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 Fournierella 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 Fournierella massiliensis EV efficacy and/or decrease drying (for example, lyophilization) cycle time.
  • lyoprotectants protect Fournierella 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 Fournierella massiliensis EVs (for example, a liquid preparation thereof) to bring the total solids to between about 2% to about 20%.
  • the Fournierella 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-D) is prepared as a stock solution in deionized water and sterile fdtered with a 0.2 mm fdter prior to use.
  • the stock solution is added to the concentrated Fournierella 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
  • 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.
  • IX PBS IX PBS
  • 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 provided herein allows a solution comprising Fournierella massiliensis EVs to be freeze dried at higher temperatures and shorter drying times.
  • the excipients and methods provided herein allow for Fournierella massiliensis 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 Fournierella 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.
  • DLS is used to measure the charge of the total particles present in a lyophilate.
  • 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. IX 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).
  • 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)
  • DLS zeta potential
  • 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,
  • DLS dynamic light scattering
  • 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 0. IX PBS).
  • Components 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
  • PBS for example, 0.1X PBS
  • lyophilates containing Fournierella massiliensis 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- 1b, 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.
  • solutions e.g., liquid mixtures
  • Fournierella massiliensis EVs e.g., EVs from a. Fournierella massiliensis strain and/ora combination of EVs from different Fournierella massiliensis strains described herein.
  • a solution includes Fournierella massiliensis EVs and an excipient that comprises a bulking agent.
  • a solution includes Fournierella massiliensis EVs and an excipient that comprises a bulking agent.
  • a solution includes Fournierella massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the disclosure provides solutions that comprise Fournierella massiliensis EVs and an excipient that comprises a bulking agent.
  • 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 one of Tables A-D.
  • a solution includes a liquid preparation containing Fournierella massiliensis EVs (for example, obtained by isolating Fournierella massiliensis EVs from a.
  • Fournierella massiliensis bacterial culture such as the supernatant or a retentate
  • an excipient that comprises a bulking agent for example, a liquid preparation containing EVs
  • an excipient stock that comprises a bulking agent for example, an excipient stock of a formula provided in one of Tables A-D, to prepare the solution.
  • a “dried form” that contains Fournierella massiliensis refers to the product resulting from drying a solution that contains Fournierella 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 ak, “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 Fournierella massiliensis EVs (for example, EVs from a Fournierella massiliensis strain and/or a combination of EVs from different Fournierella massiliensis strains described herein), and an excipient.
  • a dried form can include Fournierella massiliensis EVs and an excipient that comprises a bulking agent.
  • a dried form can include Fournierella massiliensis EVs and an excipient that comprises a bulking agent and a lyoprotectant.
  • a dried form can include Fournierella massiliensis EVs and an excipient that comprises a lyoprotectant.
  • Fournierella 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 Fournierella 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 Fournierella 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 excipient is a bulking agent comprising mannitol, sucrose, maltodextrin, dextran, Ficoll, or PVP-K30.
  • the excipient includes an additional component such as trehalose, mannitol, sucrose, sorbitol, dextran, poloxamer 188, maltodextrin, PVP-K30, Ficoll, citrate, arginine, and/or hydroxypropyl-B-cyclodextrin.
  • a dried form contains Fournierella massiliensis EVs and an excipient, for example, that comprises a bulking agent, for example, an excipient from a stock of a formula provided in one of Tables A-D.
  • 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 one of Tables A-D. In some embodiments, the Fournierella 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 Fournierella massiliensis EVs substantially or entirely free of whole bacteria (e.g., live bacteria, killed bacteria, and/or attenuated bacteria). In some embodiments, the solutions and/or dried forms comprise both Fournierella massiliensis EVs and whole bacteria (e.g., live bacteria, killed bacteria, and/or attenuated bacteria). In some embodiments, the solutions and/or dried forms comprise Fournierella massiliensis EVs from one or more Fournierella massiliensis strain. In some embodiments, the solutions and/or dried forms comprise gamma irradiated Fournierella massiliensis EVs. In some embodiments, the Fournierella massiliensis EVs are gamma irradiated after the EVs are isolated (for example, prepared).
  • NTA nanoparticle tracking analysis
  • Coulter counting Coulter counting
  • 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.
  • Fournierella massiliensis EVs are characterized by analytical methods known in the art (e.g., Jeppesen, et al. Cell 177:428 (2019)).
  • the Fournierella massiliensis EVs may be quantified based on particle count.
  • particle count of an EV preparation can be measured using NTA.
  • the Fournierella massiliensis EVs may be quantified based on the amount of protein, lipid, or carbohydrate. For example, in some embodiments, total protein content of an EV preparation is measured using the Bradford assay or BCA.
