Classifications

• • 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
• • 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
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
  • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
• • 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
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
• • 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
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
• • 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
  • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• • 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
• • 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
  • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
  • G01N33/586—Liposomes, microcapsules or cells

Definitions

• the present invention relates to a method to determine the recovery rate of sample extracellular vesicles.
• the present invention further relates to a kit comprising a control extracellular vesicle.
• the present invention also relates to methods for preparing control extracellular vesicles, a method to calibrate a device for sample extracellular vesicles analysis, and a method to evaluate the isolation of extracellular vesicles.
• Extracellular vesicles are nanometer-sized vesicles secreted by all cell types and found circulating in body fluids such as plasma, serum, urine, cerebrospinal fluid, etc.
• EVs consist of a double lipid membrane surrounding its cargo of proteins, nucleic acids and metabolites making them useful conveyors of information as the lipid membrane provides protection against degradative proteins, such as proteases and RNAses, present in biofluids.
• EVs are found to be implicated in, e.g. cancer progression. They can be secreted by cancer cells and either activate or recruit cancer or stromal cells locally or educate a premetastatic niche at a distant location, and hereby facilitating metastasis formation.
• EVs isolated from different biofluids have been found to contain specific mRNA, miRNA and protein content related to disease state for breast, urogenital, pancreatic and ovarian cancers (Sadovska et al., Anticancer Res (2015), 35: 6379-6390; Nawaz et al., Nat Rev Urol (2014), 11 : 688-701, Tang et al., Cancer Lett (2015), 367: 26-33, Melo et al., Nature (2015), 523: 177-182).
• Sadovska et al. Anticancer Res (2015), 35: 6379-6390
• Nawaz et al. Nat Rev Urol (2014), 11 : 688-701
• Tang et al. Cancer Lett (2015), 367: 26-33
• Melo et al. Nature (2015), 523: 177-182
• NTA nanotracking analysis
• these methods cannot address the central problem of analyzing complex biological or medical samples for their content and concentration of EVs. This is because physical methods fail in samples with very high protein concentration such as serum or plasma probes. As a consequence, the samples need to be processed and further purified prior to their analysis but nobody knows the quantitative loss of EVs during the downstream process of sample preparation and purification. Protocols and reference EVs are lacking.
• US20130078658 discloses a recombinant EV comprising a fusion of a type I or II membrane protein and a fluorescent protein (providing for biofluorescence) or luciferase (providing for chemiluminescence) which can be used to monitor sample EVs.
• a fluorescent protein providing for biofluorescence
• luciferase providing for chemiluminescence
• US20130078658 discloses a recombinant EV comprising a fusion of a type I or II membrane protein and a fluorescent protein (providing for biofluorescence) or luciferase (providing for chemiluminescence) which can be used to monitor sample EVs.
• the latter recombinant EVs are difficult to produce in high quantities and cannot be detected in a very sensitive manner.
• WO2019091964 discloses EVs with fusions of Gag and fluorescent proteins such as eGFP located intraluminally, i
• the present invention relates to a method to determine the recovery rate of sample extracellular vesicles, the method comprising:
• control extracellular vesicles comprise a heterologous marker wherein said marker is located (i) on the outer side or (ii) on the inner side of the control extracellular vesicle (preferably on the outer side) and which is suitable for colorimetric, photometric, and/or fluorescent detection (preferably colorimetric or photometric detection), to obtain a mixture,
• an immuno-based assay e.g., ELISA, EIA, Western Blot, ELISpot, and other suitable immune-based assays as known in the art
• a binding agent as described herein which (specifically) binds to said heterologous marker, wherein said marker is located on the outer side
• step (d) determining the recovery rate of said sample extracellular vesicles based on the ratio of the amount of control extracellular vesicles detected in step (c) after the isolation step to the known amount of control extracellular vesicles mixed with the biofluid sample prior to the isolation step.
• step (c1) is preferred for detecting the amount of control extracellular vesicles among the isolated extracellular vesicles according to step (c) of the method described and provided herein.
• the term “amount” in context with extracellular vesicles means the number of vesicles.
• the binding agent employed in step (c2) of the method described and provided herein may further comprise a heterologous marker (e.g., HRP) as described herein.
• a heterologous marker e.g., HRP
• the control extracellular vesicles may be PEGylated (i.e.
• PEG polyethylene glycol
• an immune-based assay e.g., ELISA, EIA, Western Blot, ELISpot, and other suitable immune-based assays as known in the art
• a binding agent as described herein which (specifically) binds to PEG is used.
• This approach may also be used to separate the control extracellular vesicles from the sample extracellular vesicles.
• sample extracellular vesicle refers to endogenous EVs present in a biofluid sample as described herein. Such sample EVs are already contained in a biofluid sample or are suspected to be contained in a biofluid sample.
• control extracellular vesicles refers to EVs which are not a priori present in a biofluid sample as described herein, but which are exogenously or “extra” added, e.g. mixed with sample EVs contained or suspected to be contained in a biofluid sample.
• Control EVs may; e.g. be obtainable from production cell line which is, e.g. transfected or transduced with corresponding by transfecting an appropriate host cell with corresponding constructs, e.g. an expression vector.
• extracellular vesicles with a heterologous marker suitable for colorimetric, photometric, and/or fluorescent detection allow expression of such EVs and proper subsequent assays, e.g. even after tissue fixation. That is, they allow identification of transfected cells without obscuring the intracellular ultrastructure or organelles and in particular allows identification of synaptic sites using electron microscopy.
