WO2023114293A1 - Dosages de vésicules extracellulaires - Google Patents
Dosages de vésicules extracellulaires Download PDFInfo
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- WO2023114293A1 WO2023114293A1 PCT/US2022/052838 US2022052838W WO2023114293A1 WO 2023114293 A1 WO2023114293 A1 WO 2023114293A1 US 2022052838 W US2022052838 W US 2022052838W WO 2023114293 A1 WO2023114293 A1 WO 2023114293A1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/06—Investigating concentration of particle suspensions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/06—Investigating concentration of particle suspensions
- G01N15/075—Investigating concentration of particle suspensions by optical means
Definitions
- Therapeutic compositions comprising microbial extracellular vesicles (mEVs), such as mEVs obtained and/or derived from bacteria, can have therapeutic effects and can be useful for the treatment and/or prevention of disease and/or health disorders.
- mEVs microbial extracellular vesicles
- mEVs extracellular vesicles
- a composition e.g, in a therapeutic agent (i.e., pharmaceutical agent, drug substance) or in a therapeutic composition (i.e., pharmaceutical composition, drug product)
- eTPN Equivalent Total Particle Number
- a method of determining an eTPN in a composition comprising: (a) contacting a test sample from a composition comprising mEVs with a lipophilic fluorescent dye to obtain a mixture;
- mEVs microbial extracellular vesicles
- a composition comprising mEVs (e.g., a drug product or a drug substance)
- the method comprising: (a) contacting a test sample from a composition comprising mEVs with a lipophilic fluorescent dye to obtain a mixture; (b) measuring a fluorescence signal from the mixture; and (c) quantitating the mEVs in the composition based on the fluorescence signal.
- the method can further comprise (d) determining the eTPN of the composition comprising the mEVs, e.g., by comparing the fluorescence signal from the mixture to a calibration curve (e.g., that provides a correlation between flourescence signal and particle number).
- a calibration curve e.g., that provides a correlation between flourescence signal and particle number
- a method of detecting microbial extracellular vesicles (mEVs) in a composition comprising: (a) contacting a test sample from a composition with a lipophilic fluorescent dye to obtain a mixture; (b) detecting a fluorescence signal from the mixture; and (c) determining whether mEVs are present in the composition based on the fluorescence signal.
- the method can further comprise (d) determining the eTPN of the composition comprising the mEVs, e.g., by comparing the fluorescence signal from the mixture to a calibration curve (e.g., that provides a correlation between flourescence signal and particle number).
- a method of determining the lipid content of a composition comprising microbial extracellular vesicles (mEVs) comprising: (a) contacting a test sample from a composition comprising mEVs with a lipophilic fluorescent dye to obtain a mixture; (b) measuring a fluorescence signal from the mixture; and (c) determining the lipid content of the composition comprising mEVs based on the fluorescence signal.
- mEVs microbial extracellular vesicles
- the method can further comprise (d) determining the eTPN of the composition comprising the mEVs, e.g., by comparing the fluorescence signal from the mixture to a calibration curve (e.g., that provides a correlation between flourescence signal and particle number).
- a calibration curve e.g., that provides a correlation between flourescence signal and particle number
- mEVs microbial extracellular vesicles
- a composition comprising mEVs (e.g., a drug product or a drug substance)
- the method comprising: (a) contacting a test sample from a composition comprising mEVs with a lipophilic fluorescent dye to obtain a mixture; (b) measuring a fluorescence signal from the mixture; and (c) quantitating the eTPN of mEVs in the composition e.g., by comparing the fluorescence signal from the mixture to a calibration curve (e.g., that provides a correlation between flourescence signal and particle number).
- a calibration curve e.g., that provides a correlation between flourescence signal and particle number
- a method of detecting microbial extracellular vesicles (mEVs) in a composition comprising: (a) contacting a test sample from a composition with a lipophilic fluorescent dye to obtain a mixture; (b) detecting a fluorescence signal from the mixture; and (c) determining whether mEVs are present in the composition, e.g., by comparing the fluorescence signal from the mixture to a calibration curve (e.g., that provides a correlation between flourescence signal and particle number).
- a method of determining the lipid content of a composition comprising microbial extracellular vesicles comprising: (a) contacting a test sample from a composition comprising mEVs with a lipophilic fluorescent dye to obtain a mixture; (b) measuring a fluorescence signal from the mixture; and (c) determining the eTPN of the composition comprising the mEVs, e.g., by comparing the fluorescence sigal from the mixture to a calibration curve (e.g., that provides a correlation between flourescense signal and particle number).
- mEVs microbial extracellular vesicles
- a method of determining a dose of a composition comprising: (a) contacting a test sample from a composition comprising mEVs with a lipophilic fluorescent dye to obtain a mixture; (b) measuring a fluorescence signal from the mixture; and (c) determining a dose of the composition comprising mEVs based on the fluorescence signal.
- a therapeutic agent i.e., pharmaceutical agent, drug substance
- a therapeutic composition i.e., pharmaceutical composition, drug product
- mEVs microbial extracellular vesicles
- the method can further comprise (d) determining the eTPN of the composition comprising the mEVs, e.g., by comparing the fluorescence signal from the mixture to a calibration curve (e.g., that provides a correlation between flourescence signal and particle number). In some embodiments, the method further comprises administering the dose of the composition to a subject.
- a method of determining a dose e.g., an eTPN dose of a composition (e.g, in a therapeutic agent (i.e., pharmaceutical agent, drug substance) or in a therapeutic composition (i.e., pharmaceutical composition, drug product)) comprising microbial extracellular vesicles (mEVs), the method comprising: (a) contacting a test sample from a composition comprising mEVs with a lipophilic fluorescent dye to obtain a mixture; (b) measuring a fluorescence signal from the mixture; and (c) determining an eTPNof the composition comprising the mEVs, e.g., by comparing the fluorescence signal from the mixture to a calibration curve (e.g., that provides a correlation between flourescence signal and particle number), wherein the eTPN is the dose of the composition.
- the method further comprises administering the dose of the composition to a subject.
- the method further comprises the step of separating mEVs from bacterial cells to generate the composition of step (a).
- the separation of mEVs from bacterial cells is performed using centrifugation.
- the separation of mEVs from bacterial cells is performed using filtration.
- step (b) is performed using fluorescence spectroscopy.
- step (b) is performed using a spectrafluorometer.
- step (b) is performed using a spectrafluorometer capable of reading fluorescence at Ex/Em 515/640 nm or equivalent.
- step (b) is performed using a microplate spectrafluorometer, such as a microplate spectrafluorometer capable of reading fluorescence at Ex/Em 515/640 nm or equivalent.
- step (b) is performed using high performance liquid chromatography (HPLC) with a fluorescence detector capable of reading fluorescence at Ex/Em 515/640 nm or equivalent.
- the method is performed at room temperature.
- the test sample comprises 10 9 to 10 11 mEV particles.
- composition comprising microbial extracellular vesicles (mEVs) and a lipophilic fluorescent dye.
- mEVs microbial extracellular vesicles
- the composition comprises a solution.
- the solution comprises one or more excipients, such as a bulking agent and/or lyoprotectant.
- the composition comprises a dried form.
- the dried form comprises one or more excipients, such as a bulking agent and/or lyoprotectant.
- the dried form comprises a lyophilate.
- the lyophilate comprises a lyophilized powder.
- the lyophilate comprises a lyophilized cake.
- the dried form comprises a powder.
- the powder comprises a lyophilized powder.
- the powder comprises a spray dried powder.
- the composition comprises a dried form and the dried form is resuspended, e.g., in a liquid (such as PBS or water), prior to the contacting step.
- a liquid such as PBS or water
- the dried form is resuspended in PBS prior to the contacting step.
- the dried form is resuspended in water prior to the contacting step.
- the fluorescent dye specifically binds to the lipid membrane layer of the mEVs.
- the fluorescence of the fluorescent dye increases when it binds to the lipid membrane layer of an mEV.
- the fluorescent dye is non-fluorescent in aqueous media and becomes fluorescent when it binds to the lipid membrane layer of an mEV.
- the fl orescent dye has an excitation wavelength of about 515 nm. In some embodiments, the excitation wavelength is within 20%, 15%, 10%, 5%, 1%, or 0% of 515 nanometers (e.g., the excitation wavelength is 515 nanometers).
- the fluorescent dye has an emission wavelength of about 640 nm. In some embodiments, the emission wavelength is within 20%, 15%, 10%, 5%, 1%, or 0% of 640 nanometers (e.g., the emission wavelength is 640 nanometers).
- fluorescent dyes used in the methods and compositions described herein include, but are not limited to, N-(3- triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64), a fixable analog of FM4-64 (FM4-64X (Thermo Fisher)), N-(3- Trimethylammoniumpropyl)-4-(6-(4-(Diethylamino)phenyl)hexatrienyl)Pyridinium Dibromide (FM 5-95), a slightly less lipophilic analog of FM 4-64, and SynaptoRedTM C2.
- the fluorescent dye is N-(3-triethylammoniumpropyl)- 4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64).
- the fluorescent dye is a fixable analog of FM4-64 (FM4-64X or FM4-64FX).
- the fluorescent dye is A-(3-Trimethylammoniumpropyl)-4-(6-(4- (Di ethyl ami n o)p heny 1) hexatr i eny l)Py ri di n i urn Di bromi de (F M 5-95).
- the fluorescent dye comprises the following chemical structure:
- the fluorescent dye comprises the following chemical structure:
- the fluorescent dye comprises the following chemical structure:
- the composition comprises isolated mEVs. In some embodiments, the composition comprises a therapeutically effective amount of the isolated mEVs. In certain embodiments, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the content of the composition is the isolated mEVs. In some embodiments, the mEVs are from one strain of bacteria.
- the composition comprises mEVs and bacteria. In some embodiments, the composition comprises a therapeutically effective amount of the bacteria. In some embodiments, the composition comprises a therapeutically effective amount of the mEVs. In certain embodiments, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the content of the composition is the isolated mEVs. In some embodiments, the mEVs are from one strain of bacteria. In some embodiments, the mEVs and bacteria from the same strain of bacteria.
- the mEVs comprise secreted mEVs (smEVs).
- the mEVs comprise processed mEVs (pmEVs).
- the pmEVs are produced from bacteria that have been gamma irradiated, UV irradiated, heat inactivated, acid treated, or oxygen sparged.
- the pmEVs are produced from live bacteria.
- the pmEVs are produced from dead bacteria.
- the pmEVs are produced from non-replicating bacteria.
- the mEVs are lyophilized.
- the mEVs are gamma irradiated.
- the mEVs are UV irradiated.
- the mEVs are heat inactivated (e.g., at about 50°C for at least two hours or at about 90°C for at least two hours).
- the mEVs are acid treated.
- the mEVs are oxygen sparged (e.g., at 0.1 vvm for two hours).
- the mEVs are from Gram positive bacteria. In certain embodiments, the mEVs are from Gram negative bacteria. In some embodiments, the mEVs are from aerobic bacteria. In some embodiments, the mEVs are from anaerobic bacteria. In some embodiments, the mEVs are from acidophile bacteria. In some embodiments, the mEVs are from alkaliphile bacteria. In some embodiments, the mEVs are from neutral ophile bacteria. In some embodiments, the mEVs are from fastidious bacteria. In some embodiments, the mEVs are from nonfasti di ous bacteria.
- the mEVs are from bacteria of a class, order, family, genus, species and/or strain listed in Table 1, Table 2, Table 3, or Table 4. In some embodiments, the mEVs are from a bacterial strain listed in Table 1, Table 2, Table 3, or Table 4. In some embodiments, the mEVs are from bacteria of a class, order, family, genus, species and/or strain listed in Table J. In some embodiments, the mEVs are from a bacterial strain listed in Table J.
- the mEVs are from bacteria of the genus Prevotella. In some embodiments, the mEVs are from bacteria of the species Prevotella histicola. In some embodiments, the mEVs are from a strain of bacteria comprising at least 95% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329). In some embodiments, the mEVs are from a strain of bacteria comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329). In some embodiments, the mEVs are from a from Prevotella Strain B 50329 (NRRL accession number B 50329).
- the Gram negative bacteria belong to class Negativicutes.
- the Gram negative bacteria belong to family Veillonellaceae, Selenomonadaceae, Acidaminococcaceae. or Sporomusaceae .
- the mEVs are from bacteria of the genus Megasphaera, Selenomonas, Propionospora, or Acidaminococcus .
- the mEVs are from Megasphaera sp., Selenomonas felix, Acidaminococcus intestine, o Propionospora sp. bacteria.
- the mEVs are from bacteria of the genus Lactococcus, Prevotella, Bifidobacterium, or Veillonell .
- the mEVs are from Lactococcus lactis cremoris bacteria.
- the mEVs are from Prevotella histicola bacteria.
- the mEVs are from Bifidobacterium animalis bacteria.
- the mEVs are from Veillonella parvula bacteria.
- the mEVs are from Lactococcus lactis cremoris bacteria.
- the Lactococcus lactis cremoris bacteria 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 Lactococcus lactis cremoris Strain A (ATCC designation number PTA-125368).
- the Lactococcus bacteria are from a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Lactococcus lactis cremoris Strain A (ATCC designation number PTA- 125368).
- the Lactococcus bacteria are from Lactococcus lactis cremoris Strain A (ATCC designation number PTA-125368).
- the mEVs are from Prevotella bacteria.