  • the Fournierella 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 Fournierella massiliensis EV liquid preparation obtained from the source bacteria may be fractionated into subpopulations based on the physical properties (e.g., size, density, protein content, and/or binding affinity) of the subpopulations.
  • One or more of the EV subpopulations e.g., as a liquid preparation
  • solutions and/or dried forms (and therapeutic compositions thereof) comprising Fournierella massiliensis EVs from bacteria useful for the treatment and/or prevention of disease (e.g., a cancer or a dysbiosis), 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) (e.g., for the treatment of a cancer or a dysbiosis, either alone or in combination with other therapeutics).
  • the therapeutic compositions comprise both EVs, and whole bacteria (e.g., live bacteria, killed bacteria, and/or attenuated bacteria).
  • the therapeutic compositions comprise EVs in the absence of bacteria (e.g., at least about 85%, at least about 90%, at least about 95%, or at least about 99% free of bacteria). In some embodiments, the therapeutic compositions comprise EVs and/or bacteria from one or more strain.
  • the solution and/or dried form is added to or incorporated into a food product (e.g., 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 e.g., a food or beverage
  • a food or beverage for infants e.g., a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group
  • a functional food e.g., 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,
  • 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.
  • Fournierella massiliensis EVs or a solution and/or dried form provided herein can be formulated into a therapeutic composition.
  • therapeutic compositions comprising Fournierella massiliensis EVs or a solution and/or dried form described herein.
  • the therapeutic composition comprises Fournierella massiliensis EVs or 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 Fournierella massiliensis EVs useful for the treatment and/or prevention of disease (e.g., a cancer or a dysbiosis), as well as methods of making and/or identifying such EVs, and methods of using such therapeutic compositions (e.g., for the treatment of a cancer or a dysbiosis).
  • the therapeutic compositions comprise both Fournierella massiliensis EVs and whole Fournierella massiliensis bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria).
  • the therapeutic compositions comprise Fournierella massiliensis EVs in the absence of Fournierella massiliensis bacteria (e.g., at least about 85%, at least about 90%, at least about 95%, or at least about 99% free of bacteria). In some embodiments, the therapeutic compositions comprise Fournierella 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 in order 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 (e.g., a STING agonist, a TLR agonist, or a NOD agonist).
  • an adjuvant such as an immuno-adjuvant (e.g., 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) (EPA), docosanoic acid (22:0), docosenoic acid (22: 1), docosapentaenoic acid (22:5), docosanoic acid (22:5), do
  • 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.
  • suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.
  • 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.
  • suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.
  • the therapeutic composition is a food product (e.g., 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 e.g., 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.
  • Fournierella massiliensis EVs are administered at doses e.g., of about lxlO 7 to about lxlO 15 particles, e.g., as measured by NTA.
  • the dose of EVs is about 1 x 10 5 to about 7 x 10 13 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)).
  • the dose of EVs from bacteria is about 1 x 10 10 to about 7 x 10 13 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)).
  • the dose of EVs from bacteria is about 1 x 10 11 to about l x lO 14 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)).
  • Fournierella massiliensis EVs are administered at doses e.g., of about 5 mg to about 900 mg total protein, e.g., as measured by Bradford assay.
  • EVs are administered at doses e.g., of about 5 mg to about 900 mg total protein, for example, as measured by BCA assay.
  • the dose of Fournierella massiliensis EVs is, e.g., about 2xl0 6 - about 2xl0 16 particles.
  • the dose is, e.g., about lxlO 7 - about lxlO 15 , about lxlO 8 - about lxlO 14 , about lxlO 9 - about lxlO 13 , about lxlO 10 - about lxlO 14 , or about lxlO 8 - about lxlO 12 particles.
  • the dose is, e.g., about 2xl0 6 , about 2xl0 7 , about 2xl0 8 , about 2xl0 9 , about lxlO 10 , about 2xl0 10 , about 2xlO n , about 2xl0 12 , about 2xl0 13 , about 2xl0 14 , or about lxlO 15 particles.
  • the dose is, e.g., about 2xl0 14 particles.
  • the dose is, e.g., about 2xl0 12 particles.
  • the dose is, e.g., about 2xl0 10 particles.
  • the dose is, e.g., about lxlO 10 particles. In some embodiments, the dose is, e.g., about 1 x 10 11 to about 1 x 10 14 particles. In some embodiments, the dose is, e.g., about 1 x 10 11 particles. In some embodiments, the dose is, e.g., about l x lO 12 particles. In some embodiments, the dose is, e.g., about 1 x 10 13 particles. In some embodiments, the dose is, e.g., about 1 x 10 14 particles. In some embodiments, particle count is be determined, for example, by NTA.