• a heterologous marker suitable for colorimetric, photometric, and/or fluorescent detection preferably for colorimetric or photometric detection, e.g., Horseradish-Peroxidase (HRP) or alkaline phosphate (AP)
• HRP horseradish-Peroxidase
• AP alkaline phosphate
• the method of the present invention there is no lysis of the EVs needed if said heterologous marker suitable for colorimetric, photometric, and/or fluorescent detection (preferably colorimetric or photometric detection, e.g., HRP) is located on the outer side of the control EV.
• said heterologous marker suitable for colorimetric, photometric, and/or fluorescent detection preferably colorimetric or photometric detection, e.g., HRP
• lysis of the EVs is deemed not to be required, if said heterologous marker suitable for colorimetric, photometric, and/or fluorescent detection (preferably colorimetric or photometric detection, e.g., HRP) is located on the inner side of the control EV.
• suitable substrates for colorimetric, photometric, and/or fluorescent detection preferably colorimetric or photometric detection, e.g., HRP
• suitable substrates for colorimetric, photometric, and/or fluorescent detection may be available which are able to pass or cross the membrane(s) of the EVs.
• control EVs as described herein e.g., EVs with a heterologous marker suitable for colorimetric photometric detection, e.g. HRP
• protein corona a layer of serum or plasma proteins that is believed to affect the accessibility of antibodies to EV surface proteins.
• control EVs as described herein e.g., EVs with a heterologous marker suitable for colorimetric or photometric detection, e.g.
• HRP are completely insensitive to this phenomenon and the enzymatic activity is not compromised even in undiluted serum samples spiked with control EVs as described herein (e.g., EVs with a heterologous marker suitable for colorimetric photometric detection, e.g. HRP).
• control EVs as described herein can use substrates that can be detected by colorimetric/photometric or fluorometric principles which accumulate over time and which can be measured minutes or even hours after addition of the substrate of choice such that the accumulation of the processed substrate increases the sensitivity of detection massively.
• EVs comprising a heterologous marker wherein said marker is located (i) on the outer side or (ii) on the inner side of the control extracellular vesicle and which is suitable for colorimetric, photometric, and/or fluorescent detection provide for advantageous properties as described above, which could not have been expected.
• the prior art put emphasis on EVs labelled with a marker providing for bioluminescence or chemiluminescence in order to monitor EVs (as a control) “in situ” or “live”.
• the prior art achieved this, e.g. by labelling EVs with fluorescent proteins or with luciferase.
• chemiluminescence and bioluminescence allow monitoring the quality of assays comprising EVs, they both have the disadvantage that they are not readily suitable for a quantitative analysis of assays comprising EVs. Indeed, (bio/chemi)luminescence produces light that radiates non-directionally only during the bio/chemical redox reaction.
• substrates that can be detected by colorimetric/photometric or fluorometric principles accumulate over time and can be measured minutes/hours after addition of the substrate of choice such that the accumulation of the processed substrate increases the sensitivity of detection massively.
• oxidases or peroxidases such as with HRP and other peroxidases, but also with alkaline phosphatase, for example.
• the amount of control extracellular vesicles detected in step (c) after the isolation step is quantitatively determined in step (d).
• the amount of control extracellular vesicles detected in step (c) after the isolation step is determined in step (d) at a predefined point of time after the isolation step.
• the discrete time point can be minutes (preferably 5 to 4 h, more preferably 5 to 60 min, more preferably 10 to 30 min). Also, in accordance with the present invention, accumulation of substrate may increase sensitivity with time. Accordingly, in another embodiment of the present invention, the amount of control extracellular vesicles detected in step (c) after the isolation step is determined in step (d) without lag time.
• the methods of the present invention further comprise in step (c) or step (c1) adding a fluorogenic or (preferably) chromogenic substrate. It is preferred in context of the present invention that chemiluminescent or bioluminescent substrates are excluded.
• said substrate processed by the heterologous marker accumulates over time.
• accumulation of the processed substrate is linear with time.
• processed substrate can be measured at a discrete time point after the addition of the substrate.
• the amount of EV detected in step (c) is determined 5 min to 4 hours preferably 5 to 60 min, more preferably 10 to 30 min) after the isolation step (b).
• said heterologous marker wherein said marker can be located (i) on the outer side or (ii) on the inner side of the control extracellular vesicle.
• said heterologous marker is located on the outer side of the control EV, e.g., if lysis of the EVs should be avoided in the detection assay and/or substrates are used which cannot pass through the EV membrane.
• said heterologous marker is located on the outer side of the control EV. This may for example be possible or even desirable where substrates are used that can pass the EV membrane, and/or where large amounts of proteins are present in the EVs. That is, without being bound by theory, it may be advantageous to have the heterologous marker located on the inner side of the control extracellular vesicle, if a biofluid sample to which control extracellular vesicles are mixed contains a high amount of proteins.
• step (c) of detecting the amount of control extracellular vesicles among the isolated extracellular vesicles is done using a colorimetric photometric, or fluorescent detection assay (preferably a colorimetric or photometric detection assay) as defined in step (c1) of the method described and provided herein.
• control EVs as described herein (e.g., EVs with a heterologous marker suitable for colorimetric photometric detection, e.g.
• HRP are fully compatible with immunological, antibody-based standard assays such as sandwich ELISAs and other immune-based assays as known in the art and as described herein, and said control EVs as described herein may also carry conventional surface proteins characteristic for EVs such as CD63, CD9, CD81 , and others and which are detectable by said immuno-based assays (e.g., ELISA, EIA, Western Blot, ELISpot, and other suitable immune-based assays as known in the art).
• immuno-based assays e.g., ELISA, EIA, Western Blot, ELISpot, and other suitable immune-based assays as known in the art.