- the Prevotella bacteria 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 Prevotella Strain B 50329 (NRRL accession number B 50329).
- the Prevotella bacteria are from a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
- the Prevotella bacteria are from Prevotella Strain B 50329 (NRRL accession number B 50329).
- the bacteria or the bacteria from which the mEVs are obtained and/or derived are Prevotella histicola bacteria, e.g., a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella histicola bacteria deposited as ATCC designation number PTA- 126140.
- the bacteria or the bacteria from which the mEVs are obtained and/or derived are Prevotella histicola bacteria, e.g., Prevotella histicola bacteria deposited as ATCC designation number PTA-126140.
- the mEVs are from Bifidobacterium bacteria.
- the Bifidobacterium bacteria 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 Bifidobacterium bacteria deposited as ATCC designation number PTA-125097.
- the Bifidobacterium bacteria are from a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Bifidobacterium bacteria deposited as ATCC designation number PTA-125097.
- the Bifidobacterium bacteria are from Bifidobacterium bacteria deposited as ATCC designation number PTA-125097.
- the mEVs are from Veillonella bacteria.
- the Veillonella bacteria 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 Veillonella. bacteria deposited as ATCC designation number PTA-125691.
- the Veillonella bacteria are from a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Veillonella bacteria deposited as ATCC designation number PTA-125691.
- the Veillonella bacteria are from Veillonella bacteria deposited as ATCC designation number PTA-125691.
- the mEVs are from Ruminococcus gnavus bacteria.
- the Ruminococcus gnavus bacteria 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 Ruminococcus gnavus bacteria deposited as ATCC designation number PTA- 126695.
- the Ruminococcus gnavus bacteria are from a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Ruminococcus gnavus bacteria deposited as ATCC designation number PTA- 126695.
- the Ruminococcus gnavus bacteria are from Ruminococcus gnavus bacteria deposited as ATCC designation number PTA-126695.
- the mEVs are from Megasphaera sp. bacteria.
- the Megasphaera sp. bacteria 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 Megasphaera sp. bacteria deposited as ATCC designation number PTA-126770.
- the Megasphaera sp. bacteria are from a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Megasphaera sp. bacteria deposited as ATCC designation number PTA-126770.
- the Megasphaera sp. bacteria are from Megasphaera sp. bacteria deposited as ATCC designation number PTA-126770.
- the mEVs are from Fournier ella massiliensis bacteria.
- the Fournierella massiliensis bacteria 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 bacteria deposited as ATCC designation number PTA-126696.
- the Fournierella massiliensis bacteria are from a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Fournierella massiliensis bacteria deposited as ATCC designation number PTA-126696.
- the Fournierella massiliensis bacteria are from Fournierella massiliensis bacteria deposited as ATCC designation number PTA-126696.
- the mEVs are from Harryflintia acetispora bacteria.
- the Harryflintia acetispora bacteria 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 Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694.
- the Harryflintia acetispora bacteria are from a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694.
- the Harryflintia acetispora bacteria are from Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694.
- the mEVs are from bacteria of the family Acidaminococcaceae, Alcaligenaceae, Akkermansiaceae, Bacteriodaceae, Bifidobacteriaceae, Burkholderiaceae, Catabacteriaceae, Clostridiaceae, Coriobacteriaceae, Enterobacteriaceae, Enterococcaceae, Fusobacteriaceae, Lachnospiraceae, Listeraceae, Mycobacteriaceae, Neisseriaceae, Odoribacteraceae, Oscillospiraceae, Peptococcaceae, Peptostreptococcaceae, Porphyromonadaceae, Prevotellaceae, Propionibacteraceae, Rikenellaceae, Ruminococcaceae, Selenomonadaceae, Sporomusaceae, Streptococcaceae, Streptomycetaceae, Sutterella
- the mEVs are from bacteria of the genus Akkermansia. Christensenella, Blautia, Enterococcus, Eubacterium, Roseburia, Bacteroides, Parabacter aides, or Erysipelatoclostridium.
- the mEVs are from Blautia hydrogenotrophica, Blautia slercoris, Blautia wexlerae, Eubacterium faecium, Eubacterium cantor turn, Eubacterium rectale, Enterococcus faecahs. Enterococcus durans. Enterococcus viUorum, Enterococcus gallinarum; Bifidobacterium lactis, Bifidobacterium bifidium, Bifidobacterium longum, Bifidobacterium animalis, or Bifidobacterium breve bacteria.
- the mEVs are from BCG (bacillus Calmette-Guerin), Parabacteroides, Blautia, Veillonella, Lactobacillus salivarius, Agathobaculum, Ruminococcus gnavus, Paraclostridium benzoelyticum, Turicibacter sanguinus, Burkholderia, Klebsiella quasipneumoniae ssp similpneumoniae, Klebsiella oxyloca, Tyzzerela nexilis, or Neisseria bacteria.
- BCG Bacillus Calmette-Guerin
- Parabacteroides Bacillus Calmette-Guerin
- Blautia Veillonella
- Lactobacillus salivarius Agathobaculum
- Ruminococcus gnavus Paraclostridium benzoelyticum
- Turicibacter sanguinus Burkholderia
- Klebsiella quasipneumoniae ssp similpneumoniae Klebsiella oxyloc
- the mEVs are from Blautia hydrogenotrophica bacteria.
- the mEVs are from Blautia ster coris bacteria.
- the mEVs are from Blautia wexlerae bacteria.
- the mEVs are from Enterococcus gallinarum bacteria.
- the mEVs are from Enterococcus faecium bacteria.
- the mEVs are from Bifidobacterium bifidium bacteria.
- the mEVs are from Bifidobacterium breve bacteria.
- the mEVs are from Bifidobacterium longum bacteria.
- the mEVs are from Roseburia hominis bacteria.
- the mEVs are from Bacteroides thetaiotaomicron bacteria.
- the mEVs are from Bacteroides coprocola bacteria.
- the mEVs are from Erysipelatoclostridium ramosum bacteria.
- the mEVs are from Megasphera massiliensis bacteria.
- the mEVs are from Eubacterium bacteria.
- the mEVs are from Parabacteroides distasonis bacteria.
- the mEVs are from Lactobacillus plantarum bacteria.
- the mEVs are from bacteria of the Negativi cutes class.
- the mEVs are from bacteria of the Veillonellaceae family.
- the mEVs are from bacteria of the Selenomonadaceae family. [80] In some embodiments, the mEVs are from bacteria of the Acidaminococcaceae family.
- the mEVs are from bacteria of the Sporomusaceae family.
- the mEVs are from bacteria of the Megasphaera genus.
- the mEVs are from bacteria of the Selenomonas genus.
- the mEVs are from bacteria of the Propionospora genus.
- the mEVs are from bacteria of the Acidaminococcus genus.
- the mEVs are from Megasphaera sp. bacteria.
- the mEVs are from Selenomonas felix bacteria.
- the mEVs are from Acidaminococcus intestini bacteria.
- the mEVs are from Propionospora sp. bacteria.
- the mEVs are from bacteria of the Clostridia class.
- the mEVs are from bacteria of the Oscillospriraceae family.
- the mEVs are from bacteria of the Faecalibacterium genus.
- the mEVs are from bacteria of the Fournierella genus.
- the mEVs are from bacteria of the Harryflintia genus.
- the mEVs are from bacteria of the Agathobaculum genus.
- the mEVs are from Faecalibacterium prausnitzii (e.g., Faecalibacterium prausnitzii Strain A) bacteria.
- the mEVs are from Fournierella massiliensis (e.g., Fournierella massiliensis Strain A) bacteria.
- the mEVs are from Harryflintia acetispora (e.g., Harryflintia acetispora Strain A) bacteria.
- the mEVs are from Agathobaculum sp. (e.g., Agathobaculum sp. Strain A) bacteria.
- the mEVs are from a strain of Agathobaculum sp.
- the. Agathobaculum sp. strain is a strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Agathobaculum sp.
- Strain A ATCC Deposit Number PTA-125892
- the Agathobaculum sp. strain is the Agathobaculum sp. Strain A (ATCC Deposit Number PTA- 125892).
- the mEVs are from bacteria of the class Bacteroidia [phylum Bacteroidota ⁇ . In some embodiments, the mEVs are from bacteria of order Bacteroidales. In some embodiments, the mEVs are from bacteria of the family Porphyromonoadaceae . In some embodiments, the mEVs are from bacteria of the family Prevotellaceae . In some embodiments, the mEVs are from bacteria of the class Bacteroidia wherein the cell envelope structure of the bacteria is diderm. In some embodiments, the mEVs are from bacteria of the class Bacteroidia that stain Gram negative. In some embodiments, the mEVs are from bacteria of the class Bacteroidia wherein the bacteria is di derm and the bacteria stain Gram negative.
- the mEVs are from bacteria of the class Clostridia [phylum Firmicutes , In some embodiments, the mEVs are from bacteria of the order Eubacteriales . In some embodiments, the mEVs are from bacteria of the family Oscillispiraceae . In some embodiments, the mEVs are from bacteria of the family Lachnospiraceae . In some embodiments, the mEVs are from bacteria of the family Peptostreptococcaceae . In some embodiments, the mEVs are from bacteria of the family Clostridiales family XIII/ Incertae sedis 41.
- the mEVs are from bacteria of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm. In some embodiments, the mEVs are from bacteria of the class Clostridia that stain Gram negative. In some embodiments, the mEVs are from bacteria of the class Clostridia that stain Gram positive. In some embodiments, the mEVs are from bacteria of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm and the bacteria stain Gram negative. In some embodiments, the mEVs are from bacteria of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm and the bacteria stain Gram positive.
- the mEVs are from bacteria of the class Negativicutes [phylum Firmicutes , In some embodiments, the mEVs are from bacteria of the order Veillonellales. In some embodiments, the mEVs are from bacteria of the family Veillonelloceae. In some embodiments, the mEVs are from bacteria of the order Selenomonadales. In some embodiments, the mEVs are from bacteria of the family Selenomonadaceae . In some embodiments, the mEVs are from bacteria of the family Sporomusaceae . In some embodiments, the mEVs are from bacteria of the class Negativicutes wherein the cell envelope structure of the bacteria is diderm.
- the mEVs are from bacteria of the class Negativicutes that stain Gram negative. In some embodiments, the mEVs are from bacteria of the class Negativicutes wherein the cell envelope structure of the bacteria is diderm and the bacteria stain Gram negative.
- the mEVs are from bacteria of the class Synergistia [phylum Synergistota ⁇ . In some embodiments, the mEVs are from bacteria of the order Synergistales . In some embodiments, the mEVs are from bacteria of the family Synergistaceae . In some embodiments, the mEVs are from bacteria of the class Synergistia wherein the cell envelope structure of the bacteria is diderm. In some embodiments, the mEVs are from bacteria of the class Synergistia that stain Gram negative. In some embodiments, the mEVs are from bacteria of the class Synergistia wherein the cell envelope structure of the bacteria is diderm and the bacteria stain Gram negative.
- the mEVs are from bacteria that produce metabolites, e.g., the bacteria produce butyrate, iosine, proprionate, or tryptophan metabolites.
- the mEVs are from bacteria that produce butyrate.
- the bacteria are from the genus Blautia; Christensella; Copracoccus; Eubacterium; Lachnosperacea; Megasphaera; or Roseburia.
- the mEVs are from bacteria that produce iosine.
- the bacteria are from the genus Bifidobacterium; Lactobacillus; or Olsenella.
- the mEVs are from bacteria that produce proprionate.
- the bacteria are from the genus Akkermansia; Bacteriodes; Dialister; Eubacterium; Megasphaera; Parabacteriodes; Prevotella; Ruminococcus; or Veillonella.
- the mEVs are from bacteria that produce tryptophan metabolites.
- the bacteria are from the genus Lactobacillus or Peptostreptococcus .
- the mEVs are from bacteria that produce inhibitors of histone deacetylase 3 (HDAC3).
- HDAC3 histone deacetylase 3
- the bacteria are from the species Bariatricus massiliensis, Faecalibacterium prausnitzii, Megasphaera massiliensis or Roseburia intestinalis.
- the mEVs are from bacteria of the genus Alloiococcus,'
- Exiguobacterium ' Faecalibacterium Geobacillus,' Methylobacterium,' Micrococcus,' Morganella,' Proteus,' Pseudomonas,' Rhizobium,' or Sphingomonas.
- the mEVs are from bacteria of the genus Cutibacterium.
- the mEVs are from bacteria of the species Cutibacterium avidum.
- the mEVs are from bacteria of the genus Lactobacillus.
- the mEVs are from bacteria of the species Lactobacillus gasseri.
- the mEVs are from bacteria of the genus Dysosmobacter .
- the mEVs are from bacteria of the species Dysosmobacter welbionis.
- the mEVs are from bacteria of the genus Leuconostoc.
- the mEVs are from bacteria of the genus Lactobacillus.
- the mEVs are from bacteria of the genus Akkermansia,
- the mEVs are from Leuconostoc holzapfelii bacteria.
- the mEVs are from Akkermansia muciniphikr, Cupriavidus metallidurans,' Faecalibacterium prausnitzii,' Lactobacillus casei,' Lactobacillus plantarum, ' Lactobacillus paracasei,' Lactobacillus plantarum, ' Lactobacillus rhamnosus,' Lactobacillus sakei,' or Streptococcus pyogenes bacteria.
- the mEVs are from Lactobacillus casei,' Lactobacillus plantarum, ' Lactobacillus paracasei,' Lactobacillus plantarum, ' Lactobacillus rhamnosus,' or Lactobacillus sakei bacteria.