  • the dose of Fournierella massiliensis EVs is, e.g., based on total protein. In some embodiments, the dose is, e.g., about 5 mg to about 900 mg total protein. In some embodiments, the dose is, e.g., about 20 mg to about 800 mg, about 50 mg to about 700 mg, about 75 mg to about 600 mg, about 100 mg to about 500 mg, about 250 mg to about 750 mg, or about 200 mg to about 500 mg total protein.
  • the dose is, e.g., about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, or about 750 mg total protein.
  • the total protein is determined, for example, by Bradford assay or BCA assay.
  • the dose of Fournierella massiliensis EVs is, e.g., about lxlO 6 - about lxlO 16 particles.
  • the dose is, e.g., about lxlO 7 - about lxlO 15 , about lxlO 8 - about lxlO 14 , about lxlO 9 - about lxlO 13 , about lxlO 10 - about lxlO 14 , or about lxlO 8 - about lxlO 12 particles.
  • the dose is, e.g., about 2xl0 6 , about 2xl0 7 , about 2xl0 8 , about 2xl0 9 , about lxlO 10 , about 2xl0 10 , about 2xlO n , about 2xl0 12 , about 2xl0 13 , about 2xl0 14 , or about lxlO 15 particles.
  • the dose is, e.g., about lxlO 15 particles.
  • the dose is, e.g., about 2x10 14 particles.
  • the dose is, e.g., about 2x10 13 particles.
  • the dose is, e.g., about 1 x 10 11 to about 1 x 10 14 particles. In some embodiments, the dose is, e.g., about 1 x 10 11 particles. In some embodiments, the dose is, e.g., about 1 x 10 12 particles. In some embodiments, the dose is, e.g., about 1 x 10 13 particles. In some embodiments, the dose is, e.g., about 1 x 10 14 particles. In some embodiments, particle count is determined, for example, by NTA.
  • the dose of Fournierella massiliensis EVs is, e.g., about 5 mg to about 900 mg total protein.
  • the dose is, e.g., about 20 mg to about 800 mg, about 50 mg to about 700 mg, about 75 mg to about 600 mg, about 100 mg to about 500 mg, about 250 mg to about 750 mg, or about 200 mg to about 500 mg total protein.
  • the dose is, e.g., about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, or about 750 mg total protein.
  • the dose is, e.g., about 700 mg total protein.
  • the dose is, e.g., about 350 mg total protein.
  • the dose is, e.g., about 175 mg total protein.
  • total protein is determined, for example, by Bradford assay or BCA assay.
  • the dose is comprised in a therapeutic composition and/or dose form described herein.
  • 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.
  • thesolid dose form can comprise 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 Fournier ella massiliensis EVs.
  • the Fournierella 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 (e.g., 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, 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) (e.g., 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 1mm 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.
  • 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 form 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
  • the Fournierella massiliensis EVs are in a buffer, for example, a pharmaceutically acceptable buffer, e.g., saline or PBS.
  • a therapeutic composition comprising a solution and/or dried form (for example, that comprises Fournierella 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 Fournierella massiliensis EVs in the solution or dried form are isolated Fournierella massiliensis EVs.
  • the Fournierella massiliensis EVs in the suspension are gamma irradiated.
  • a solid dosage form e.g., capsule, tablet or minitablet
  • 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 Fournierella massiliensis 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 (e.g., 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 e.g., 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 (e.g., 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 coplymer, for example, a Eudragit L (e.g., Eudragit L 100-55; Eudragit L 30 D-55), a Eudragit S, a Eudragit RL, a Eudragit RS, a Eudragit E, or a Eudragit FS (e.g., Eudragit FS 30 D).
  • a Eudragit L e.g., Eudragit L 100-55; Eudragit L 30 D-55
  • Eudragit S e.g., Eudragit L 100-55; Eudragit L 30 D-55
  • Eudragit S e.g., Eudragit S
  • RL Eudragit RL
  • Eudragit RS Eudragit RS
  • Eudragit E a Eudragit E
  • Eudragit FS e.g., Eudragit FS 30 D
  • enteric coating examples include those described in, e.g., U.S. 6312728; U.S. 6623759; U.S. 4775536; U.S. 5047258; U.S. 5292522; U.S. 6555124; U.S. 6638534; U.S. 2006/0210631; U.S. 2008/200482; U.S. 2005/0271778; U.S. 2004/0028737; WO 2005/044240.
  • methacrylic acid copolymers include: poly(methacrylic acid, methyl methacrylate) 1: 1 sold, for example, under the Eudragit El 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-100P trade name; and poly(methacrylic acid, methyl methacrylate) 1:2 sold, for example, under the Eudragit SI 00 trade name.