• binding means in accordance with this invention that the recognition molecule is capable of specifically interacting with and/or binding to at least two, preferably at least three, more preferably at least four amino acids of an epitope as defined herein. Such binding may be exemplified by the specificity of a "lock-and-key-principle".
• the term “specifically” in this context means that the recognition molecule binds to a given target epitope but does not essentially bind to another protein.
• another protein includes any protein including proteins closely related to or being homologous to the epitope against which the recognition molecule is directed to.
• cross-species recognition or "interspecies specificity” as used herein means binding of a binding domain described herein to the same target molecule in humans and non-human species.
• cross-species specificity or “interspecies specificity” is to be understood as an interspecies reactivity to the same epitope expressed in different species, but not to another molecule other than X.
• a “binding agent” as used herein can be any molecule which is capable of (specifically) binding to a polypeptide.
• a binding agent can be an antibody or a fragment thereof (e.g., Fab, Fab’, Fv, scFv, F(ab’)2).
• An “antibody” as used herein is a protein comprising one or more polypeptides (comprising one or more binding domains, preferably antigen binding domains) substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
• immunoglobulin Ig
• the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
• an “antibody” when used herein is typically tetrameric glycosylated proteins composed of two light (L) chains of approximately 25 kDa each and two heavy (H) chains of approximately 50 kDa each. Two types of light chain, termed lambda and kappa, may be found in antibodies. Depending on the amino acid sequence of the constant domain of heavy chains, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1, and lgA2.
• IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons.
• antibody as used herein includes antibodies that compete for binding to the same epitope as the epitope bound by the antibodies of the present invention, preferably obtainable by the methods for the generation of an antibody as described herein elsewhere.
• antibody also includes but is not limited to polyclonal, monoclonal, monospecific, polyspecific such as bispecific, non-specific, humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies, with a polyclonal antibody being preferred. Said term also includes domain antibodies (dAbs) and nanobodies. Accordingly, the term “antibody” also relates to a purified serum, i.e. a purified polyclonal serum. Accordingly, said term preferably relates to a serum, more preferably a polyclonal serum and most preferably to a purified (polyclonal) serum.
• antibody variants include humanized variants of non- human antibodies, "affinity matured” antibodies (see, e.g., Hawkins et al., J Mol Biol (1992), 254, 889-896; and Lowman et al., Biochemistry (1991), 30: 10832- 10837) and antibody mutants with altered effector function (s) (see, e.g., US Patent 5, 648, 260).
• monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e.
• the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post- translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts.
• Monoclonal antibodies are highly specific, being directed against a single antigenic site.
• each monoclonal antibody is directed against a single determinant on the antigen.
• the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
• the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
• the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature (1975), 256: 495, or may be made by recombinant DNA methods (see, e.g., U. S. Patent No. 4,816, 567).
• the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature (1991), 352: 624- 628; and Marks et al., J Mol Biol (1991), 222: 581-597, for example.
• the heterologous marker is a protein.
• the heterologous marker is selected from the group consisting of peroxidase, alkaline phosphatase beta-galactosidase, glycosidase, caspase, granzyme B, HIV protease, elastase, collagenase, chloramphenicol acetyltransferase, and lipase.
• the heterologous marker is a peroxidase, preferably Horseradish Peroxidase (HRP) or any isoenzyme thereof as known in the art (e.g., Shannon et al., J Biol Chem (1966), 241 : 2166-2172).
• HRP Horseradish Peroxidase
• Preferred substrates for alkaline phosphatase include fluorescein disphosphate, dimethylacradinone phosphate, methylumbelliferyl phosphate, ELF 97 phosphate, NBT/BCIP, and Amplex red.
• Preferred substrates for beta-galactosidase include difluorescein digalactoside, resorufin galactoside, DDAO galactoside, methylumbelliferyl galactoside, 5- chloromethylfluorescein di-P-D-galactopyranoside (CMFDG), and 5-(Pentafluorobenzoylamino)fluorescein di-p-D- galactopyranoside (PFB-FDG).
• HRP provides for a superior readout, as it is a highly active enzyme (turnover of substrate leads to accumulation of the product) and its presence can be easily monitored. Accordingly, in context of the methods of the present invention, at each step after spiking a sample, HRP- EVs can be traced and quantitated by simply adding substrate followed by a short incubation time (e.g. minutes) and a photometric/colori metric readout. Emplying the method described in provided in context with the present invention allows a much more sensitive approach, e.g. detecting as few as app. 10 5 to 10 6 control EVs among 1O 10 to 10 11 sample EVs. In contrast, in the prior art (e.g.
• identification is based on the presence of GFP in the EVs and ratio of nanotracking analysis (NTA) versus fluorescent NTA (fNTA) signals needs to be determined using a specialized nanotracking analysis instrument. Also, that technology cannot be adapted to high throughput processes as it is slow, unprecise and needs constant supervision. Besides, using bioluminescence or chemiluminescence needs a very high input of, e.g. EVs with fluorescent proteins, such as gag-eGFP-EVs or EVs labelled with, e.g.
• HRP-EVs can be removed at each step of analysis or prior to further sample processing, if required.
• gag-eGFP-EVs need to be modified - PEGylated EVs - to carry an external tag that can be used in combination with PEG-specific antibodies for their removal.
• gag-eGFP-EVs is high [in some experiments in the order of 10 to 20%] removal is a must for certain readouts.
• HRP-EVs are fully compatible with immunological, antibody-based standard assays such as sandwich ELISAs, because the HRP-EVs carry also conventional surface proteins characteristic of EV such as CD63, CD9, CD81 , etc.. Immobilization or capture of our HRP-EVs together with EVs in the sample to be analyzed is possible and compatible with the HRP readout (substrate turnover) in this assay format.