- the mEVs are from Megasphaera sp. bacteria (e.g., from the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387).
- the mEVs are from Megasphaera massiliensis bacteria (e.g., from the strain with accession number NCIMB 42787, NCIMB 43388 or NCIMB 43389). [126] In some embodiments, the mEVs are from Megasphaera massiliensis bacteria (e.g., from the strain with accession number DSM 26228).
- the mEVs are from Bacillus amyloliquefaciens bacteria (e.g., from the strain with accession number NCIMB 43088, NCIMB 43087, or NCIMB 43086).
- the mEVs are from Parabacteroides distasonis bacteria (e.g., from the strain with accession number NCIMB 42382).
- the mEVs are from Megasphaera massiliensis bacteria (e.g., from the strain with accession number NCIMB 43388 or NCIMB 43389), or a derivative thereof. See, e.g., WO 2020/120714.
- the Megasphaera massiliensis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of Megasphaera massiliensis bacteria from the strain with accession number NCIMB 43388 or NCIMB 43389.
- the Megasphaera massiliensis bacteria is the strain with accession number NCIMB 43388 or NCIMB 43389.
- the mEVs are from Megasphaera massiliensis bacteria strain deposited under accession number NCIMB 42787, or a derivative thereof. See, e.g., WO 2018/229216.
- the Megasphaera massiliensis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of the Megasphaera massiliensis bacteria strain deposited under accession number NCIMB 42787.
- the Megasphaera massiliensis bacteria is the strain deposited under accession number NCIMB 42787.
- the mEVs are from Megasphaera spp. bacteria from the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387, or a derivative thereof. See, e.g., WO 2020/120714. In some embodiments, the Megasphaera sp.
- bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of the Megasphaera sp. from a strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387.
- the Megasphaera sp. bacteria is the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387.
- the mEVs are from Parabacteroides distasonis bacteria deposited under accession number NCIMB 42382, or a derivative thereof. See, e.g., WO 2018/229216.
- the Parabacteroides distasonis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of the Parabacteroides distasonis bacteria deposited under accession number NCIMB 42382.
- the Parabacteroides distasonis bacteria is the strain deposited under accession number NCIMB 42382.
- the mEVs are from Megasphaera massiliensis bacteria deposited under accession number DSM 26228, or a derivative thereof. See, e.g., WO 2018/229216.
- the Megasphaera massiliensis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of Megasphaera massiliensis bacteria deposited under accession number DSM 26228.
- the Megasphaera massiliensis bacteria is the strain deposited under accession number DSM 26228.
- the mEVs are from Bacillus amyloliquefaciens bacteria (e.g., from the strain with accession number NCIMB 43088, NCIMB 43087, or NCIMB 43086, or a derivative thereof. See, e.g., WO 2019/236806.
- the Bacillus amyloliquefaciens bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of Bacillus amyloliquefaciens bacteria from the strain with accession number NCIMB 43088, NCIMB 43087, or NCIMB 43086.
- 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 Bacillus amyloliquefaciens bacteria is the strain with accession number NCIMB 43088, NCIMB 43087, or NCIMB 43086. In some embodiments, the Bacillus amyloliquefaciens bacteria is the strain with accession number NCIMB 43088.
- the EVs obtained and/or derived from bacteria that have been selected based on certain desirable properties, such as reduced toxicity and adverse effects (for example, by removing or deleting lipopolysaccharide (LPS)), enhanced oral delivery (for example, by improving acid resistance, muco-adherence and/or penetration and/or resistance to bile acids, resistance to anti-bacterial peptides and/or antibody neutralization), target desired cell types (for example, M-cells, goblet cells, enterocytes, dendritic cells, macrophages), improved bioavailability systemically or in an appropriate niche (for example, mesenteric lymph nodes, Peyer’s patches, lamina intestinal, tumor draining lymph nodes, and/or blood), enhanced immunomodulatory and/or therapeutic effect (for example, either alone or in combination with another therapeutic agent), enhanced immune activation , and/or manufacturing attributes (for example, growth characteristics, yield, greater stability, improved freeze-thaw tolerance, shorter generation times).
- LPS lipopolysaccharide
- the EVs are from engineered bacteria that are modified to enhance certain desirable properties.
- the engineered bacteria are modified so that EVs produced therefrom will have reduced toxicity and adverse effects (for example, by removing or deleting lipopolysaccharide (LPS)), enhanced oral delivery (for example, by improving acid resistance, muco-adherence and/or penetration and/or resistance to bile acids, resistance to anti-microbial peptides and/or antibody neutralization), target desired cell types (for example, M-cells, goblet cells, enterocytes, dendritic cells, macrophages), improved bioavailability systemically or in an appropriate niche (for example, mesenteric lymph nodes, Peyer’s patches, lamina intestinal, tumor draining lymph nodes, and/or blood), enhanced immunomodulatory and/or therapeutic effect (for example, either alone or in combination with another therapeutic agent), enhanced immune activation, and/or improved manufacturing attributes (for example, growth characteristics, yield, greater stability, improved freeze-thaw tolerance
- LPS lipopolys
- Figure 1 is an exemplary graph showing a calibration curve generated according to exemplary methods disclosed herein.
- Figure 2 is an exemplary graph showing liposome calibration curve generated according to exemplary methods disclosed herein.
- Figure 3 is a flow chart for EV quantitation.
- compositions for detecting, quantifying and/or dosing mEVs in a composition (e.g, in a therapeutic agent (i.e., pharmaceutical agent, drug substance) or in a therapeutic composition (i.e., pharmaceutical composition, drug product)) using a lipophilic fluorescent dye.
- a composition e.g, in a therapeutic agent (i.e., pharmaceutical agent, drug substance) or in a therapeutic composition (i.e., pharmaceutical composition, drug product)
- the composition can be a solution or a dried form.
- a dried form can be, for example, a powder or lyophilate.
- the composition can comprise one or more excipients, such as a bulking agent and/or lyoprotectant.
- bulking agents include, but are not limited to: sucrose, mannitol, polyethylene glycol (PEG, such as PEG 6000), cyclodextrin, maltodextrin, and dextran (such as dextran 40k).
- a bulking agent can make dried forms (such as powders and/or lyophilates) easier to handle after drying.
- the bulking agent is or comprises mannitol.
- lyoprotectants include, but are not limited to: trehalose, sucrose, and lactose.
- thelyoprotectant is or comprises trehalose.
- a lyoprotectant can protect the mEVs during drying, such as freeze-drying or spray drying.
- the excipient functions to decrease drying cycle time. In some embodiments, the excipient functions to maintain therapeutic efficacy of the mEVs.
- Equivalent Total Particle Number is a method by which the quantification of mEV content is based on measurement of mEV lipid quantity by use of a fluorescent dye and comparison of measured fluorescence against a standard curve of an mEV reference standard.
- a key component of mEVs is a lipid bilayer.
- the source of lipid in a composition is associated with the mEVs.
- This method utilizes a fluorescent-labeled dye (such as FM4-64) that specifically interacts with the lipid bilayer of the mEVs.
- the resulting fluorescent signal intensity correlates to the lipid content of mEVs and can be quantified by spectroscopy.
- a representative mEV reference standard with known correlation between the lipid content (based on fluorescence signal intensity) and mEV particle numbers is used to generate a standard curve using the same fluorescent lipid assay.
- a sample fluorescence signal representing lipid content
- a calibration curve e.g., that provides a correlation between flourescense signal and particle number
- a method of determining the lipid content of a composition comprising mEVs can include the steps of: (a) contacting a test sample from a composition comprising the mEVs with a lipophilic fluorescent dye (such as FM4-64) to obtain a mixture; (b) measuring a fluorescence signal from the mixture; and (c) determining the lipid content of the composition comprising the mEVs based on the fluorescence signal.
- a lipophilic fluorescent dye such as FM4-64
- the method can further include (d) determining the eTPN of the composition comprising the mEVs, e.g., by comparing the fluorescence sigal from the mixture to a calibration curve (e.g., that provides a correlation between flourescense signal and particle number).
- a calibration curve e.g., that provides a correlation between flourescense signal and particle number
- a method of determining a dose of a composition comprising mEVs can include the steps of: (a) contacting a test sample from a composition comprising the mEVs with a lipophilic fluorescent dye (such as FM4-64) to obtain a mixture; (b) measuring a fluorescence signal from the mixture; and (c) determining a dose of the composition comprising the mEVs based on the fluorescence signal.
- a lipophilic fluorescent dye such as FM4-64
- the method can further include (d) determining the eTPN of the composition comprising the mEVs, e.g., by comparing the fluorescence sigal from the mixture to a calibration curve (e.g., that provides a correlation between flourescense signal and particle number), wherein dose by eTPN.
- a calibration curve e.g., that provides a correlation between flourescense signal and particle number
- fluorescent dyes used in the methods include, but are not limited to, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64), a fixable analog of FM4-64 (FM4-64X (Thermo Fisher)), V-(3- Trimethylammoniumpropyl)“4 ⁇ (6-(4”(Diethylamino)phenyl)hexatrienyl)Pyridinium Dibromide (FM 5-95), a slightly less lipophilic analog of FM 4-64, and SynaptoRedTM C2.
- the fluorescent dye used in the methods is N-(3- triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64).
- administering broadly refers to a route of administration of a composition (for example, a pharmaceutical composition) to a subject.
- routes of administration include oral administration, rectal administration, topical administration, inhalation (nasal) or injection.
- Administration by injection includes intravenous (IV), intramuscular (IM), intratumoral (IT) and subcutaneous (SC) administration.
- a therapeutic composition described herein is administered in any form by any effective route, including but not limited to intratumoral, oral, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal (for example, using any standard patch), intradermal, ophthalmic, (intra)nasally, local, non-oral, such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, (trans)rectal, vaginal, intra-arterial, and intrathecal, transmucosal (for example, sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (for example, trans- and perivaginally), implanted, intravesical, intrapulmonary, intraduodenal, intragastrical, and intrabronchial.
- any effective route including but not limited to intratumoral, oral, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal (for example, using any standard patch), intradermal, ophthalmic
- a therapeutic composition described herein is administered orally, rectally, intratumorally, topically, intravesically, by injection into or adjacent to a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously.
- a therapeutic composition described herein is administered orally, intratumorally, or intravenously.
- a therapeutic composition described herein is administered orally.
- a “carbohydrate” refers to a sugar or polymer of sugars.
- saccharide a sugar or polymer of sugars.
- oligosaccharide a sugar or polymer of sugars.
- 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 CnEbnOn.
- a carbohydrate may be a monosaccharide, a disaccharide, tri saccharide, oligosaccharide, or polysaccharide.
- the most basic carbohydrate is a monosaccharide, such as glucose, galactose, mannose, ribose, arabinose, xylose, and fructose.
- Disaccharides are two joined monosaccharides. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose. Typically, an oligosaccharide includes between three and six monosaccharide units (for example, raffinose, stachyose), and polysaccharides include six or more monosaccharide units. Exemplary polysaccharides include starch, glycogen, and cellulose.
- Carbohydrates may contain modified saccharide units such as 2’-deoxyribose wherein a hydroxyl group is removed, 2’ -fluororibose wherein a hydroxyl group is replaced with a fluorine, or N-acetylglucosamine, a nitrogen-containing form of glucose (for example, 2’-fluororibose, deoxyribose, and hexose).
- Carbohydrates may exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.
- 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 mEVs from one source strain with another agent, for example, another mEV (for example, from another strain), with bacteria (for example, of the same or different strain that the mEV was obtained and/or derived 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 coadministration or co-localization of the mEVs 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.
- a “dried form” that contains microbial extracellular vesicles (mEVs) refers to the product resulting from drying a solution that contains mEVs.
- the drying is performed by freeze drying (lyophilization) or spray drying.
- the dried form is a powder.
- a powder refers to a type of dried form and includes a lyophilized powder, but includes powders, such as spray-dried powders, obtained and/or derived by methods such as spray drying.
- freeze drying lyophilization
- 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.
- 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.
- the term “gene” is used broadly to refer to any nucleic acid associated with a biological function.
- the term “gene” applies to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence.
- “Identity” as between nucleic acid sequences of two nucleic acid molecules can be determined as a percentage of identity using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., et al., J Molec Biol 215:403 (1990); Guide to Huge Computers, Martin J.
- isolated or “enriched” encompasses a microbe (such as a bacterium), an mEV (such as an smEV and/or pmEV) or other entity or substance that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by the hand of man.
- Isolated microbes or mEVs may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated.
- isolated microbes or mEVs are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
- a substance is “pure” if it is substantially free of other components.
- the terms “purify,” “purifying” and “purified” refer to a microbe or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production.
- a microbe or a microbial population or mEVs may be considered purified if it is isolated at or after production, such as from a material or environment containing the microbe or microbial population, and a purified microbe or microbial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “isolated.”
- purified microbes or microbial population or mEVs are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
- the one or more microbial types present in the composition can be independently purified from one or more other microbes produced and/or present in the material or environment containing the microbial type.
- Microbial compositions and the microbial components thereof are generally purified from residual habitat products.
- 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).
- Microbial extracellular vesicles can be obtained and/or derived from microbes such as bacteria, archaea, fungi, microscopic algae, protozoans, and parasites. In some embodiments, the mEVs are obtained and/or derived from bacteria. mEVs include secreted microbial extracellular vesicles (smEVs) and processed microbial extracellular vesicles (pmEVs). “Secreted microbial extracellular vesicles” (smEVs) are naturally- produced vesicles derived from microbes.