  • the solid dose form (for example, a capsule) comprises a single layer coating, for example, a non-enteric coating such as HPMC (hydroxyl propyl methyl cellulose) or gelatin.
  • a non-enteric coating such as HPMC (hydroxyl propyl methyl cellulose) or gelatin.
  • the disclosure also provides methods of preparing solutions of Fournierella 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,
  • 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.
  • a liquid preparation of Fournierella massiliensis EVs and an excipient that comprises a bulking agent are combined to prepare a solution.
  • a liquid preparation of Fournierella massiliensis EVs (for example, obtained by isolating Fournierella massiliensis EVs from abacterial culture (such as a supernatant or a retentate)) and an excipient that comprises a bulking agent, for example, an excipient stock of a formula provided in Tables A-D, are combined to prepare a solution.
  • a bacteria culture such as a supernatant or a retentate
  • a liquid preparation containing Fournierella massiliensis EVs for example, obtained by isolating Fournierella massiliensis EVs from a bacterial culture (such as a supernatant or a retentate)) and an excipient that comprises a bulking agent are combined
  • a liquid preparation containing Fournierella massiliensis EVs for example, obtained by isolating Fournierella massiliensis EVs from a bacterial culture ((such as a supernatant or a retentate)
  • an excipient that comprises a bulking agent such as mannitol or an excipient of an excipient stock of a formula provided in Tables A-D
  • the disclosure also provides methods of preparing dried forms of
  • 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 Fournierella massiliensis EVs (for example, obtained by isolating Fournierella massiliensis EVs from a bacterial culture (such as a supernatant or a retentate)) is combined with an excipient that comprises a bulking agent, such as mannitol or an excipient stock of a formula provided in Tables A-D; and dried (e.g., by lyophilization or spray drying) to thereby prepare a dried form.
  • a bulking agent such as mannitol or an excipient stock of a formula provided in Tables A-D
  • 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.
  • 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-D.
  • the Fournierella massiliensis EVs comprise about l%to about 99% of the total solids by weight of the dried form.
  • 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 Fournierella massiliensis EVs includes: combining a liquid preparation that comprises Fournierella massiliensis EVs with an excipient that comprises a bulking agent, thereby preparing the solution.
  • a method of preparing a solution that comprises Fournierella massiliensis EVs includes: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs includes: combining a liquid preparation that comprises Fournierella massiliensis EVs with an excipient that comprises a lyoprotectant, thereby preparing the solution.
  • a method of preparing a solution that comprises Fournierella massiliensis EVs includes: combining a liquid preparation that comprises Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D, thereby preparing a solution.
  • the Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A-D, 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 Fournierella 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-D, 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A-D, 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A-D, thereby preparing a solution; and drying the solution, thereby preparing the powder.
  • the disclosure provides a method of preparing a powder that comprises Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A-D, 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A-D, 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 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A-D, 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D, 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, or D, 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A-D, 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 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 Fournierella massiliensis EVs, the method comprising: combining a liquid preparation that comprises Fournierella massiliensis EVs with a stock comprising one or more excipients, wherein the stock comprises a formula provided in Tables A-D, 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 can be, e.g., 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 Fournierella massiliensis EVs and an excipient that comprises a bulking agent.
  • the dried form comprises Fournierella massiliensis EVs and an excipient that comprises a lyoprotectant.
  • the dried form comprises Fournierella 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, e.g., 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 (e.g., 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;
  • a method of making the solid dosage form includes: compressing a dried form 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 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 (e.g., a buffer, juice, or water) to prepare a suspension.
  • a liquid e.g., a buffer, juice, or water
  • a therapeutic composition comprising a solution is resuspended (for example, diluted) in a liquid (e.g., a buffer, juice, or water) to prepare a suspension.
  • a liquid e.g., a buffer, juice, or water
  • powders and frozen biomass are gamma-irradiated.
  • powders for example, of Fournierella massiliensis EVs
  • powders are gamma-irradiated at 17.5 kGy radiation unit at ambient temperature.
  • frozen biomasses for example, of Fournierella 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 Fournierella massiliensis EVs is administered to the subject before the additional therapeutic agent is administered (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
  • the therapeutic composition comprising Fournierella massiliensis EVs is administered to the subject after the additional therapeutic agent is administered (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours after or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days after).
  • the therapeutic composition comprising Fournierella 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).
  • the methods provided herein include the administration of a therapeutic composition described herein in combination with one or more additional therapeutic agents. In some embodiments, the methods disclosed herein include the administration of two therapeutic agents.
  • 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 a cancer therapeutic.