• gag-eGFP-EVs disables the direct readout of particle numbers [ratio of EVs enumerated by NTA versus EVs enumerated by fNTA], As a substitute, the gag domain can be used, but its detection requires the lysis of gag-eGFP-EVs and the subsequent immobilization of gag to be analyzed in a sandwich ELISA.
• HRP-EVs provide for unexpected superiority versus EVs labelled, e.g. with fluorescent or bioluminescent markers.
• polypeptide is equally used herein with the term “protein”. Proteins (including fragments thereof, preferably biologically active fragments, and peptides, usually having fewer than 30 amino acids) comprise one or more amino acids coupled to each other via a covalent peptide bond (resulting in a chain of amino acids).
• polypeptide as used herein describes a group of molecules which typically comprise more than 15 amino acids. Polypeptides may further form multimers such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule.
• control extracellular vesicles further comprise a membrane protein, and said heterologous marker is connected to said membrane protein.
• Said heterologous marker can be connected to the membrane protein in any way as known in the art.
• said heterologous marker can be directly fused to said membrane protein (preferably recombinantly, via genetic fusion and joint translation), covalently linked to said membrane protein, linked via click chemistry, or connected to said membrane protein via a linker.
• Such linker can be of any kind known in the art, e.g., flexible linkers, rigid linkers or in vivo cleavable linkers (see, e.g., Chen et al., Adv Drug Deliv Rev (2013), 65(10): 1357-1369).
• linkers as used in context with the present invention are in vivo non- cleavable.
• Examples of linkers usable in context with the present invention include short peptide linkers (e.g., peptides comprising only 2-6, preferably 2-4 amino acids) or (poly)peptide linkers and chemical linkers, preferably short peptide linkers or (poly)peptide linkers.
• the linker can also serve as docking-site for other binding agents.
• a linker could also be, e.g., streptavidin or another polypeptide serving as docking-site for biotin.
• said heterologous marker can be connected to the membrane protein via chemical coupling from the outside as known in the art and described in, e.g., Henna Matero’s Master’s Thesis “HRP-labeling of bacterial extracellular vesicles for transmission electron microscopy imaging” (2022).
• control extracellular vesicles further comprise a membrane protein
• said membrane protein may direct its own release through vesicles as a membrane-bound protein (self-assembling; cf. US20130078658).
• said heterologous marker protein can penetrate a lipid bilayer.
• the membrane protein is a cellular membrane protein, for example, an exosomal membrane protein.
• the membrane protein can be a portion or fragment of a full-length membrane protein sufficient to introduce the fusion protein into the exosomes, particularly the lipid membrane of the exosome.
• the membrane protein can comprise an N- terminus or C-terminus region of the membrane protein (e.g., about 5 or more, about 10 or more, about 15 or more, about 20 or more, or about 25 or more contiguous amino acids from the C-terminus or N-terminus of a full-length membrane protein).
• origins of membrane proteins can be, e.g., metazoan or protozoan cells, alternatively, enveloped viruses or bacteriophages.
• said membrane protein is selected from the group consisting of transmembrane proteins, Gi-coupled proteins, membrane-anchored receptors, tetraspanins, type I or II or III transmembrane proteins, and single or multipass transmembrane proteins.
• said membrane protein is selected from the group consisting of Gag (HIV) and other retroviral specific antigen (variations), M1 (Influenza), Arrdcl (non-viral; human), VP40 (Ebola), Arc (non-viral; human), M protein (VSV), EpCAM, CD63, CD37, CD53, CD81 , CD82, CD54 (ICAM1), CD9, CD151 , TSPAN-8 (tetraspanins), Lactadherin (MFGE8), Hsc70, MHC I, Tsg101 , calnexin, gp96, L1 , integrins such as alpha-3, -5, -V, -6 and beta-1 and -3, and type I or II membrane proteins.
• said membrane protein is selected from the group consisting of CD63 as it is enriched in EVs and Lactadherin (MFGE8).
• said heterologous marker is HRP
• said detection step (c) is carried out in the presence of a substrate of HRP.
• said substrate is preferably a fluorogenic substrate or chromogenic substrate.
• substrates for colorimetric or photometric assays can be advantageous in context of the present invention as such substrates, i.e., their processed, enzymatically converted derivatives, accumulate with time while the enzyme still works on processing remaining substrate molecules.
• Enzymatically converted substrate derivatives can be detected by colorimetric/photometric principles minutes or even hours after addition of the substrate of choice such that the accumulation of the processed substrate increases the sensitivity of detection.
• control extracellular vesicles further comprise
• streptavidin or biotin preferably streptavidin as biotin can be present in small amounts in biofluids located on the outer side of the control extracellular vesicle.
• control extracellular vesicles can comprise streptavidin or biotin (preferably streptavidin) located on the outer side of the control extracellular vesicle either apart from the heterologous marker, or connected to the heterologous marker as described herein.
• streptavidin or biotin preferably streptavidin
• biotin preferably biotin
• the presence of streptavidin or biotin (preferably streptavidin) can serve as docking-site for biotin or streptavidin (preferably biotin), respectively, thus making the control extracellular vesicle extremely versatile as only one type of streptavidin- or biotin- (preferably streptavidin-) comprising type of extracellular vesicle needs to be prepared which can then be coupled in a second step with almost covalent bond strength with, e.g. an streptavidin-coupled enzyme of choice such as HRP or another peroxidase.
• an streptavidin-coupled enzyme of choice such as HRP or another peroxidase.
• said biofluid sample comprising sample extracellular vesicles can be of any suitable origin.