- smEVs are comprised of microbial lipids and/or microbial proteins and/or microbial nucleic acids and/or microbial carbohydrate moieties, and are isolated from culture supernatant.
- the natural production of these vesicles can be artificially enhanced (e.g., increased) or decreased through manipulation of the environment in which the bacterial cells are being cultured (e.g., by media or temperature alterations).
- smEV compositions may be modified to reduce, increase, add, or remove microbial components or foreign substances to alter efficacy, immune stimulation, stability, immune stimulatory capacity, stability, organ targeting (e.g., lymph node), absorption (e.g., gastrointestinal), and/or yield (e.g., thereby altering the efficacy).
- purified smEV composition or “smEV composition” refers to a preparation of smEVs that have been separated from at least one associated substance found in a source material (e.g., separated from at least one other microbial component) or any material associated with the smEVs in any process used to produce the preparation.
- microbial extracellular vesicles are a non-naturally-occurring collection of microbial membrane components that have been purified from artificially lysed microbes (e.g., bacteria) (e.g., microbial membrane components that have been separated from other, intracellular microbial cell components), and which may comprise particles of a varied or a selected size range, depending on the method of purification.
- artificially lysed microbes e.g., bacteria
- microbial membrane components e.g., microbial membrane components that have been separated from other, intracellular microbial cell components
- a pool of pmEVs is obtained and/or derived by chemically disrupting (e.g., by lysozyme and/or lysostaphin) and/or physically disrupting (e.g., by mechanical force) microbial cells and separating the microbial membrane components from the intracellular components through centrifugation and/or ultracentrifugation, or other methods.
- chemically disrupting e.g., by lysozyme and/or lysostaphin
- physically disrupting e.g., by mechanical force
- the resulting pmEV mixture contains an enrichment of the microbial membranes and the components thereof (e.g., peripherally associated or integral membrane proteins, lipids, glycans, polysaccharides, carbohydrates, other polymers), such that there is an increased concentration of microbial membrane components, and a decreased concentration (e.g., dilution) of intracellular contents, relative to whole microbes.
- the microbial membranes and the components thereof e.g., peripherally associated or integral membrane proteins, lipids, glycans, polysaccharides, carbohydrates, other polymers
- concentration e.g., dilution
- pmEVs may be modified to increase purity, to adjust the size of particles in the composition, and/or modified to reduce, increase, add or remove, microbial components or foreign substances to alter efficacy, immune stimulation, stability, immune stimulatory capacity, stability, organ targeting (e.g., lymph node), absorption (e.g., gastrointestinal), and/or yield (e.g., thereby altering the efficacy).
- pmEVs can be modified by adding, removing, enriching for, or diluting specific components, including intracellular components from the same or other microbes.
- the term “purified pmEV composition” or “pmEV composition” refers to a preparation of pmEVs that have been separated from at least one associated substance found in a source material (e.g., separated from at least one other microbial component) or any material associated with the pmEVs in any process used to produce the preparation. It can also refer to a composition that has been significantly enriched for specific components.
- Extracellular vesicles smEVs and/or pmEVs
- the methods provided herein may be used with mammalian cells or extracellular vesicles obtained and/or derived from mammalian cells.
- Extracellular vesicles may also be obtained and/or derived from a microbe, e.g., a microbe that is an archaea, bacteria, fungi, microscopic algae, protozoan, or parasite.
- the methods provided herein may be used with microbial cells or extracellular vesicles obtained and/or derived from microbial cells, e.g., where the microbial cells are cells of an archaea, bacteria, fungi, microscopic algae, protozoan, or parasite.
- the methods provided herein may be used with mammalian cells or extracellular vesicles obtained and/or derived from bacteria.
- “Modified” in reference to a bacteria broadly refers to a bacteria that has undergone a change from its wild-type form.
- Bacterial modification can result from engineering bacteria. Examples of bacterial modifications include genetic modification, gene expression modification, phenotype modification, formulation modification, chemical modification, and dose or concentration. Examples of improved properties are described throughout this specification and include, for example, attenuation, auxotrophy, homing, or antigenicity.
- Phenotype modification might include, by way of example, bacteria growth in media that modify the phenotype of a bacterium such that it increases or decreases virulence.
- Oncotrophic or “oncophilic” microbes and bacteria are microbes that are highly associated or present in a cancer microenvironment. They may be preferentially selected for within the environment, preferentially grow in a cancer microenvironment or hone to a said environment.
- “Operational taxonomic units” and “OTU(s)” refer to a terminal leaf in a phylogenetic tree and is defined by a nucleic acid sequence, for example, the entire genome, or a specific genetic sequence, and all sequences that share sequence identity to this nucleic acid sequence at the level of species.
- the specific genetic sequence may be the 16S sequence or a portion of the 16S sequence.
- the entire genomes of two entities are sequenced and compared.
- select regions such as multilocus sequence tags (MLST), specific genes, or sets of genes may be genetically compared.
- OTUs that share > 97% average nucleotide identity across the entire 16S or some variable region of the 16S are considered the same OTU. See for example, Claesson MJ, Wang Q, O’Sullivan O, Greene-Diniz R, Cole JR, Ross RP, and O’Toole PW. 2010. Comparison of two 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.
- MLSTs 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 for example, 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 Lond B Biol Sci 361 : 1929-1940. OTUs are frequently defined by comparing sequences between organisms.
- OTUs may also be characterized by any combination of nucleotide markers or genes, in particular highly conserved genes (for example, “house-keeping” genes), or a combination thereof.
- Operational Taxonomic Units (OTUs) with taxonomic assignments made to, for example, genus, species, and phylogenetic clade are provided herein.
- the term “pharmaceutical agent” is used interchangeably with “therapeutic agent” and “drug substance” and refers to an agent for therapeutic use.
- a pharmaceutical agent is a composition comprising mEVs that can be used to treat and/or prevent a disease and/or condition.
- a medicinal product, medical food, a food product, or a dietary supplement comprises a pharmaceutical agent.
- the pharmaceutical agent is a powder that contains the bacteria and/or mEVs. The powder can include one or more additional components in addition to the bacteria and/or mEVs, such as a cryoprotectant.
- 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.
- preventing refers to administering to the subject to a pharmaceutical treatment, e.g., the administration of one or more agents (e.g., pharmaceutical agent), such that onset of at least one symptom of the disease or condition is delayed or prevented.
- a pharmaceutical treatment e.g., the administration of one or more agents (e.g., pharmaceutical agent)
- agents e.g., pharmaceutical agent
- a substance is “pure” if it is substantially free of other components.
- the terms “purify,” “purifying” and “purified” refer to an EV (such as an EV from bacteria) preparation or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (for example, whether in nature or in an experimental setting), or during any time after its initial production.
- An EV preparation or compositions may be considered purified if it is isolated at or after production, such as from one or more other bacterial components, and a purified microbe or bacterial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “purified.”
- purified EVs are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
- EV compositions (or preparations) are, for example, purified from residual habitat products.
- the term “purified mEV composition” or “mEV composition” refers to a preparation that includes mEVs (such as smEVs and/or pmEVs) that have been separated from at least one associated substance found in a source material (e.g., separated from at least one other bacterial component) or any material associated with the mEVs (such as smEVs and/or pmEVs) in any process used to produce the preparation. It also refers to a composition that has been significantly enriched or concentrated. In some embodiments, the mEVs (such as smEVs and/or pmEVs) are concentrated by 2 fold, 3-fold, 4-fold, 5-fold, 10- fold, 100-fold, 1000-fold, 10,000-fold or more than 10,000 fold.
- 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.
- strains may be differentiated by selection or counter-selection using an antibiotic or nutrient/metabolite, respectively.
- subject refers to any mammal.
- a subject or a patient described as “in need thereof’ refers to one in need of a treatment (or prevention) for a disease.
- Mammals i.e., mammalian animals
- mammals include humans, laboratory animals (for example, primates, rats, mice), livestock (for example, cows, sheep, goats, pigs), and household pets (for example, dogs, cats, rodents).
- the subject may be a human.
- the subject may be a non-human mammal including but not limited to of a dog, a cat, a cow, a horse, a pig, a donkey, a goat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, a gorilla or a chimpanzee.
- the subject may be healthy, or may be suffering from a cancer at any developmental stage, wherein any of the stages are either caused by or opportunistically supported of a cancer associated or causative pathogen, or may be at risk of developing a cancer, or transmitting to others a cancer associated or cancer causative pathogen.
- a subject has lung cancer, bladder cancer, prostate cancer, plasmacytoma, colorectal cancer, rectal cancer, Merkel Cell carcinoma, salivary gland carcinoma, ovarian cancer, and/or melanoma.
- the subject may have a tumor.
- the subject may have a tumor that shows enhanced macropinocytosis with the underlying genomics of this process including Ras activation.
- the subject has another cancer.
- the subject has undergone a cancer therapy.
- a therapeutic agent refers to an agent for therapeutic use.
- a therapeutic agent is a composition comprising mEVs (“an mEV 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 lyophilized cake or powder.
- the dried form of the therapeutic agent is a spray-dried powder.
- the term “therapeutic composition” or “pharmaceutical composition” or “drug product” refers to a composition that comprises a therapeutically effective amount of a therapeutic agent (for example an mEV composition described herein).
- the therapeutic composition is (or is present in) a medicinal product, medical food, a food product, or a dietary supplement.
- mEVs Microbial Extracellular Vesicles
- compositions comprising microbial extracellular vesicles (mEVs).
- compositions comprising microbial extracellular vesicles (mEVs) and bacteria (bacterial cells).
- the mEVs are from one strain of bacteria. In some embodiments, the mEVs and bacteria from the same strain of bacteria.
- the bacteria from which the mEVs are obtained and/or derived are modified to reduce toxicity or other adverse effects, to enhance delivery) (e.g., oral delivery) (e.g., by improving acid resistance, muco-adherence and/or penetration and/or resistance to bile acids, digestive enzymes, resistance to anti-microbial peptides and/or antibody neutralization), to target desired cell types (e.g., M-cells, goblet cells, enterocytes, dendritic cells, macrophages), to enhance their immunomodulatory and/or therapeutic effect of the bacteria and/or mEVs (e.g., either alone or in combination with another pharmaceutical agent), and/or to enhance immune activation or suppression by the bacteria and/or mEVs (such as smEVs and/or pmEVs) (e.g., through modified production of polysaccharides, pili, fimbriae, adhesins).
- delivery e.g., oral delivery
- target desired cell types e.g., M
- the engineered bacteria described herein are modified to improve bacteria and/or mEV (such as smEV and/or pmEV) manufacturing (e.g., higher oxygen tolerance, stability, improved freeze-thaw tolerance, shorter generation times).
- the engineered bacteria described include bacteria harboring one or more genetic changes, such change being an insertion, deletion, translocation, or substitution, or any combination thereof, of one or more nucleotides contained on the bacterial chromosome or endogenous plasmid and/or one or more foreign plasmids, wherein the genetic change may result in the overexpression and/or underexpression of one or more genes.
- the engineered bacteria may be produced using any technique known in the art, including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, or any combination thereof.
- taxonomic groups e.g., class, order, family, genus, species or strain
- mEVs such as smEVs and/or pmEVs
- Table J examples of taxonomic groups (e.g., class, order, family, genus, species or strain) of bacteria that can be used as a source of mEVs (such as smEVs and/or pmEVs) for a composition described herein are provided herein (e.g., listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification (e.g., Table J)).
- the bacterial strain is a bacterial strain having a genome that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to a strain listed herein.
- the bacteria from which the mEVs are obtained and/or derived are oncotrophic bacteria.
- the bacteria from which the mEVs are obtained and/or derived are immunomodulatory bacteria.
- the bacteria from which the mEVs are obtained and/or derived are immunostimulatory bacteria.
- the bacteria from which the mEVs are obtained and/or derived are immunosuppressive bacteria. In some embodiments, the bacteria from which the mEVs are obtained and/or derived are immunomodulatory bacteria. In certain embodiments, the bacteria from which the mEVs are obtained and/or derived are generated from a combination of bacterial strains provided herein. In some embodiments, the combination is a combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 bacterial strains.
- the combination includes the bacteria from which the mEVs are obtained and/or derived are from bacterial strains listed herein and/or bacterial strains having a genome that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to a strain listed herein (e.g., listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification (e.g., Table J)).
- a strain listed herein e.g., listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification (e.g., Table J)).
- the bacteria from which the mEVs are obtained and/or derived are generated from a bacterial strain provided herein.
- the bacteria from which the mEVs are obtained and/or derived are from a bacterial strain listed herein (e.g., listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification (e.g., Table J))and/or a bacterial strain having a genome that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to a strain listed herein (e.g., listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification (e.g., Table J)).
- the bacteria from which the mEVs are obtained and/or derived are Gram negative bacteria.
- the Gram negative bacteria belong to the class Negativicutes.
- the Negativicutes represent a unique class of microorganisms as they are the only diderm members of the Firmicutes phylum. These anaerobic organisms can be found in the environment and are normal commensals of the oral cavity and GI tract of humans. Because these organisms have an outer membrane, the yields of EVs from this class were investigated. It was found that on a per cell basis these bacteria produce a high number of vesicles (10-150 EVs/cell). The EVs from these organisms are broadly stimulatory and highly potent in in vitro assays. Investigations into their therapeutic applications in several oncology and inflammation in vivo models have shown their therapeutic potential.