  • the therapeutic composition comprising Fournierella massiliensis EVs is administered to the subject before the additional therapeutic agent is administered (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • the therapeutic composition comprising EVs is administered to the subject after the additional therapeutic agent is administered (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours after or at least
  • the therapeutic composition comprising 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 EVs is administered to the subject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days before).
  • an antibiotic is administered to the subject after therapeutic composition comprising EVs is administered to the subject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days after).
  • the therapeutic composition comprising 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; callystatin; CC- 1065 (including its adozelesin,
  • the cancer therapeutic is a cancer immunotherapy agent.
  • Immunotherapy refers to a treatment that uses a subject’s immune system to treat cancer, e.g., 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 (e.g., 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- A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0010718C (avelumab), AUR-012 and STI-A1010.
  • the immune checkpoint inhibitor is a CTLA-4 inhibitor.
  • the immune checkpoint inhibitor is a PD-1 inhibitor.
  • the immune checkpoint inhibitor is a PD-L1 inhibitor.
  • the immune checkpoint inhibitor is an antibody.
  • 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 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
  • a 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”), ARTC1, 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
  • PBF pml-RARalpha fusion protein
  • PPP1R3B polymorphic epithelial mucin
  • PRAME polymorphic epithelial mucin
  • PRDX5 PSA, PSMA, PTPRK, RAB38/NY-MEL-1
  • RAGE-1 RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secemin 1, SIRT2, SNRPD1, SOXIO, Spl7, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, Telomerase, TGF-betaRII, TPBG, TRAG-3, Triosephosphate isomerase, TRP- l/gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE- lb/GAGE
  • the immunotherapy agent is a cancer vaccine and/or a component of a cancer vaccine (e.g 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 (e.g., 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, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTC1, 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, EphA3, epithelial tumor antigen (“ETA”), ETV6-AML1 fusion protein, EZH2, FGF5, FLT3-IT
  • 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, b-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 enter
  • 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
  • Monocyte chemoattractant protein 2 (“MCP-2”), Monocyte chemoattractant protein 3 (“MCP-3”), Monocyte chemoattractant protein 4 (“MCP-4"), Macrophage-derived chemokine (“MDC”), Macrophage migration inhibitory factor (“MIF”), Chemokine (C-C motif) ligand 20 (“MIP-3 alpha”), C-C motif chemokine 19 (“MIP-3 beta”), Chemokine (C-C motif) ligand 23 (“MPIF-1”), Macrophage stimulating protein alpha chain (“MSPalpha”), Nucleosome assembly protein 1-like 4 (“NAP-2”), Secreted phosphoprotein 1 (“Osteopontin”), Pulmonary and activation-regulated cytokine (“PARC”), Platelet factor 4 (“PF4"), Stroma cell-derived factor- 1 alpha (“SDF-1 alpha”), Chemokine (C-C motif) ligand 17 (“TARC”), Thymus-expressed chemokine (“
  • SOST Heparan sulfate proteoglycan
  • TACI Tumor necrosis factor receptor superfamily member 13B
  • TFPI Tissue factor pathway inhibitor
  • TSP-1 Tumor necrosis factor receptor superfamily member 10b
  • TRANCE TRANCE
  • Troponin I Urokinase Plasminogen Activator
  • uPA Urokinase Plasminogen Activator
  • Cadherin 5 type 2 or VE- cadherin (vascular endothelial) also known as CD 144
  • WISP-1 WNT1 -inducible signaling pathway protein 1
  • RANK Receptor Activator of Nuclear Factor k B
  • 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 (Afmitor®), Exemestan
  • Pralatrexate Fluorescence Fluorescence (Folotyn®), Regorafenib (Stivarga®), Rituximab (Rituxan®), Romidepsin (Istodax®), Sorafenib tosylate (Nexavar®), Sunitinib malate (Sutent®), Tamoxifen, Temsirolimus (Torisel®), Toremifene (Fareston®), Tositumomab and 1311- tositumomab (Bexxar®), Trastuzumab (Herceptin®), Tretinoin (Vesanoid®), Vandetanib (Caprelsa®), Vemurafenib (Zelboraf®), Vorinostat (Zolinza®), and Ziv-aflibercept (Zaltrap®).
  • 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®).
  • anti-cancer compounds that increase anti-tumor immune response are Rituximab (Rituxan®), Alemtuzumab (Campath®), Ofatumumab (Arzerra®), and Ipilimumab (YervoyTM).
  • anti-cancer compounds that deliver toxic agents to cancer cells are Tositumomab and 1311-tositumomab (Bexxar®)and Ibritumomab tiuxetan (Zevalin®), Denileukin diftitox (Ontak®), and Brentuximab vedotin (Adcetris®).
  • exemplary anti-cancer compounds are small molecule inhibitors and conjugates thereof of, e.g., Janus kinase, ALK, Bel -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 (e.g., Gram-negative vs. Gram-positive bacteria, aerobic vs.