• the biofluid sample is selected from the group consisting of plasma sample, serum sample, urine sample, saliva sample, tear drop sample, cerebrospinal fluid sample, spinal fluid sample, peritoneal sample, ascites sample, pleural sample, and joint fluid sample, preferably serum and/or plasma samples.
• the sample is of human origin.
• said control extracellular vesicles may further comprise a protein of interest whose presence or overexpression is considered to represent a medical condition.
• protein of interest may be a tumor marker protein which is considered to be indicative for the presence of or increased risk for developing a certain tumor.
• the method to determine the recovery rate of sample extracellular vesicles as described and provided herein may further comprise a step of detecting said protein of interest as described herein.
• the detection of said protein of interest may be conducted in any suitable way known in the art, including, e.g., employing an immune-based assay such as ELISA, EIA, Western Blot, ELISpot, and other.
• kits comprising a control extracellular vesicle as described herein above and below.
• a kit may further comprise a fluorogenic substrate or chromogenic substrate.
• said substrate can be selected from the group consisting of 2,2'- Azino-bis(3-ethylbenzthiazoline-6-sulfonis acid (ABTS), 3-amino-9-ethylcarbazole (AEC), 4- chloro-1 -naphthol (4CN), 3,3-Diaminobenzidine (DAB), 3,3',5,5-Tetramethylbenzidine (TMB), o-Phenylenediamine (OPD), Amplex Red (10-acetyl-3,7-dihy- droxyphenoxazine), tyramine, homovanillic acid (HVA), and 4-Hydroxyphenylee acetic acid, preferably from the group consisting of 2,
• the kit may further comprise a binding agent as described herein which (specifically) binds to said heterologous marker comprised by the control extracellular vesicle.
• the binding agent itself may further comprise a heterologous marker (e.g., HRP) as described herein.
• the present invention further relates to a method for preparing control extracellular vesicles as described herein, comprising the steps
• introduction of the expression system encoding said heterologous marker into a host cell in step (i) can be done in any suitable manner known in the art.
• Examples for introducing said system into a host cell include, e.g., (stable) transduction, transfection, or transformation of the expression system into the host cell.
• an “expression system” is preferably a nucleic acid molecule.
• nucleic acid molecules may comprise inter alia DNA molecules, RNA molecules, oligonucleotide thiophosphates, substituted ribo-oligonucleotides or PNA molecules.
• nucleic acid molecule may refer to DNA or RNA or hybrids thereof or any modification thereof that is known in the art incl. LNAs (locked nucleic acids) (see, e.g., US 5525711 , US 471 1955, US 5792608 or EP 302175 for examples of modifications).
• the polynucleotide sequence may be single- or double- stranded, linear or circular, natural or synthetic, and without any size limitation.
• the polynucleotide sequence may be genomic DNA, cDNA, mitochondrial DNA, mRNA, antisense RNA, ribozymal RNA or a DNA encoding such RNAs or chimeroplasts (Gamper, Nucleic Acids Research, 2000, 28, 4332 - 4339).
• Said polynucleotide sequence may be in the form of a vector, plasmid or of viral DNA or RNA.
• nucleic acid molecules which are complementary to the nucleic acid molecules described above and nucleic acid molecules which are able to hybridize to nucleic acid molecules described herein.
• a nucleic acid molecule described herein may also be a fragment of the nucleic acid molecules in context of the present invention. Particularly, such a fragment is a functional fragment. Examples for such functional fragments are nucleic acid molecules which can serve as primers.
• the heterologous marker can thus be provided by direct transfer into producer cells using cell-transfection procedures known to a person skilled in the art.
• this molecule can be supplied either in its native form (i.e. unprotected) or in a protected form.
• Protected RNA relates to modifications in the RNA molecule that enhance the stability of the molecule by reducing RNase activity (see Fisher et al., Nucleic Acids Res (1993), 21 : 3857-3865). Examples of modifications are phosphorothioate bonds, 2'-0-methyl bases and 2'fluoro bases.
• the heterologous RNA molecule can be transcribed from an expression cassette, either transfected in cells as a double stranded DNA fragment, as part of a plasmid, or after integration of the expression cassette in the genome.
• an expression cassette consists of a promoter, a sequence that is transcribed and a sequence that terminates transcription. Additional elements can be added to improve the transcription process: 1) The transcribed DNA fragment can be fused directly to the DNA that encodes the self-assembling protein; 2) The transcribed DNA encodes a heterologous marker protein as described herein that is translated as a fusion protein to the self-assembling protein.
• the heterologous RNA marker molecule can be recruited to the vesicles by a-specific binding to the self-assembling protein (see Comas-Garcia et al., eLife (2017), 6: 1-27) or by specific recruitment to the self-assembling protein via the retroviral packaging signal psi (see Comas- Garcia et al., loc cit).
• the heterologous marker is recruited by a nucleotide- binding polypeptide that recognizes a specific nucleotide target sequence.
• the nucleotide-binding polypeptide can be fused directly to the self-assembling protein.
• Nonlimiting examples of RNA-binding polypeptides are Lambda N, RNA-binding domain of MS2, NS3 and Cas9.
• the nucleotide target sequence can be used directly as heterologous marker, or can be fused to the heterologous marker sequence.
• Non-limiting examples of nucleotide target sequences are Nutl, MS2, NS3 aptamers and gRNAs.
• suitable host cells into which said expression system is introduced include HEK293 and HEK293T, whereinHEK293 is preferred for stable expression and HEK293T is preferred for transient expression (particularly using plasmids carrying SV40 origins such as, e.g., pcDNA3.1(+)).
