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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230601	1/1	Escherichia coli	BL21 DE3 Delta_msbB	(d)(U)C Filtration UF ExoLutE (SL Bigen) Dialysis	Won S	2023	63%
Study summary Full title Mass-produced gram-negative bacterial outer membrane vesicles activate cancer antigen-specific stem-like CD8 T cells which enables an effective combination immunotherapy with anti-PD-1. All authors Won S, Lee C, Bae S, Lee J, Choi D, Kim MG, Song S, Lee J, Kim E, Shin H, Basukala A, Lee TR, Lee DS, Gho YS Journal J Extracell Vesicles Abstract Despite the capability of extracellular vesicles (EVs) derived from Gram-negative and Gram-positive (show more...)Despite the capability of extracellular vesicles (EVs) derived from Gram-negative and Gram-positive bacteria to induce potent anti-tumour responses, large-scale production of bacterial EVs remains as a hurdle for their development as novel cancer immunotherapeutic agents. Here, we developed manufacturing processes for mass production of Escherichia coli EVs, namely, outer membrane vesicles (OMVs). By combining metal precipitation and size-exclusion chromatography, we isolated 357 mg in total protein amount of E. coli OMVs, which was equivalent to 3.93 × 10 particles (1.10 × 10 particles/μg in total protein amounts of OMVs) from 160 L of the conditioned medium. We show that these mass-produced E. coli OMVs led to complete remission of two mouse syngeneic tumour models. Further analysis of tumour microenvironment in neoantigen-expressing tumour models revealed that E. coli OMV treatment causes increased infiltration and activation of CD8 T cells, especially those of cancer antigen-specific CD8 T cells with high expression of TCF-1 and PD-1. Furthermore, E. coli OMVs showed synergistic anti-tumour activity with anti-PD-1 antibody immunotherapy, inducing substantial tumour growth inhibition and infiltration of activated cancer antigen-specific stem-like CD8 T cells into the tumour microenvironment. These data highlight the potent anti-tumour activities of mass-produced E. coli OMVs as a novel candidate for developing next-generation cancer immunotherapeutic agents. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles outer membrane vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Ultrafiltration ExoLutE (SL Bigen) Dialysis Protein markers EV: OmpA/ CD63 non-EV: FtsZ Proteomics yes Show all info Study aim Function/New methodological development/Identification of content (omics approaches) Sample Species Escherichia coli Sample Type Cell culture supernatant EV-producing cells BL21 DE3 Delta_msbB EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type Polysulfone Commercial kit ExoLutE (SL Bigen) Other Name other separation method ExoLutE (SL Bigen) Other Name other separation method Dialysis Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins OmpA Not detected EV-associated proteins CD63 Not detected contaminants FtsZ Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 20.24 NTA EV concentration Yes Particle yield particles per milliliter of starting sample: 2.45E+10 EM EM-type Transmission-EM Image type Wide-field

	EV230601	2/1	Escherichia coli	BL21 DE3 Delta_msbB	(d)(U)C Filtration UF DG	Won S	2023	11%
Study summary Full title Mass-produced gram-negative bacterial outer membrane vesicles activate cancer antigen-specific stem-like CD8 T cells which enables an effective combination immunotherapy with anti-PD-1. All authors Won S, Lee C, Bae S, Lee J, Choi D, Kim MG, Song S, Lee J, Kim E, Shin H, Basukala A, Lee TR, Lee DS, Gho YS Journal J Extracell Vesicles Abstract Despite the capability of extracellular vesicles (EVs) derived from Gram-negative and Gram-positive (show more...)Despite the capability of extracellular vesicles (EVs) derived from Gram-negative and Gram-positive bacteria to induce potent anti-tumour responses, large-scale production of bacterial EVs remains as a hurdle for their development as novel cancer immunotherapeutic agents. Here, we developed manufacturing processes for mass production of Escherichia coli EVs, namely, outer membrane vesicles (OMVs). By combining metal precipitation and size-exclusion chromatography, we isolated 357 mg in total protein amount of E. coli OMVs, which was equivalent to 3.93 × 10 particles (1.10 × 10 particles/μg in total protein amounts of OMVs) from 160 L of the conditioned medium. We show that these mass-produced E. coli OMVs led to complete remission of two mouse syngeneic tumour models. Further analysis of tumour microenvironment in neoantigen-expressing tumour models revealed that E. coli OMV treatment causes increased infiltration and activation of CD8 T cells, especially those of cancer antigen-specific CD8 T cells with high expression of TCF-1 and PD-1. Furthermore, E. coli OMVs showed synergistic anti-tumour activity with anti-PD-1 antibody immunotherapy, inducing substantial tumour growth inhibition and infiltration of activated cancer antigen-specific stem-like CD8 T cells into the tumour microenvironment. These data highlight the potent anti-tumour activities of mass-produced E. coli OMVs as a novel candidate for developing next-generation cancer immunotherapeutic agents. (hide) EV-METRIC 11% (30th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles outer membrane vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Ultrafiltration Density gradient Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function/New methodological development/Identification of content (omics approaches) Sample Species Escherichia coli Sample Type Cell culture supernatant EV-producing cells BL21 DE3 Delta_msbB EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 50% Orientation Bottom-up Speed (g) 200000 Duration (min) 120 Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type Polysulfone Other Name other separation method Characterization: Protein analysis None Protein Concentration Method Bradford Protein Yield (µg) per milliliter of starting sample Characterization: Lipid analysis No Characterization: Particle analysis DLS

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EV-TRACK ID	EV230601
species	Escherichia coli
sample type	Cell culture
cell type	BL21 DE3 Delta msbB
condition	Control condition
separation protocol	dUC/ Filtration/ Ultrafiltration/ ExoLutE (SL Bigen)/ Dialysis	dUC/ Filtration/ Ultrafiltration/ Density gradient
Exp. nr.	1	2
EV-METRIC %	63	11