  • 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 Fournierella massiliensis EVs. In some embodiments, antibiotics are administered before therapeutic composition comprising Fournierella 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, b-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, gly copeptides, 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 coli, 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 Gram positive and Gram-negative bacteria as broad-spectrum antibiotics. Carbapenems are believed to inhibit bacterial cell wall synthesis.
  • Cephalosporins include, but are not limited to, Cefadroxil, 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
  • MERS A 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 5 OS 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.
  • Oxazobdonones include, but are not limited to, Linezobd, Posizobd, Radezolid, and Torezolid. Oxazobdonones are believed to be protein synthesis inhibitors.
  • Penicillins include, but are not limited to, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicilbn, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Temocillin and Ticarcilbn. Penicillins are effective, for example, against Gram-positive bacteria, facultative anaerobes, e.g., 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 Ticarcilbn/clavulanate .
  • Polypeptide antibiotics include, but are not limited to, Bacitracin, Colistin, and Polymyxin B and E.
  • Polypeptide Antibiotics are effective, for example, against Gram-negative 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 counter ions.
  • 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.
  • Quinolones/Fluoroquinolone are effective, e.g., 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, Demeclocycline,
  • 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 PI, 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, ole
  • 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, lomoxicam, 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 inhibitor
  • a method of delivering a therapeutic composition described herein for example, a therapeutic composition comprising Fournierella massiliensis EVs, 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 Fournierella massiliensis EVs, a solution, or dried form described herein is co-formulated with the additional therapeutic agent.
  • the therapeutic composition comprising Fournierella massiliensis EVs, 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 Fournierella massiliensis EVs, a solution, or dried form described herein (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes before, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours before, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days before).
  • the additional therapeutic agent is administered to the subject after administration of the therapeutic composition comprising Fournierella massiliensis EVs, a solution, or dried form described herein (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
  • the same mode of delivery is used to deliver both the therapeutic composition comprising Fournierella massiliensis EVs, a solution, or dried form described herein and the additional therapeutic agent.
  • different modes of delivery are used to administer the therapeutic composition comprising Fournierella massiliensis EVs, a solution, or dried form described herein and the additional therapeutic agent.
  • the therapeutic composition comprising Fournierella massiliensis EVs, a solution, or dried form described herein is administered orally while the additional therapeutic agent is administered via injection (e.g., an intravenous, intratumoral and/or intramuscular injection).
  • injection e.g., an intravenous, intratumoral and/or intramuscular 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 Fournierella massiliensis EVs, 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 Fournierella massiliensis EVs, 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 (e.g., a cancer or a dysbiosis,), delay its onset, or slow or stop its progression, or relieve one or more symptoms of the disease.
  • disease e.g., a cancer or a dysbiosis
  • 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 range-finding 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 therapeutic 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 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 of cancer.
  • any cancer can be treated using the methods described herein.
  • cancers that may treated by methods and therapeutic 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
  • the methods and therapeutic 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
  • the methods and therapeutic compositions provided herein relate to the treatment of a carcinoma.
  • 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, Abemethy'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 (e.g., triple negative breast cancer). In some embodiments, the cancer comprises triple negative breast cancer. In some embodiments, the cancer comprises metastatic triple negative breast cancer.
  • the cancer comprises colorectal cancer (e.g., microsatellite stable (MSS) colorectal cancer).
  • MSS microsatellite stable
  • the cancer comprises renal cell carcinoma.
  • the cancer comprises lung cancer (e.g., non small cell lung cancer). In some embodiments, the cancer comprises non small cell lung cancer.
  • the cancer comprises bladder cancer.
  • the cancer comprises gastroesophageal cancer.
  • the cancer comprises a solid tumor.
  • 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, hypemephroid adenocarcinoma, bile duct carcinoma, chor
  • Cancers treated in certain embodiments also include precancerous lesions, e.g., 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 e.g., actinic keratosis (solar keratosis), moles (dysplastic nevi), acitinic chelitis (farmer's lip), cutaneous horns, Barrett's esophagus, atrophic gastritis, dyskeratosis congenita, sideropenic dysphagia, lichen
  • Cancers treated in some embodiments include non-cancerous or benign tumors, e.g., 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.
  • non-cancerous or benign tumors e.g., of endodermal, ectodermal or mesenchymal origin, including, but not limited to cholangioma, colonic
  • 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, https://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 (e.g., 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.