- the Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidaminococcaceae. and Sporomusaceae .
- the Negativicutes class includes the genera Megasphaera, Selenomonas, Propionospora, and Acidaminococcus.
- Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestine, and Propionospora sp.
- the bacteria from which the mEVs are obtained and/or derived are Gram positive bacteria.
- the bacteria from which the mEVs are obtained and/or derived are aerobic bacteria.
- the bacteria from which the mEVs are obtained and/or derived are anaerobic bacteria.
- the anaerobic bacteria comprise obligate anaerobes.
- the anaerobic bacteria comprise facultative anaerobes.
- the bacteria from which the mEVs are obtained and/or derived are acidophile bacteria.
- the bacteria from which the mEVs are obtained and/or derived are alkaliphile bacteria.
- the bacteria from which the mEVs are obtained and/or derived are neutralophile bacteria.
- the bacteria from which the mEVs are obtained and/or derived are fastidious bacteria.
- the bacteria from which the mEVs are obtained and/or derived are nonfastidious bacteria.
- the bacteria from which the mEVs are obtained and/or derived or the mEVs themselves are lyophilized.
- the bacteria from which the mEVs are obtained and/or derived or the mEVs themselves are gamma irradiated (e.g., at 17.5 or 25 kGy).
- the bacteria from which the mEVs are obtained and/or derived or the mEVs themselves are UV irradiated.
- the bacteria from which the mEVs are obtained and/or derived or the mEVs themselves are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).
- the bacteria from which the mEVs are obtained and/or derived or the mEVs themselves are acid treated.
- the bacteria from which the mEVs are obtained and/or derived or the mEVs themselves 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 mEVs produced by bacteria.
- mEVs can be isolated, e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
- the bacteria from which the mEVs are obtained and/or derived from obligate anaerobic bacteria examples include gram-negative rods (including the genera of Bacleroides, Prevotella, Porphyromonas, Fusobacterium, Bilophila and Sutter ella spp. gram-positive cocci (primarily Peptostreptococcus spp. gram-positive spore-forming (Clostridium spp. nonspore-forming bacilli (Actinomyces, Propionibacterium, Eubacterium, Lactobacillus and Bifidobacterium spp. and gram-negative cocci (mainly Veillonella spp.).
- gram-negative rods including the genera of Bacleroides, Prevotella, Porphyromonas, Fusobacterium, Bilophila and Sutter ella spp.
- gram-positive cocci primarily Peptostreptococcus spp. gram-positive spore-forming (Clos
- the obligate anaerobic bacteria are of a genus selected from the group consisting of Agathobaculum, Atopobium, Blautia, Burkholderia, Dielma, Longicatena, Paraclostridium, Turicibacter, and Tyzzerella.
- the Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidaminococcaceae, and Sporomusaceae .
- the Negativicutes class includes the genera Megasphaera, Selenomonas, Propionospora, and Acidaminococcus.
- Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestini, and Propionospora sp.
- the bacteria from which the mEVs are obtained and/or derived are of the Negativicutes class.
- the bacteria from which the mEVs are obtained and/or derived are of the Veillonellaceae family.
- the bacteria from which the mEVs are obtained and/or derived are of the Selenomonadaceae family.
- the bacteria from which the mEVs are obtained and/or derived are of the Acidaminococcaceae family.
- the bacteria from which the mEVs are obtained and/or derived are of the Sporomusaceae family.
- the bacteria from which the mEVs are obtained and/or derived are of the Megasphaera genus.
- the bacteria from which the mEVs are obtained and/or derived are of the Selenomonas genus.
- the bacteria from which the mEVs are obtained and/or derived are of the Propionospora genus.
- the bacteria from which the mEVs are obtained and/or derived are of the Acidaminococcus genus.
- the bacteria from which the mEVs are obtained and/or derived are Megasphaera sp. bacteria.
- the bacteria from which the mEVs are obtained and/or derived are Selenomonas felix bacteria.
- the bacteria from which the mEVs are obtained and/or derived are Acidaminococcus intestini bacteria.
- the bacteria from which the mEVs are obtained and/or derived are Propionospora sp. bacteria.
- the bacteria from which the mEVs are obtained and/or derived are of the Clostridia class.
- the bacteria from which the mEVs are obtained and/or derived are of the Oscillospriraceae family.
- the bacteria from which the mEVs are obtained and/or derived are of the Faecalibacterium genus.
- the bacteria from which the mEVs are obtained and/or derived are of the Fournierella genus.
- the bacteria from which the mEVs are obtained and/or derived are of the Harryflintia genus.
- the bacteria from which the mEVs are obtained and/or derived are of the Agathobaculum genus.
- the bacteria from which the mEVs are obtained and/or derived are Faecalibacterium prausnitzii (e.g., Faecalibacterium prausnitzii Strain A) bacteria.
- the bacteria from which the mEVs are obtained and/or derived are Fournierella massiliensis (e.g., Fournierella massiliensis Strain A) bacteria.
- the bacteria from which the mEVs are obtained and/or derived are Harryflintia acetispora (e.g., Harryflintia acetispora Strain A) bacteria.
- the bacteria from which the mEVs are obtained and/or derived are Agathobaculum sp. (e.g., Agathobaculum sp. Strain A) bacteria.
- the bacteria from which the mEVs are obtained and/or derived are bacteria of a genus selected from the group consisting of Escherichia, Klebsiella, Lactobacillus, Shigella, and Staphylococcus.
- the bacteria from which the mEVs are obtained and/or derived are a species selected from the group consisting of Blautia massiliensis, Paraclostridium benzoelyticum, Dielma fastidiosa, Longicatena caecimuris, Lactococcus lactis cremoris, Tyzzerella nexilis, Hungatella effluvia, Klebsiella quasipneumoniae subsp. Similipneumoniae, Klebsiella oxytoca, and Veillonella tobetsuensis.
- the bacteria from which the mEVs are obtained and/or derived are a Prevotella bacteria selected from the group consisting of Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea,
- the bacteria from which the mEVs are obtained and/or derived are a strain of bacteria comprising a genomic sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the genomic sequence of the strain of bacteria deposited with the ATCC Deposit number as provided in Table 3.
- 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 bacteria from which the mEVs are obtained and/or derived are a strain of bacteria comprising a 16S sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the 16S sequence of the strain of bacteria deposited with the ATCC Deposit number as provided in Table 3.
- 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 Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidaminococcaceae, and Sporomusaceae .
- the Negativicutes class includes the genera Megasphaera, Selenomonas, Propionospora, and Acidaminococcus.
- Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestini, and Propionospora sp.
- the bacteria from which the mEVs are obtained and/or derived are of the Negativicutes class.
- the bacteria from which the mEVs are obtained and/or derived are of the Veillonellaceae family.
- the bacteria from which the mEVs are obtained and/or derived are of the Selenomonadaceae family.
- the bacteria from which the mEVs are obtained and/or derived are of the Acidaminococcaceae family.
- the bacteria from which the mEVs are obtained and/or derived are of the Sporomusaceae family.
- the bacteria from which the mEVs are obtained and/or derived are of the Megasphaera genus.
- the bacteria from which the mEVs are obtained and/or derived are of the Selenomonas genus.
- the bacteria from which the mEVs are obtained and/or derived are of the Propionospora genus.
- the bacteria from which the mEVs are obtained and/or derived are of the Acidaminococcus genus.
- the bacteria from which the mEVs are obtained and/or derived are Megasphaera sp. bacteria.
- the bacteria from which the mEVs are obtained and/or derived are Selenomonas felix bacteria.
- the bacteria from which the mEVs are obtained and/or derived are Acidaminococcus intestini bacteria.
- the bacteria from which the mEVs are obtained and/or derived are Propionospora sp. bacteria.
- the Oscillospriraceae family within the Clostridia class of microorganisms are common commensal organisms of vertebrates.
- the bacteria from which the mEVs are obtained and/or derived are of the Clostridia class.
- the bacteria from which the mEVs are obtained and/or derived are of the Oscillospriraceae family.
- the bacteria from which the mEVs are obtained and/or derived are of the Faecalibacterium genus.
- the bacteria from which the mEVs are obtained and/or derived are of the Fournierella genus.
- the bacteria from which the mEVs are obtained and/or derived are of the Harryflintia genus.
- the bacteria from which the mEVs are obtained and/or derived are of the Agathobaculum genus.
- the bacteria from which the mEVs are obtained and/or derived are Faecalibacterium prausnitzii (e.g., Faecalibacterium prausnitzii Strain A) bacteria.
- the bacteria from which the mEVs are obtained and/or derived are Fournierella massiliensis (e.g., Fournierella massiliensis Strain A) bacteria.
- the bacteria from which the mEVs are obtained and/or derived are Harryflintia acetispora (e.g., Harryflintia acetispora Strain A) bacteria.
- the bacteria from which the mEVs are obtained and/or derived are Agathobaculum sp. (e.g., Agathobaculum sp. Strain A) bacteria.
- the bacteria from which the mEVs are obtained and/or derived are a strain of Agathobaculum sp.
- the Agathobaculum sp. strain is a strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Agathobaculum sp.
- Strain A ATCC Deposit Number PTA- 125892
- the Agathobaculum sp. strain is the Agathobaculum sp. Strain A (ATCC Deposit Number PTA- 125892).
- the bacteria from which the mEVs are obtained and/or derived are of the class Bacteroidia [phylum Bacteroidota ⁇ . In some embodiments, the bacteria from which the mEVs are obtained and/or derived are bacteria of order Bacteroidales. In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the family Porphyromonoadaceae . In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the family Prevotellaceae . In some embodiments, the bacteria from which the mEVs are obtained and/or derived are bacteria of the class Bacteroidia wherein the cell envelope structure of the bacteria is diderm.
- the bacteria from which the mEVs are obtained and/or derived are bacteria of the class Bacteroidia that stain Gram negative. In some embodiments, the bacteria from which the mEVs are obtained and/or derived are bacteria of the class Bacteroidia wherein the bacteria is diderm and the bacteria stain Gram negative.
- the bacteria from which the mEVs are obtained and/or derived are bacteria of the class Clostridia [phylum Firmicutes , In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the order Eubacteriales. In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the family Oscillispiraceae . In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the family Lachnospiraceae . In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the family Peptostreptococcaceae .
- the bacteria from which the mEVs are obtained and/or derived are of the family Clostridiales family XIII/ Incertae sedis 41. In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm. In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the class Clostridia that stain Gram negative. In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the class Clostridia that stain Gram positive.
- the bacteria from which the mEVs are obtained and/or derived are of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm and the bacteria stain Gram negative. In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm and the bacteria stain Gram positive.
- the bacteria from which the mEVs are obtained and/or derived are of the class Negativicutes [phylum Firmicutes , In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the order Veillonellales. In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the family Veillonelloceae. In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the order Selenomonadales . In some embodiments, the bacteria from which the mEVs are obtained and/or derived are bacteria of the family Selenomonadaceae .
- the bacteria from which the mEVs are obtained and/or derived are of the family Sporomusaceae . In some embodiments, t the bacteria from which the mEVs are obtained and/or derived are of the class Negativicutes wherein the cell envelope structure of the bacteria is diderm. In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the bacteria from which the mEVs are obtained and/or derived are the EVs are from bacteria of the class Negativicutes wherein the cell envelope structure of the bacteria is diderm and the bacteria stain Gram negative.
- the bacteria from which the mEVs are obtained and/or derived are of the class Synergistia [phylum Synergistota ⁇ . In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the order Synergistales. In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the family Synergistaceae . In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the class Synergistia wherein the cell envelope structure of the bacteria is diderm.
- the bacteria from which the mEVs are obtained and/or derived are of the class Synergistia that stain Gram negative. In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the class Synergistia wherein the cell envelope structure of the bacteria is diderm and the bacteria stain Gram negative.
- the bacteria from which the mEVs are obtained and/or derived are from one strain of bacteria, e.g., a strain provided herein.
- the bacteria from which the mEVs are obtained and/or derived are from one strain of bacteria (e.g., a strain provided herein) or from more than one strain provided herein.
- the bacteria from which the mEVs are obtained and/or derived are Lactococcus lactis cremoris bacteria, e.g., a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Lactococcus lactis cremoris Strain A (ATCC designation number PTA-125368).
- the bacteria from which the mEVs are obtained and/or derived are Lactococcus bacteria, e.g., Lactococcus lactis cremoris Strain A (ATCC designation number PTA- 125368).
- the bacteria from which the mEVs are obtained and/or derived are Prevotella bacteria, e.g., a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
- the bacteria from which the mEVs are obtained and/or derived are Prevotella bacteria, e.g., Prevotella Strain B 50329 (NRRL accession number B 50329).
- the bacteria or the bacteria from which the mEVs are obtained and/or derived are Prevotella histicola bacteria, e.g., a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella histicola bacteria deposited as ATCC designation number PTA- 126140.
- the bacteria or the bacteria from which the mEVs are obtained and/or derived are Prevotella histicola bacteria, e.g., Prevotella histicola bacteria deposited as ATCC designation number PTA-126140.
- the bacteria from which the mEVs are obtained and/or derived are Bifidobacterium bacteria, e.g., a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Bifidobacterium bacteria deposited as ATCC designation number PTA-125097.
- the bacteria from which the mEVs are obtained and/or derived are Bifidobacterium bacteria, e.g., Bifidobacterium bacteria deposited as ATCC designation number PTA-125097.