• cultivation of the cells into which said expression system has been introduced according to step (ii) can be done in any medium suitable for the respective host cells.
• both introduction of said expression system into the cells according to step (i) and cultivation if said host cells according to step (ii) is done in a manner to allow constant release of control extracellular vesicles into the medium.
• such medium in which the cells are cultivated according to step (ii) of the method provided herein is free of fetal calf serum (FCS).
• FCS fere medium has the benefit that it is free of markerless (bovine) exogenous EVs.
• an expression system encoding a membrane protein as defined herein is introduced into said host cell.
• said expression system encoding a membrane protein may either be the same as that encoding said heterologous marker, or be another expression system, i.e. the molecules (nucleic acid molecules) encoding said heterologous marker and said membrane protein may be the same molecule or be two different molecules.
• the present invention further relates to an alternative method for preparing control extracellular vesicles as described herein, comprising the steps
• said heterologous marker can be chemically conjugated by any method known in the art, e.g., as described in Henna Matero’s Master’s Thesis “HRP-labeling of bacterial extracellular vesicles for transmission electron microscopy imaging” (2022).
• said control extracellular vesicles may further comprise a membrane protein as defined herein.
• the cells from which the EVs are derived from may naturally encode such proteins.
• an expression system encoding such membrane protein has been introduced (e.g., via (stable) transduction, transfection or transformation) to the cells releasing said EVs.
• the present invention further relates to a method to calibrate a device for sample extracellular vesicles analysis, the method comprising:
• control extracellular vesicles comprise: a heterologous marker wherein said marker is located (i) on the outer side or (ii) on the inner side (preferably on the outer side) of the control extracellular vesicle and which is suitable for photometric, colorimetric, and/or fluorescent detection (preferably colorimetric or photometric detection), to obtain a mixture,
• the term 'to calibrate' as used herein means to define the appropriate settings of a device to analyze sample EV.
• the term 'a property' as used herein means the concentration and/or a specific characteristic like the size, the morphology, the presence of EV markers, the zeta potential etc., of EV.
• the method to calibrate a device for sample extracellular vesicles analysis further comprises in step (c) adding a fluorogenic or chromogenic substrate.
• the present invention further relates to a method to evaluate the isolation of extracellular vesicles, the method comprising: (a) mixing a biofluid sample as described herein comprising sample extracellular vesicles with a known amount of control extracellular vesicles, wherein the control extracellular vesicles comprise: a heterologous marker wherein said marker is located (i) on the outer side or (ii) on the inner side (preferably on the outer side) of the control extracellular vesicle and which is suitable for photometric, colorimetric, and/or fluorescent detection (preferably colorimetric or photometric detection), to obtain a mixture,
• the method to evaluate the isolation of extracellular vesicles further comprises in step (c) adding a fluorogenic or chromogenic substrate. It is preferred in context of the present invention that chemiluminescent or bioluminescent substrates are excluded.
• Figure 1 shows the structure of three HRP fusion proteins.
• the structure of three HRP fusion proteins A, B and C is shown with domains derived from the human CD63 protein and the two Epstein-Barr virus glycoproteins gH (encoded by BXLF2) and gp350 (encoded by BLLF1), respectively.
• the domains are fused via ‘linker’ sequences composed of the amino acids (aa) serine and glycine. Scale numbers indicate aa residues.
• the illustrations on the right represent the presumed orientation of the fusion proteins in the EV membrane.
• TM transmembrane domain
• cyto tail cytoplasmic domain of gp350
• SP signal peptide
• EX extraluminal
• IN intraluminal
• C C-terminus
• N N-terminus.
• Figure 2 shows the expression of HRP on the surface of a modified HEK293 suspension cell line, HEK293-HRP, as revealed by flow cytometry.
• a retroviral transduction system was used to achieve stable expression of HRP as a fusion protein according to Construct A in Figure 1.
• a stable HEK293 suspension cell line expressing HRP was generated by retroviral transduction followed by column-based magnetic cell sorting for cell membrane anchored HRP. After multiple passages of the sorted cells in serum-free synthetic cell culture medium, HRP surface expression in the retrovirally transduced cells (right panel) was measured by flow cytometry using an anti-HRP Alexa647- coupled antibody and compared to parental HEK293 cells (left panel).
• SSC-A area of sideward scatter; numbers within the gates: percentage of HRP- positive cells.
• Figure 3 shows in (A) the concentration and in (B) size distribution of EVs derived from HEK293-HRP suspension cells.
• C shows the HRP enzymatic activity of conditioned cell culture medium obtained from this cell line, and
• D shows an HRP-specific colorimetric ELISA assay.
• the HEK293-HRP suspension cell line was cultivated for three days in serum-free medium supplemented with 1 pM hemin (bovine origin; H9039 by Sigma-Aldrich).
• the conditioned medium (CM) containing HRP-positive extracellular vesicles (HRP + EVs) was harvested and centrifuged (10 min at 300 g, 20 min at 5,000 g). Nanoparticle Tracking Analysis was used to determine EV concentration (A) and size distribution (B).
• HRP activity was measured as a function of volume directly by colorimetric detection (C) or after antibody-mediated immobilization of extracellular vesicles (D).
• C TMB substrate was added directly to different volumes of conditioned cell culture medium. The reaction was terminated after 15 min by adding 0.1 M sulfuric acid and absorbance was measured at 450 nm using a plate reader.