  • host immune cells e.g., T cells, dendritic cells, mast cells, NK cells, intestinal epithelial lymphocytes (IEC), macrophages and phagocytes
  • 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
  • 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 (e.g., systemic lupus erythematosus (SLE)) or inflammatory disorders (e.g., functional gastrointestinal disorders such as inflammatory bowel disease (IBD), ulcerative colitis, and Crohn’s disease), neuroinflammatory diseases (e.g., multiple sclerosis), transplant disorders (e.g., 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 e.g., systemic lupus erythematosus (SLE)
  • inflammatory disorders e.g., functional gastrointestinal disorders such as inflammatory bowel disease (IBD), ulcerative colitis, and Crohn’s disease
  • neuroinflammatory diseases
  • 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, modulating 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 Fournierella massiliensis EVs. 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 Fournierella massiliensis EVs. 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 Fournierella massiliensis bacteria are administered (e.g., 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 cytokine secretion, modulating 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, e.g., subpopulations of T cells, immune lymphoid cells, dendritic cells, NK cells and other immune cells, or the function thereof, in the recipient.
  • host immune cell subpopulations e.g., 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 Fournierella massiliensis EVs capable of altering the relative proportions of immune cell subpopulations, e.g., T cell subpopulations, immune lymphoid cells, NK cells and other immune cells, or the function thereof, in the recipient subject.
  • immune cell subpopulations e.g., 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 Fournierella massiliensis EVs.
  • 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 (e.g., anti-inflammatory bacterial cells) or a population of Fournierella massiliensis EVs.
  • therapeutic compositions useful for treatment of disorders associated with a dysbiosis stimulate secretion of one or more anti inflammatory cytokines by host immune cells.
  • Anti-inflammatory cytokines include, but are not limited to, IL-10, IL-13, IL-9, IL-4, IL-5, TGF , and combinations thereof.
  • therapeutic 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, IFNy, IL-12p70, IL-la, IL-6, IL-8, MCP1, MIPla, MIRIb, 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 (e.g., orally administering) to the subject a therapeutic composition in the form of a probiotic or medical food comprising Fournierella massiliensis 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 Fournierella 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 Fournierella massiliensis EVs (e.g., either alone or in combination with another therapeutic agent), to reduce toxicity and/or to improve bacterial and/or EV manufacturing (e.g., higher oxygen tolerance, improved freeze-thaw tolerance, shorter generation times).
  • the engineered bacteria may be produced using any technique known in the art, including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, CRISPR Cas9, or any combination thereof.
  • 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 (e.g., by exposure to chemical mutagens and/or UV radiation) or exposing them to a therapeutic agent (e.g., antibiotic) followed by an assay to detect bacteria having the desired phenotype (e.g., an in vivo assay, an ex vivo assay, or an in vitro assay).
  • a therapeutic agent e.g., antibiotic
  • Example 1 Cultivation and storage conditions for Fournierella massiliensis strains Anaerobic Tryptic Soy Broth (TSB) medium supplemented with Hemoglobin:
  • Reducing agent 100 preparation a. Dissolve 5 g of F-Cysteine-HCl in 100 ml of H2O. b. Add 0.5 g of FeCh. c. Store solution under anaerobic conditions.
  • Hemoglobin solution 100 preparation a. Dissolve 2 g of Hemoglobin in 100 ml of 0.01N NaOH. b. Autoclave the solution. c. Store the solution protected from direct light at 4°C. d. Add the solution to sterilized medium.
  • Anaerobic glycerol as crvoprotectant Anaerobic glycerol as crvoprotectant.
  • Anaerobe Systems #AS-9045 Composition (g/L):
  • Example 2A EV isolation and enumeration
  • 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 Fiberbte 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. Isolation of EVs using Ultracentrifugation
  • 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 lxPBS.
  • the resuspended purified EV samples are stored at 4°C or at -80°C.
  • Example 2B EV isolation and enumeration Equipment required:
  • Microbes must be pelleted and filtered away from supernatant in order to recover EVs and not microbes.
  • a. Pellet Microbial Culture i. Use Sorvall RC-5C centrifuge with the SLA-3000 rotor and centrifuge culture for a minimum of 15min at a minimum of 7,000 rpm.
  • b. Supernatant Filtration i. Filter supernatant through 0.2 pum filter.
  • ii For supernatants with poor filterability (less than 300 ml of supernatant pass through filter) attach a 0.45 pum capsule filter ahead of the 0.2 pum vacuum filter.
  • iii. Store ‘filtered’ supernatant at 4°C.
  • Filtered supernatant can then be concentrated using TFF.
  • Centrifuging concentrated supernatant in the ultracentrifuge will pellet EVs isolating the EVs from smaller biomolecules.
  • i. Set speed for 200,000 g, time for 1 hour, and temperature at 4°C.
  • iii. When rotor has stopped, remove tubes from ultracentrifuge and gently pour off the supernatant.