- the bacteria from which the mEVs are obtained and/or derived are Veillonella bacteria, e.g., a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Veillonella bacteria deposited as ATCC designation number PTA-125691.
- the bacteria from which the mEVs are obtained and/or derived are Veillonella bacteria, e.g., Veillonella bacteria deposited as ATCC designation number PTA-125691.
- the bacteria from which the mEVs are obtained and/or derived are Ruminococcus gnavus bacteria.
- the Ruminococcus gnavus bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Ruminococcus gnavus bacteria deposited as ATCC designation number PTA-126695.
- the Ruminococcus gnavus bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Ruminococcus gnavus bacteria deposited as ATCC designation number PTA-126695.
- the Ruminococcus gnavus bacteria are Ruminococcus gnavus bacteria deposited as ATCC designation number PTA-126695.
- the bacteria from which the mEVs are obtained and/or derived are Megasphaera sp. bacteria.
- the Megasphaera sp. bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Megasphaera sp. bacteria deposited as ATCC designation number PTA-126770.
- the bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Megasphaera sp. bacteria deposited as ATCC designation number PTA-126770.
- the Megasphaera sp. bacteria are Megasphaera sp. bacteria deposited as ATCC designation number PTA-126770.
- the bacteria from which the mEVs are obtained and/or derived are Fournierella massiliensis bacteria.
- the Fournierella massiliensis bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Fournierella massiliensis bacteria deposited as ATCC designation number PTA-126696.
- the Fournierella massiliensis bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Fournierella massiliensis bacteria deposited as ATCC designation number PTA-126696.
- the Fournierella massiliensis bacteria are Fournierella massiliensis bacteria deposited as ATCC designation number PTA-126696.
- the bacteria from which the mEVs are obtained and/or derived are Harryflintia acetispora bacteria.
- the Harryflintia acetispora bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694.
- the Harryflintia acetispora bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694.
- the Harryflintia acetispora bacteria are Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694.
- the bacteria from which the mEVs are obtained and/or derived are bacteria that produce metabolites, e.g., the bacteria produce butyrate, iosine, proprionate, or tryptophan metabolites.
- the bacteria from which the mEVs are obtained and/or derived are bacteria that produce butyrate.
- the bacteria are from the genus Blautia; Christensella; Copracoccus; Eubacterium; Lachnosperacea; Megasphaera; or Roseburia.
- the bacteria from which the mEVs are obtained and/or derived are bacteria that produce iosine. In some embodiments, the bacteria are from the genus Bifidobacterium; Lactobacillus; or Olsenella.
- the bacteria from which the mEVs are obtained and/or derived are bacteria that produce proprionate.
- the bacteria are from the genus Akkermansia; Bacteriodes; Dialister; Eubacterium; Megasphaera; Parabacteriodes; Prevotella; Ruminococcus; or Veillonella.
- the bacteria from which the mEVs are obtained and/or derived are bacteria that produce tryptophan metabolites.
- the bacteria are from the genus Lactobacillus or Peptostreptococcus .
- the bacteria from which the mEVs are obtained and/or derived are bacteria that produce inhibitors of histone deacetylase 3 (HDAC3).
- the bacteria are from the species Bariatricus massiliensis, Faecalibacterium prausnitzii, Megasphaera massiliensis or Roseburia intestinalis.
- the bacteria are from the genus AHoiococcus Bacillus; Catenibacterium; Corynebacterium; Cupriavidus; Enhydrobacter; Exiguobacterium;
- the bacteria are from the genus Cutibacterium. In some embodiments, the bacteria are from the species Cutibacterium avidum. In some embodiments, the bacteria are from the genus Lactobacillus. In some embodiments, the bacteria are from the species Lactobacillus gasseri. In some embodiments, the bacteria are from the genus Dysosmobacter . In some embodiments, the bacteria are from the species Dysosmobacter welbionis.
- the bacteria from which the mEVs are obtained and/or derived are of the genus AHoiococcus; Bacillus; Catenibacterium; Corynebacterium;
- Methylobacterium Methylobacterium; Micrococcus; Morganella; Proteus; Pseudomonas; Rhizobium; or Sphingomonas.
- the bacteria from which the mEVs are obtained and/or derived are of the Cutibacterium genus. In some embodiments, the bacteria from which the mEVs are obtained and/or derived are Cutibacterium avidum bacteria. [272] In some embodiments, the bacteria from which the mEVs are obtained and/or derived are of the genus Leuconostoc.
- the bacteria from which the mEVs are obtained and/or derived are of the genus Lactobacillus.
- the bacteria from which the mEVs are obtained and/or derived are of the genus Akkermansia Bacillus,' Blaulia Cupriavidus; laihydrobacler: Faecahbaclerium: Lactobacillus,' Lactococcus,' Micrococcus,' Morganella;
- Propionibacterium Proteus,' Rhizobiunr, or Streptococcus.
- the bacteria from which the mEVs are obtained and/or derived are Leuconostoc holzapfelii bacteria.
- the bacteria from which the mEVs are obtained and/or derived are Akkermansia muciniphila; Cupriavidus metallidurans; Faecalibacterium prausnitzii; Lactobacillus casei; Lactobacillus plantarum; Lactobacillus paracasei;
- Lactobacillus plantarum Lactobacillus rhamnosus; Lactobacillus sakei; or Streptococcus pyogenes bacteria.
- the bacteria from which the mEVs are obtained and/or derived are Lactobacillus casei; Lactobacillus plantarum; Lactobacillus paracasei;
- Lactobacillus plantarum Lactobacillus rhamnosus; or Lactobacillus sakei bacteria.
- the bacteria from which the mEVs are obtained and/or derived are Megasphaera sp. bacteria (e.g., from the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387).
- the bacteria from which the mEVs are obtained and/or derived are Megasphaera massiliensis bacteria (e.g., from the strain with accession number NCIMB 42787, NCIMB 43388 or NCIMB 43389).
- the bacteria from which the mEVs are obtained and/or derived are Megasphaera massiliensis bacteria (e.g., from the strain with accession number DSM 26228).
- the bacteria from which the mEVs are obtained and/or derived are Bacillus amyloliquefaciens bacteria (e.g., from the strain with accession number NCIMB 43088, NCIMB 43087, or NCIMB 43086).
- the bacteria from which the mEVs are obtained and/or derived are Parabacteroides distasonis bacteria (e.g., from the strain with accession number NCIMB 42382).
- the bacteria from which the mEVs are obtained and/or derived are Megasphaera massiliensis bacteria (e.g., from the strain with accession number NCIMB 43388 or NCIMB 43389), or a derivative thereof See, e.g., WO 2020/120714.
- the Megasphaera massiliensis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of Megasphaera massiliensis bacteria from the strain with accession number NCIMB 43388 or NCIMB 43389.
- the Megasphaera massiliensis bacteria is the strain with accession number NCIMB 43388 or NCIMB 43389.
- the bacteria from which the mEVs are obtained and/or derived are Megasphaera massiliensis bacteria strain deposited under accession number NCIMB 42787, or a derivative thereof. See, e.g., WO 2018/229216.
- the Megasphaera massiliensis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of the Megasphaera massiliensis bacteria strain deposited under accession number NCIMB 42787.
- the Megasphaera massiliensis bacteria is the strain deposited under accession number NCIMB 42787.
- the bacteria from which the mEVs are obtained and/or derived are Megasphaera spp. bacteria from the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387, or a derivative thereof. See, e.g., WO 2020/120714. In some embodiments, the Megasphaera sp.
- bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of the Megasphaera sp. from a strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387. In some embodiments, the Megasphaera sp.
- 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
- bacteria is the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387.
- the bacteria from which the mEVs are obtained and/or derived are Parabacteroides distasonis bacteria deposited under accession number NCIMB 42382, or a derivative thereof. See, e.g., WO 2018/229216.
- the Parabacteroides distasonis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of the Parabacteroides distasonis bacteria deposited under accession number NCIMB 42382.
- the Parabacteroides distasonis bacteria is the strain deposited under accession number NCIMB 42382.
- the bacteria from which the mEVs are obtained and/or derived are Megasphaera massiliensis bacteria deposited under accession number DSM 26228, or a derivative thereof. See, e.g., WO 2018/229216.
- the Megasphaera massiliensis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of Megasphaera massiliensis bacteria deposited under accession number DSM 26228.
- the Megasphaera massiliensis bacteria is the strain deposited under accession number DSM 26228.
- the bacteria from which the mEVs are obtained and/or derived are Bacillus amyloliquefaciens bacteria (e.g., from the strain with accession number NCIMB 43088, NCIMB 43087, or NCIMB 43086, or a derivative thereof. See, e.g., WO 2019/236806.
- the Bacillus amyloliquefaciens bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, and/or CRISPR sequence) of Bacillus amyloliquefaciens bacteria from the strain with accession number NCIMB 43088, NCIMB 43087, or NCIMB 43086.
- 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 Bacillus amyloliquefaciens bacteria is the strain with accession number NCIMB 43088, NCIMB 43087, or NCIMB 43086. In some embodiments, the Bacillus amyloliquefaciens bacteria is the strain with accession number NCIMB 43088.
- the mEVs (such as smEVs and/or pmEVs) described herein are modified such that they comprise, are linked to, and/or are bound by a therapeutic moiety.
- the therapeutic moiety is a cancer-specific moiety.
- the cancer-specific moiety has binding specificity for a cancer cell (e.g., has binding specificity for a cancer-specific antigen).
- the cancerspecific 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 (e.g., 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 mEV (e.g., 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 pharmaceutical agent (either in combination or in separate administrations) increases the targeting of the pharmaceutical agent to the cancer cells.
- CAR chimeric antigen receptor
- the mEVs described herein can be modified such that they comprise, are linked to, and/or are bound by a magnetic and/or paramagnetic moiety (e.g., a magnetic bead).
- 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 mEV-binding moiety that binds to the mEV.
- the mEV-binding moiety is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP.
- the mEV-binding moiety has binding specificity for the mEV (e.g., by having binding specificity for a bacterial antigen).
- the mEV-binding moiety comprises an antibody or antigen binding fragment thereof.
- the mEV-binding moiety comprises a T cell receptor or a chimeric antigen receptor (CAR).
- the mEV-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 mEVs can be used to increase the targeting of the mEVs (e.g., to cancer cells and/or a part of a subject where cancer cells are present).
- mEVs Processed Microbial Extracellular Vesicles
- the pmEVs described herein can be prepared using any method known in the art.
- the 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 (e.g., using an antibiotic described herein).
- the bacteria are killed using UV irradiation.
- the pmEVs described herein are purified from one or more other bacterial components. Methods for purifying pmEVs from bacteria (and optionally, other bacterial components) are known in the art. In some embodiments, pmEVs are prepared from bacterial cultures using methods described in Thein, et al. (J. Proteome Res. 9(12):6135-6147 (2010)) or Sandrini, et al. (Bio-protocol 4(21): el287 (2014)), each of which is hereby incorporated by reference in its entirety. In some embodiments, the bacteria are cultured to high optical density and then centrifuged to pellet bacteria (e.g., at 10,000- 15,000 x g for 10-15 min at room temperature or 4°C).
- 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 hr 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.
- pmEVs are obtained and/or derived by methods adapted from Sandrini et al, 2014.
- bacterial cultures are centrifuged at 10, GOO- 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 MgC12 to a final concentration of 100 mM.
- samples are sonicated using a QSonica Q500 sonicator with 7 cycles of 30 sec on and 30 sec off.
- debris and unlysed cells are pelleted by centrifugation at 10,000 x g for 15 min. at 4°C. In some embodiments, supernatants are then centrifuged at 110,000 x g for 15 min at 4°C.
- 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 (e.g., preparing) isolated bacterial pmEVs comprises the steps of: (a) centrifuging a bacterial culture, thereby forming a first pellet and a first supernatant, wherein the first pellet comprises cells; (b) discarding the first supernatant; (c) resuspending the first pellet in a solution; (d) lysing the cells; (e) centrifuging the lysed cells, thereby forming a second pellet and a second supernatant; (f) discarding the second pellet and centrifuging the second supernatant, thereby forming a third pellet and a third supernatant; (g) discarding the third supernatant and resuspending the third pellet in a second solution, thereby forming the isolated bacterial pmEVs.
- the method further comprises the steps of: (h) centrifuging the solution of step (g), thereby forming a fourth pellet and a fourth supernatant; (i) discarding the fourth supernatant and resuspending the fourth pellet in a third solution. In some embodiments, the method further comprises the steps of: (j) centrifuging the solution of step (i), thereby forming a fifth pellet and a fifth supernatant; and (k) discarding the fifth supernatant and resuspending the fifth pellet in a fourth solution.
- the centrifugation of step (a) is at 10,000 x g. In some embodiments the centrifugation of step (a) is for 10-15 minutes. In some embodiments, the centrifugation of step (a) is at 4°C or room temperature. In some embodiments, step (b) further comprises freezing the first pellet at -80°C.
- the solution in step (c) is lOOmM Tris-HCl, pH 7.5 supplemented with Img/ml DNasel. In some embodiments, the solution in step (c) is lOmM Tris-HCl, pH 8.0, ImM 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 1.6mg/ml. In some embodiments, step (c) further comprises adding MgChto a final concentration of lOOmM. In some embodiments, the cells are lysed in step (d) via homogenization. In some embodiments, the cells are lysed in step (d) via emulsiflex C3. In some embodiments, the cells are lysed in step (d) via sonication.