• D Wells of an ELISA plate were coated with 50 pL of a solution (5 pg/mL) of an HRP-specific antibody overnight. On the next day, the plate was washed with ELISA wash buffer (PBS/0.05% Tween-20) and blocked with 5% (w/v) milk powder in PBS for two hours at 37 °C. Different indicated aliquots of conditioned medium were diluted in PBS (final volume 50 pL) and added to the wells at 37 °C for two hours. The wells were washed with wash buffer and TMB substrate was added to measure HRP activity. The reaction was stopped after 15 min with sulfuric acid as in A. CM: conditioned medium; OD: optical density.
• NTA nanoparticle tracking analysis
• Figure 5 shows the separation of HRP + EVs from conditioned medium by sizeexclusion chromatography.
• 500 pL of conditioned medium from HEK293-HRP suspension cells were loaded on a 10 mL Sepharose column (Izon Inc.) and separated by size exclusion chromatography according to the manufacturer’s protocol. The enzymatic activity in each fraction was measured by adding TMB substrate.
• Figure 6 shows the calculation of the enzymatic activity by using a HRP standard as an external reference and the HRP activity of purified HRP + EVs as function of absolute EV numbers.
• a serial dilution of a commercially available, purified HRP enzyme with known HRP activity (263 U/mL, Thermo Fisher) was used to establish a calibration curve that shows the direct correlation between the absorbance of the HRP processed TMB substrate and the enzyme concentration. A trendline was calculated using linear regression function (y 4.14' 5 x). HRP was added to 100 pL PBS and HRP activity was measured by adding TMB substrate stopping the reaction after 10 min with sulfuric acid.
• HRP + EVs contained in 30 mL conditioned medium from HEK293-HRP suspension cells were pelleted by ultracentrifugation (100,000 g, 2h, 4 °C). The particle concentration was determined by nanoparticle tracking analysis and discrete particle numbers were dilured in 100 pL PBS. HRP activity was determined by adding TMB substrate and stopping the reaction after 10 min with sulfuric acid. OD: optical density; one enzyme unit catalyzes the production of 1 mg of purpurogallin from pyrogallol at 20°C and pH 6.0 in 20 seconds.
• Figure 7 shows the stability of detecting HRP in human serum spiked with HRP + EVs.
• 100 pL of a serum pool (mixture of three human sera) and 50 pL of conditioned medium from HRP-expressing HEK293 cells containing approximately 7x10 7 EVs were mixed and incubated at 4 °C for 0, 24, 48 or 72 hours as indicated.
• EVs were isolated by size exclusion chromatography (SEC) using a Sepharose 4B-packed spin column.
• SEC size exclusion chromatography
• the HRP activities in the isolated EV fractions from the different serum/EV mixtures were measured as a function of volume (1 , 2, 5, 10 and 20 pL) in a total volume of 100 pL topped with PBS.
• the reaction with TMB substrate was stopped after 10 min with sulfuric acid.
• OD optical density
• SEC size exclusion chromatography.
• Figure 8 shows an example of how HRP + EVs can be used as an internal reference to back-calculate the original concentration of serum EVs after their preparation with a commonly used EV isolation method.
• 200 pL of a serum pool (mix of three human sera) and 50 pL of conditioned medium from HRP-expressing HEK293 cells containing approximately 7x10 7 EVs (corresponding to approx. 50 pU HRP) were mixed and incubated for 10 min. EVs were isolated using the ExoQuick EV isolation kit according to the manufacturer's protocol.
• the HRP enzymatic activity was measured in 5, 10 and 20 pL of the serum/HRP + EV mix prior to ExoQuick isolation (‘Input’) and after isolation (‘Output’).
• Output fraction of the ExoQuick eluate was measured by NTA.
• a method to determine the recovery rate of sample extracellular vesicles comprising:
• control extracellular vesicles comprise a heterologous marker wherein said marker is located (i) on the outer side or (ii) on the inner side of the control extracellular vesicle and which is suitable for colorimetric, photometric, and/or fluorescent detection, to obtain a mixture
• step (d) determining the recovery rate of said sample extracellular vesicles based on the ratio of the amount of control extracellular vesicles detected in step (c) after the isolation step to the known amount of control extracellular vesicles mixed with the biofluid sample prior to the isolation step.
• heterologous marker is a protein.
• said control extracellular vesicles further comprise a membrane protein, and wherein said heterologous marker is connected to said membrane protein.
• membrane protein is selected from the group consisting of transmembrane proteins, Gi-coupled proteins, membrane-anchored receptors, tetraspanins, type I or II or III transmembrane proteins, and single or multipass transmembrane proteins.
• heterologous marker is selected from the group consisting of peroxidase, alkaline phosphatase and beta-galactosidase.
• heterologous marker is horseradish peroxidase (HRP).
• step (9) The method according to any one of items 1 to 8, wherein the amount of control extracellular vesicles detected in step (c) after the isolation step is quantitatively determined in step (d).
• step (10) The method according to any one of items 1 to 8, wherein the amount of control extracellular vesicles detected in step (c) after the isolation step is determined in step (d) at a predefined point of time after the isolation step.
• step (c) The method according to any one of items 1 to 8, wherein the amount of control extracellular vesicles detected in step (c) after the isolation step is determined in step (d) without lag time.
• step (c) The method according to any one of items 12 to 14, wherein the amount of EV detected in step (c) is determined 5 min to 4 hours after the isolation step (b).
• TMB Tetramethylbenzidine
• OPD o-Phenylenediamine
• HVA homovanillic acid
• control extracellular vesicles further comprise
• biofluid sample is selected from the group consisting of plasma sample, serum sample, urine sample, saliva sample, tear drop sample, cerebrospinal fluid sample, spinal fluid sample, peritoneal sample, ascites sample, pleural sample, and joint fluid sample.
• control extracellular vesicles further comprise a protein of interest whose presence or overexpression is considered to represent a medical condition.