  • iii. Add more supernatant, balance, and centrifuge tubes again.
  • the pellets generated are referred to as ‘crude’ EV pellets.
  • 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.
  • a. Preparation of Density Medium i. 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 lxPBS. ii. The 45% gradient layer should contain the crude EV sample.
  • Example 3 Pro-inflammatory cytokine release from dendritic cells
  • the Fournierella massiliensis EVs are from Fournierella massiliensis Strain A (ATCC Deposit Number PTA- 126696).
  • Example 4 Preparation of lvophilates
  • excipient stocks with the formulas provided in Table A to Table D are prepared (amounts shown are percentages of each component in the formula) as solutions.
  • the excipient stock solutions are mixed with a liquid preparation of extracellular vesicles.
  • the resulting solutions are freeze-dried and analyzed.
  • the extracellular vesicles are isolated from a strain of Fournierella massiliensis ( Fournierella massiliensis Strain A). Fournierella massiliensis EVs are dried, such as by freeze drying or spray drying, using one of the stocks provided in Table A to Table D.
  • Table A Excipient stocks for stabilizing extracellular vesicles during lyophilization. The numerical values given are on a weight percent basis in the solution.
  • Table B Certain example excipient stocks including polymers for stabilizing extracellular vesicles during lyophilization. The numerical values given are on a weight percent basis in the solution.
  • Table C Certain example excipient stocks including polymers for stabilizing extracellular vesicles during lyophilization. The numerical values given are on a weight percent basis in the solution.
  • Table D Certain example excipient stocks including polymers for stabilizing extracellular vesicles during lyophilization. The numerical values given are on a weight percent basis in the solution.
  • Example 5 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 fdtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If fdtration was used to concentrate the fdtered 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 fdtered supernatants, pellets are resuspended in 45% Optiprep in PBS. If fdtration was used to concentrate the fdtered 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 6 Manipulating bacteria through stress to produce various amounts of
  • 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 microcins 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.
  • 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 fdtration, 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 mM) 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 deoxycholate.
  • detergent such as sodium dodecyl sulfate (SDS) or deoxycholate.
  • 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.
  • 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 (e.g., Silver staining, Coomassie Blue, or Gel Code Blue) for visualization of bands.
  • a gel for example a Bolt Bis-Tris Plus 4-12% gel (Thermo-Fisher Scientific)
  • EV proteins are separated by SDS-PAGE as described above and subjected to Western blot analysis (Cvjetkovic et ak, Sci. Rep. 6, 36338 (2016)) and are quantified via ELISA.
  • EV proteins present in EVs 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 lplex Label Reagents (Thermo Fischer Scientific, San Jose, CA, USA).
  • iTRAQ Reagent-8plex Multiplex Kit Applied Biosystems, Foster City, CA
  • TMT lOplex and 1 lplex 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). Media samples or other complex metabolic mixtures ( ⁇ 10 pL) are extracted using nine volumes of 74.9:24.9:0.2 (v/v/v) acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (valine-d8, Isotec; and phenylalanine- d8, Cambridge Isotope Laboratories). Standards may be adjusted or modified depending on the metabolites of interest.
  • the samples are centrifuged (10 minutes, 9,000 x g, 4°C), and the supernatants (10 pL) are submitted to LCMS by injecting the solution onto the HILIC column (150 x 2.1 mm, 3 pm particle size).
  • the column is eluted by flowing a 5% mobile phase [10 mM 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 are quantified using FM4-64 (Life Technologies), by methods similar to those described by A.J. McBroom et al. J Bacteriol 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. Lipidomics can be used to identify the lipids present in the EVs.
  • 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
  • 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 (Bio-Rad), 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 acid analysis Nucleic acids are extracted from EVs and quantified using a Qubit fluorometer. Size distribution is assessed using a BioAnalyzer and the material is sequenced.
  • the zeta potential of different preparations are measured using instruments such as the Zetasizer ZS (Malvern Instruments).
  • 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°Cto 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.
  • nitrogen or gas mixtures may be used in order to establish anaerobic conditions.
  • 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 (for example, exponential growth, stationary growth, unstressed, or 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.
  • the state of bacteria at the end of a fermentation and during harvesting may impact cell survival and activity.

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Abstract

La présente invention concerne des vésicules extracellulaires de Fournierella massiliensis, et des solutions et une forme séchée (et des compositions thérapeutiques de celles-ci) de vésicules extracellulaires de Fournierella massiliensis qui peuvent être utiles en tant qu'agents thérapeutiques, et leurs procédés d'utilisation.
PCT/US2022/024243 2021-04-12 2022-04-11 Préparations de vésicules extracellulaires de fournierella WO2022221183A1 (fr)

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