- the cells are sonicated in 7 cycles, wherein each cycle comprises 30 seconds of sonication and 30 seconds without sonication.
- the centrifugation of step (e) is at 10,000 x g. In some embodiments, the centrifugation of step (e) is for 15 minutes. In some embodiments, the centrifugation of step (e) is at 4°C or room temperature.
- the centrifugation of step (f) is at 120,000 x g. In some embodiments, the centrifugation of step (f) is at 110,000 x g. In some embodiments, the centrifugation of step (f) is for 1 hour. In some embodiments, the centrifugation of step (f) is for 15 minutes. In some embodiments, the centrifugation of step (f) is at 4°C or room temperature.
- the second solution in step (g) is 100 mM sodium carbonate, pH 11. In some embodiments, the second solution in step (g) is lOmM 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 lOOmM 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 lOOmM Tris-HCl, pH 7.5 or PBS.
- pmEVs obtained and/or derived 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 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 pmEVs are isolated and enriched by chromatography and binding surface moi eties on pmEVs.
- select pmEVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.
- the pmEVs can be analyzed, e.g., as described in Jeppesen, et al. Cell 177:428 (2019).
- pmEVs are lyophilized.
- pmEVs are gamma irradiated (e.g., at 17.5 or 25 kGy).
- pmEVs are UV irradiated.
- pmEVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).
- pmEVs are acid treated.
- pmEVs are oxygen sparged (e.g., at 0.1 vvm for two hours).
- pmEVs can be isolated, e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
- the smEVs described herein can be prepared using any method known in the art.
- the smEVs are prepared without an smEV purification step.
- bacteria described herein are killed using a method that leaves the smEVs intact and the resulting bacterial components, including the smEVs, are used in the methods and compositions described herein.
- the bacteria are killed using an antibiotic (e.g., using an antibiotic described herein).
- the bacteria are killed using UV irradiation.
- the bacteria are heat-killed.
- the smEVs described herein are purified from one or more other bacterial components.
- Methods for purifying smEVs from bacteria are known in the art.
- smEVs are prepared from bacterial cultures using methods described in S. Bin Park, et al. PLoS ONE. 6(3):el7629 (2011) or G. Norheim, et al. PLoS ONE. 10(9): e0134353 (2015) or Jeppesen, et al. Cell 177:428 (2019), each of which is hereby incorporated by reference in its entirety.
- the bacteria are cultured to high optical density and then centrifuged to pellet bacteria (e.g., at 10,000 x g for 30 min at 4°C, at 15,500 x g for 15 min at 4°C).
- 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 bacterial smEVs (e.g., at 100,000-150,000 x g for 1-3 hours at 4°C, at 200,000 x g for 1-3 hours at 4°C).
- the smEVs are further purified by resuspending the resulting smEV pellets (e.g., in PBS), and applying the resuspended smEVs to an Optiprep (iodixanol) gradient or gradient (e.g., a 30-60% discontinuous gradient, a 0- 45% discontinuous gradient), followed by centrifugation (e.g., at 200,000 x g for 4-20 hours at 4°C).
- Optiprep iodixanol gradient or gradient
- centrifugation e.g., at 200,000 x g for 4-20 hours at 4°C.
- smEV bands can be collected, diluted with PBS, and centrifuged to pellet the smEVs (e.g., at 150,000 x g for 3 hours at 4°C, at 200,000 x g for 1 hour at 4°C).
- the purified smEVs can be stored, for example, at -80°C or -20°C until use.
- the smEVs are further purified by treatment with DNase and/or proteinase K.
- cultures of bacteria can be centrifuged at 11,000 x g for 20-40 min at 4°C to pellet bacteria. Culture supernatants may be passed through a 0.22 pm filter to exclude intact bacterial cells.
- Filtered supernatants may then be concentrated using methods that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration.
- 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
- filtered supernatants can be centrifuged at 100,000-200,000 x g for 1-16 hours at 4°C.
- the pellet of this centrifugation contains bacteria smEVs and other debris such as large protein complexes.
- 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.
- smEVs can be obtained and/or derived 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., smEVs, 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 smEVs 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. smEVs collected by this method may be further purified and/or concentrated by ultracentrifugation or filtration as described above for filtered supernatants.
- smEVs obtained and/or derived by methods provided herein may be further purified by size-based column chromatography, by affinity chromatography, by ion-exchange chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0.
- the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3- 24 hours at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in PBS and 3 volumes of 60% Optiprep are added to the sample.
- the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep.
- Samples are applied to a 0-45% discontinuous Optiprep gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C, e.g., 4-24 hours at 4°C.
- smEVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 pm filter to exclude intact cells. To further increase purity, isolated smEVs may be DNase or proteinase K treated.
- smEVs used for in vivo injections purified smEVs are processed as described previously (G. Norheim, et al. PLoS ONE. 10(9): e0134353 (2015)). Briefly, after sucrose gradient centrifugation, bands containing smEVs are resuspended to a final concentration of 50 pg/mL in a solution containing 3% sucrose or other solution suitable for in vivo injection known to one skilled in the art. This solution may also contain adjuvant, for example aluminum hydroxide at a concentration of 0-0.5% (w/v).
- smEVs in PBS are sterile-filtered to ⁇ 0.22 pm.
- 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 smEV preparations can be confirmed by plating a portion of the smEVs onto agar medium used for standard culture of the bacteria used in the generation of the smEVs and incubating using standard conditions.
- select smEVs are isolated and enriched by chromatography and binding surface moi eties on smEVs.
- select smEVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.
- smEVs can be analyzed, e.g., as described in Jeppesen, et al. Cell 177:428 (2019). [324] In some embodiments, smEVs are lyophilized.
- smEVs are gamma irradiated (e.g., at 17.5 or 25 kGy).
- smEVs are UV irradiated.
- smEVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).
- smEVs s are acid treated.
- smEVs 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 smEVs produced by bacteria.
- smEVs can be isolated, e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
- the growth environment e.g., culture conditions
- the yield of smEVs can be increased by an smEV inducer, as provided in Table 5.
- the method can optionally include exposing a culture of bacteria to an smEV inducer prior to isolating smEVs from the bacterial culture.
- the culture of bacteria can be exposed to an smEV inducer at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
- a the mEVs disclosed herein are formulated into a therapeutic composition.
- therapeutic compositions comprising a therapeutic agent described herein.
- the therapeutic composition comprises a therapeutic agent provided herein and a pharmaceutically acceptable carrier.
- the therapeutic composition comprises a pharmaceutically acceptable excipient, such as a glidant, lubricant, and/or diluent.
- the therapeutic composition comprises at least one carbohydrate.
- the therapeutic composition comprises at least one lipid.
- the lipid comprises at least one fatty acid selected from lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16: 1), margaric acid (17:0), heptadecenoic acid (17: 1), stearic acid (18:0), oleic acid (18: 1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20: 1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EP A), docosanoic acid (22:0), docosenoic acid (22: 1), docosapentaenoic acid (22:5), dococosanoic acid (22:0
- the therapeutic composition comprises at least one supplemental mineral or mineral source.
- supplemental mineral or mineral source examples include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium.
- Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.
- the therapeutic composition comprises at least one supplemental vitamin.
- the at least one vitamin can be fat-soluble or water soluble vitamins.
- Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin.
- Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.
- the therapeutic composition comprises an excipient, such as a pharmaceutically acceptable excipient.
- excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.
- the excipient is a buffering agent.
- suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.
- the excipient comprises a preservative.
- suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.
- the therapeutic composition comprises a binder as an excipient.
- suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof.
- the therapeutic composition comprises a lubricant as an excipient.
- Non-limiting examples of 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 (for example, a food or beverage) such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.
- a food product for example, a food or beverage
- a food or beverage such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.
- the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, and Chinese soups; soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products, including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, carb
- the therapeutic composition is a food product for animals, including humans.
- the animals, other than humans, are not particularly limited, and the composition can be used for various livestock, poultry, pets, experimental animals, and the like.
- Specific examples of the animals include pigs, cattle, horses, sheep, goats, chickens, wild ducks, ostriches, domestic ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, and the like, but the animals are not limited thereto.
- a composition e.g, in a therapeutic agent (i.e., pharmaceutical agent, drug substance) or in a therapeutic composition (i.e., pharmaceutical composition, drug product)
- a lipophilic fluorescent dye i.e., in a composition
- a lipophilic fluorescent dye i.e., in a composition with a lipophilic fluorescent dye
- provied herein is a method of quantitating microbial extracellular vesicles (mEVs) in a composition comprising EVs (e.g., a drug product or a drug substance), the method comprising: (a) contacting a test sample from a composition comprising mEVs with a lipophilic fluorescent dye to obtain a mixture; (b) measuring a fluorescence signal from the mixture; and (c) quantitating the mEVs in the composition based on the fluorescence signal.
- mEVs extracellular vesicles
- provied herein is a method of detecting microbial extracellular vesicles (mEVs) in a composition (e.g., a drug product or a drug substance), the method comprising: (a) contacting a test sample from a composition with a lipophilic fluorescent dye to obtain a mixture; (b) detecting a fluorescence signal from the mixture; and (c) determining whether mEVs are present in the composition based on the fluorescence signal.
- a composition e.g., a drug product or a drug substance
- provied herein is a method of determining the lipid content of a composition comprising microbial extracellular vesicles (mEVs) (e.g., a drug product or a drug substance), the method comprising: (a) contacting a test sample from a composition comprising mEVs with a lipophilic fluorescent dye to obtain a mixture; (b) measuring a fluorescence signal from the mixture; and (c) determining the lipid content of the composition comprising mEVs based on the fluorescence signal.
- mEVs microbial extracellular vesicles
- provied herein is a method of determining a dose (e.g., a therapeutically effective dose) of a composition (e.g., a drug product or a drug substance) comprising microbial extracellular vesicles (mEVs), the method comprising: (a) contacting a test sample from a pharmaceutical composition comprising mEVs with a lipophilic fluorescent dye to obtain a mixture; (b) measuring a fluorescence signal from the mixture; and (c) determining a dose of the pharmaceutical composition comprising mEVs based on the fluorescence signal.
- the method further comprises administering the dose of the drug product to a subject.
- the dried form is resuspended, e.g., in a liquid (such as PBS or water), prior to the contacting step.
- a liquid such as PBS or water
- the dried form is resuspended in PBS prior to the contacting step.
- the PBS is PBS without CaC12 and MgC12.
- the PBS is PBS pH 6.8.
- the PBS is PBS pH 7.2.
- the dried form is resuspended in water prior to the contacting step.
- the method further comprises the step of separating mEVs from bacterial cells to generate the composition of step (a).
- the separation of mEVs from bacterial cells is performed using centrifugation.
- the separation of mEVs from bacterial cells is performed using filtration.
- step (b) is performed using fluorescence spectroscopy.
- step (b) is performed using a spectrafluorometer.
- step (b) is performed using a spectrafluorometer capable of reading fluorescence at Ex/Em 515/640 nm or equivalent.
- step (b) is performed using a microplate spectrafluorometer, such as a microplate spectrafluorometer capable of reading fluorescence at Ex/Em 515/640 nm or equivalent.
- step (b) is performed using high performance liquid chromatography (HPLC) with a fluorescence detector capable of reading fluorescence at Ex/Em 515/640 nm or equivalent.
- the method is performed at room temperature.
- the test sample comprises 10 9 to 10 11 mEV particles.
- the fluorescent dye can be any lipophilic fluorescent dye.
- the fluorescent dye specifically binds to the lipid membrane layer of the mEVs.
- the fluorescent dye preferentially binds to the lipid membrane layer of mEVs over the lipid membrane layers of whole cells (e.g., bacterial cells).
- the fluorescence of the fluorescent dye increases when it binds to the lipid membrane layer of an mEV (e.g., increases by at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, or more).
- the fluorescent dye is non-fluorescent in aqueous media and becomes fluorescent when it binds to the lipid membrane layer of an mEV.
- the flourescent dye has an excitation wavelength of about 515 nm. In some embodiments, the excitation wavelength is within 20%, 15%, 10%, 5%, 1%, or 0% of 515 nanometers (e.g., the excitation wavelength is 515 nanometers). In some embodiments, the fluorescent dye has an emission wavelength of about 640 nm. In some embodiments, the emission wavelength is within 20%, 15%, 10%, 5%, 1%, or 0% of 640 nanometers (e.g., the emission wavelength is 640 nanometers).
- fluorescent dyes used in the methods and compositions described herein include, but are not limited to, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64), a fixable analog of FM4-64 (FM4-64X (Thermo Fisher)), JV ⁇ (3 ⁇ Trimethyl ammoniumpropyl)-4-(6 ⁇ (4-(Diethylamino)phenyl)hexatrienyl)Pyridinium Dibromide (FM 5-95), and SynaptoRedTM C2.
- the fluorescent dye is N-(3- triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64).
- the fluorescent dye is a fixable analog of FM4-64 (FM4- 64X or FM4-64FX).
- the fluorescent dye is N-(3- Trimethylammoniumpropyl)-4-(6-(4-(Diethylamino)phenyl)hexatrienyl)Pyridinium Dibromide (FM 5-95).
- the fluorescent dye comprises the following chemical structure:
- the fluorescent dye comprises the following chemical structure: [362] In certain embodiments of the methods provided herein, the fluorescent dye comprises the following chemical structure:
- the fluorescent dye comprises one of the above chemical structures but with a different counter-ion.
- step (c) comprises determining a lipid level for the test sample based on the fluorescence signal and one or more calibration signals obtained and/or derived from a calibrator standard.
- the calibrator standard comprises a reconstituted vesicle liposome (RV).
- the calibrator standard comprises an E. coli lipid.
- the method further comprises generating a calibration curve from the one or more calibration signals.
- the calibrator standard comprises mEVs, e.g., a preparation thereof.
- the calibrator standard comprises mEVs from a bacterial strain, e.g., a preparation thereof.
- the calibrator standard comprises mEVs (e.g., a preparation thereof) from a bacterial strain that is the same strain as the mEVs in a composition from which a test sample is obtained and/or derived.
- the calibrator standard comprises mEVs (e.g., a preparation thereof) for which Total Particle Number (TPN) is obtained and/or derived using Scatter NTA Analysis.
- TPN Total Particle Number
- the TPN is obtained and/or derived using Scatter NTA Analysis and is generated as an average of more than one (such as 2-20, 5-15, 10, or 12) replicates of mEVs (e.g., a preparation thereof).
- the calibrator standard comprises mEVs (e.g., a preparation thereof) from a bacterial strain that is the same strain as the mEVs in a composition from which a test sample is obtained and/or derived, and for which Total Particle Number (TPN) is obtained and/or derived using Scatter NTA Analysis.
- TPN Total Particle Number
- the TPN is obtained and/or derived using Scatter NTA Analysis and is generated as an average of more than one (such as 2-20, 5-15, 10, or 12) replicates of the mEVs.
- [366] A method of quantitating microbial extracellular vesicles (mEVs) in a composition comprising mEVs, the method comprising: (a) contacting a test sample from a composition comprising mEVs with a lipophilic fluorescent dye to obtain a mixture; (b) measuring a fluorescence signal from the mixture; and (c) quantitating the mEVs in the composition based on the fluorescence signal, and optionally (d) determining the eTPN of the composition comprising the mEVs, e.g., by comparing the fluorescence signal from the mixture to an mEV reference standard curve (e.g., that provides a correlation between flourescence signal and mEV particle number).
- mEVs extracellular vesicles
- [367] 2 A method of detecting microbial extracellular vesicles (mEVs) in a composition, the method comprising: (a) contacting a test sample from a composition with a lipophilic fluorescent dye to obtain a mixture; (b) detecting a fluorescence signal from the mixture; and (c) determining whether mEVs are present in the composition based on the fluorescence signal, and optionally (d) determining the eTPN of the composition comprising the mEVs, e.g., by comparing the fluorescence signal from the mixture to an mEV reference standard curve (e.g., that provides a correlation between flourescence signal and mEV particle number).
- an mEV reference standard curve e.g., that provides a correlation between flourescence signal and mEV particle number
- a method of determining a dose of a composition comprising: (a) contacting a test sample from a composition comprising mEVs with a lipophilic fluorescent dye to obtain a mixture; (b) measuring a fluorescence signal from the mixture; and (c) determining a dose of the composition comprising mEVs based on the fluorescence signal, and optionally (d) determining the eTPN of the composition comprising the mEVs, e.g., by comparing the fluorescence signal from the mixture to an mEV reference standard curve (e.g., that provides a correlation between flourescence signal and mEV particle number), wherein the eTPN comprises the dose.
- mEV reference standard curve e.g., that provides a correlation between flourescence signal and mEV particle number
- step (b) is performed using fluorescence spectroscopy.
- composition 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the content of the composition is the isolated mEVs.
- [416] 50 The method of embodiment 49, wherein the mEVs are from a bacterial strain listed in Table 1, Table 2, Table 3, or Table 4.
- [421] 55 The method of any one of embodiments 1 to 48, wherein the mEVs are from a strain of bacteria comprising at least 95% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
- [424] 58 A composition comprising mEVs and a lipophilic fluorescent dye.
- composition of embodiment 58 wherein the composition comprises a drug substance.
- composition of embodiment 58, wherein the composition comprises a drug product comprises a drug product.
- composition of any one of embodiments 58-65, wherein the fluorescent dye is V-(3-Trimethylammoniumpropyl)-4-(6-(4- (Diethylamino)phenyl)hexatrienyl)Pyridinium Dibromide (FM 5-95)
- composition of embodiment 89, wherein the mEVs are from a bacterial strain listed in Table 1, Table 2, Table 3, or Table 4.
- composition of embodiment 91, wherein the mEVs are from a bacterial strain listed in Table J.
- composition of embodiment 93, wherein the mEVs are from bacteria of the species Prevotella histicola.
- composition of any one of embodiments 58-88, wherein the mEVs are from a strain of bacteria comprising at least 95% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
- composition of any one of embodiments 58-88, wherein the mEVs are from a strain of bacteria comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
- Example 1 eTPN conversion using Total Lipid Content method
- This method describes the quantification of equivalent Total Particle Number (eTPN) of microbial extracellular vesicles (mEVs) by using a total lipid content method by fluorescent dye FM4-64. Scope
- Lipophilic FM4-64 dye is non-fluore scent in aqueous media; when inserted into the lipid membrane layer of an EV it becomes intensely fluorescent at Ex/Em 515/640 nm.
- TPN Total Particle Number
- the relative fluorescent unit (RFU) readouts of the test samples are plotted against a trendline of TPN/mg DS for a calibration curve. Since TPN per mg DS is well characterized, the RFLT is plotted against the TPN to determine the particle count of the sample.
- Figure 1 provides an graph showing a calibration curve generated with this method.
- FM4-64 fluorescent dye solution 150 pg/mL o 7.2.1.
- FM4-64 dye powder dissolve powder in 500 pL PBS by vortexing for 30 seconds, transfer dye solution to a 15-mL conical tube and keep protected from light. Rinse tube with 500 pL PBS and combine with dye solution. Add 5670 pL PBS to bring total volume to 6670 pL. Mix well by vortexing tube for 10 seconds.
- o 7.2.2 Aliquot dye solution in 500 pL aliquots (for partial 96-well plate) and 1 mL aliquots (for full plate) and stored at -20°C.
- Step 1 Measure 50 mg of pilot batch control and transfer to a 1.5 mL microcentrifuge tube. Add 500 pL PBS. Vortex on high for at least 10 seconds until no clumps observed.
- Step 2 Take 100 pL of the suspension from step 1 and add to 900 pL PBS. Vortex on high for at least 10 seconds. This is the 1 :100 dilution.
- Step 3 Take 200 pL of the 1 :100 dilution and add in 800 pL PBS. Vortex on high for at least 10 seconds. This is the 1 :500 dilution.
- ⁇ Strength C 1 : 10,000 ⁇ Weigh ⁇ 50 mg sample and dilute in 450 pL PBS. This is the 1 : 10 dilution.
- ⁇ 1 10 dilution: Weigh ⁇ 50 mg sample and dilute in 450 pL PBS.
- ⁇ 1 100 dilution: Add 100 pL of 1 : 10 dilution into 900 pL PBS.
- ⁇ 1 10,000 dilution: Add 100 pL of 1 : 1000 dilution into 900 pL PBS. o 7.6.2. Each DP test sample should be tested in 3 independent preparations, starting with 3 DP aliquots. Each preparation is also tested in 3 replicates. Average of all three aliquots and all three replicates will be taken and reported as the final value. o 7.6.3. Dilution can be adjusted accordingly so that the sample falls within the qualified range of the curve.
- Table 8 o 7.7.8. Suggested plate loading configuration for In-process samples (Table 9): Table 9 o 7.7.9. Use multi-channel pipette add 10 pL FM4-64 dye to each well, mix by pipetting for at least 8 up-down cycles. o NOTE: To ensure well mixing by taping the side of the plate several times with gentle shaking until visually the dye is well mixed in the wells. o 7.7.10. Cover plate with plate sealer, incubate for 10 minutes protected from light at room temperature. o 7.7.11. Mix by gentle tapping on side of the plate. o 7.7.12. Read plate at Ex/Em 515/640 nm.
- the aim of this work is to establish limits and conditions for the generation of equivalent Total Particle Number (eTPN) of EV derived from a bacterial strain of Prevotellaceae Drug Substance and Drug Product using the Lipid Content method and nanoparticle tracking analysis (NTA).
- eTPN equivalent Total Particle Number
- NTA nanoparticle tracking analysis
- Total lipid content assay utilizes lipid binding fluorescent dye FM4-64.
- Lipophilic FM4-64 dye is non-fluore scent in aqueous media; when inserted into the lipid membrane layer it becomes intensely fluorescent at Ex/Em 515/640 nm.
- TPN Total Particle Number
- the relative fluorescent unit (RFU) readouts of the test samples are plotted against a trendline of TPN/mg DS for a calibration curve. Since TPN per mg DS is well characterized, the RFU is plotted against the TPN to determine the particle count of the sample.
- the method describes quantification of total lipid content of EV derived from a bacterial strain of Prevotellaceae extracellular vesicles (EV) by lipid binding fluorescent dye FM4-64.
- Lipophilic FM4-64 dye is nonfluorescent in aqueous media; when inserted into the lipid membrane layer of the EV it becomes intensely fluorescent at Ex/Em 515/640 nm.
- Reconstituted vesicle liposome (RV) is used as calibrator standard.
- the relative fluorescent unit (RFU) readouts of the test samples are plotted against a trendline of RV liposome calibration curve to determine the lipid content in the sample.
- the protocol is suitable for in-process control (IPC), drug substance and related samples.
- dilute liposome stock (lOmg/mL) 100-fold with PBS.
- the relative fluorescent unit (RFU) readout of the samples is plotted against a trendline of liposome calibration curve to calculate the lipid content in the test samples.
- Example 4 Representative Strains As Sources for EVs
- smEVs secreted microbial extracellular vesicles
- Bacteria of the taxonomic groups listed in Table J can be used in the methods and compositions described herein.
- mEVs of bacteria of the taxonomic groups listed in Table J can be used in the methods and compositions described herein.
- Table J Strains from which extracellular vesicles (EVs) were isolated
- Example 5 EV quantitation in Microbe Drug Substance and Drug Product by eTPN conversion using Total Lipid Content method
- the method describes the quantification of extracellular vesicles (EV) in microbe drug substance and drug product material by equivalent Total Particle Number (eTPN) using total lipid content method by fluorescent dye FM4-64.
- a Prevotella histicola-based active pharmaceutical ingredient contains freeze-dried P. histicola bacterial cells. Bacteria are known to naturally release extracellular vesicles (EVs) that contain components from their mother cells. Product characterization work suggests that the drug substance material contains some EVs from the process and product related residuals from bacterial culture harvest supernatant.
- EVs extracellular vesicles
- Lipophilic FM4-64 dye is non- fluorescent in aqueous media; when inserted into the lipid membrane layer of the EV it becomes intensely fluorescent at Ex/Em 515/640 nm.
- the relative fluorescent unit (RFU) readouts of the test samples are plotted against a trendline of total particle number/mg D S for the batch calibration curve. Since TPN per mg DS is well characterized, the RFU is plotted against the TPN to determine the particle count of the sample.
- TPN Total Particle Number
- the protocol is suitable for drug substance and drug product use.
- Source material DS or DP 250 mg in duplicates are suspended in 50 ml PBS pH 6.8 (extraction buffer), 200-fold buffer/powder ratio
- the calibration curve was prepared in Example 1 and shown in Figure 1.
- FM4-64 dye powder dissolve powder in 500 pL PBS by vortexing for 30 seconds, transfer dye solution to a 15-mL conical tube and keep protected from light. Rinse tube with 500 pL PBS and combine with dye solution. Add 5670 pL PBS to bring total volume to 6670 pL. Mix well by vortexing tube for 10 seconds. [Adjust dilution accordingly when other sizes of FM4-64 dye powder are used. For example, in a lOOug size vial add 667 pL PBS], .
- Step 1 Measure 50 mg of PP01 HS batch control and transfer to a 1.5 mL microcentrifuge tube. Add 500 pL PBS. Vortex on high for at least 10 seconds until no clumps observed. 0.2.2.
- Step 2. Take 100 pL of the suspension from step 1 and add to 900 pL PBS. Vortex on high for at least 10 seconds. This is the 1 : 100 dilution. 0.2.3.
- Step 3. Take 200 pL of the 1:100 dilution and add in 800 pL PBS. Vortex on high for at least 10 seconds. This is the 1 :500 dilution.
- test samples Prepare test samples according to Sections 7.6.2, if applicable. 5. In a 96- well black plate with clear bottom, transfer 100 pL calibration standard series (in duplicates), assay control (3 preparations of 1 DS aliquot), and EV supernatant test samples load neat in triplicates. 6. Use single channel pipette to add 10 pL FM4-64 dye to each well, mix by pipetting for at least 4 up-down cycles. [For high number of samples multichannel pipette may be used], 2.6.1. NOTE: To ensure well mixing by taping the side of the plate several times with gentle shaking until visually the dye is well mixed in the wells. 7. Cover plate with plate sealer, incubate for 10 minutes protected from light at room temperature. 7.12.8. Mix by gentle tapping on side of the plate before read plate at Ex/Em 515/640 nm.
- the relative fluorescent unit (RFU) readout of the samples is plotted against a trendline of eTPN calibration curve to calculate the eTPN / g DS in the test samples.
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Abstract
L'invention concerne des procédés et des compositions associés à la quantification et à la qualification de vésicules extracellulaires (EVs).
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