• said method further comprising a step of detecting said protein of interest.
• kits comprising a control extracellular vesicle as defined in any one of the preceding items.
• ABTS 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonis acid
• AEC 3-amino-9- ethylcarbazole
• DAB 3,3'-Diaminobenzidine
• TMB 3,3',5,5'-
• step (i) an expression system encoding a membrane protein as defined in any one of items 3 to 6 is introduced into said host cell.
• control extracellular vesicles with conjugated heterologous markers from said medium.
• said control extracellular vesicles further comprise a membrane protein as defined in any one of items 3 to 6.
• a method to calibrate a device for sample extracellular vesicles analysis comprising:
• control extracellular vesicles comprise: a heterologous marker wherein said marker is located (i) on the outer side or (ii) on the inner side of the control extracellular vesicle and which is suitable for photometric, colorimetric, and/or fluorescent detection, to obtain a mixture,
• a method to evaluate the isolation of extracellular vesicles comprising:
• control extracellular vesicles comprise: a heterologous marker wherein said marker is located (i) on the outer side or (ii) on the inner side of the control extracellular vesicle and which is suitable for photometric, colorimetric, and/or fluorescent detection, to obtain a mixture,
• HRP horseradish peroxidase
• a HEK293 suspension cell line stably expressing HRP was generated by retroviral transduction followed by column-based magnetic cell sorting (MACS MicroBeads Technology, Miltenyi Biotec). High levels of transgene expression on the cell surface were measured by flow cytometry using a HRP-sepcific antibody (ab10183, Abeam; Figure 2). To avoid contamination of EVs released from cells with EVs that stem from fetal calf serum (FCS, a constituent of standard cell culture media), the HRP + EV-producing cells were adapted to grow in serum-free, synthetic medium (LV-MAX Production medium, Thermo Fisher). Furthermore, 1 pM of hemin (oxidized form of heme which functions as a cofactor in the HRP enzymatic active center; H9039, Sigma-Aldrich) was added to the medium.
• FCS fetal calf serum
• EVs secreted by HEK293-HRP cells into the cell culture medium were characterized ( Figure 3).
• Cells were seeded at a concentration of 0.5x10 6 cells/mL and incubated in synthetic medium supplemented with 1 pM hemin for 72 h. The supernatant was harvested, centrifuged and the EV number was enumerated by nanoparticle tracking analysis (ZetaView PMX110, Particle Metrix; Figure 3A). In parallel the size distribution was determined, showing a mean particle size of 150 nm ( Figure 3B).
• the HRP activity in the conditioned cell culture medium (CM) was furthermore quantified by using two independent read-out methods.
• TMB (3,3’,5,5’-tetramethylbenzidine, BD OptEIA, BD Biosciences) was added as HRP substrate to the conditioned medium and substrate turnover was measured by colorimetric detection (plate reader Clariostar, BMG Labtech). Based on absorbance measured in a microplate reader, a linear correlation was shown in a defined concentration range of 6x10 6 - 6x10 7 EVs (correlates to 1-10 pL of CM in Figure 3C).
• an ELISA plate was coated with an HRP-specific antibody (5 ⁇ g/mL, ab10183, Abeam).
• the yield (recovery) of EVs was evaluated in a commonly used EV purification method (ExoQuick, System Biosciences) that is often used to prepare EVs from e.g. serum samples.
• a serum sample was spiked with a certain number of HRP + EVs corresponding to 50 pU HRP enzymatic activity.
• the HRP signal was measured prior to (Input) and after EV purification (Output).
• the slopes of the linear regression lines were determined and the ratio of the Output HRP signal divided by Input HRP signal resulted in a factor of 0.42 (Figure 8).
• the recovery rate of the ExoQuick was approx. 42%. This correction factor furthermore allows the accurate enumeration of EVs in biological samples as described in the legend of Figure 8.
• HRP horseradish peroxidase
• the publicly retrievable aa sequence encompasses a signal peptide leader (aa 1-30), the enzymatic moiety (aa 31-338) and a carboxy-terminal pro-peptide (aa 339-353).
• a single enzyme subunit binds two Ca ++ ions and one heme b molecule (heme b (iron(ll)- protoporphyrin IX) as cofactors which are essential for HRP's enzymatic activity.
• Construct A (internal database entries 7516, 7518, 7520):
• the first amino-terminal transmembrane domain of human CD63 (P08962; MLA1 , TSPAN30; https://www.uniprot.org/uniprotkb/P08962/entry) encompassing aa 1-30 of CD63 (MAVEGGMKCVKFLLYVLLLAFCACAVGLIA; SEQ ID NO: 2) was fused to the enzymatic moiety of HRP, which was coupled to a flexible linker peptide (GGGGSGGGGGS; SEQ ID NO: 3) followed by the transmembrane domain and the cytoplasmic domain (LSMLVLQWASLAVLTLLLLLVMADCAFRRNLSTSHTYTTPPYDDAETYV; SEQ ID NO: 4) of the glycoprotein gp350 of Epstein-Barr virus (EBV) (https://www.uniprot.org/uniprotkb/P03200/entry).
• EBV Epstein-Barr virus
• Construct A The amino acid sequence of Construct A was reverse transcribed, codon-optimized and optimized for efficient translation following standard rules and algorithms.
• the deduced DNA nucleotide sequence was equipped with a close-to-perfect human Kozak consensus sequence, synthesized and molecularly cloned in E. coli using a standard vector plasmid.
• the DNA fragment was cloned into an appropriate expression plasmid (pcDNA3.1+) and, alternatively, into a basic retrovirus vector encoding the CD63:HRP:gp350 gene fusion, only.
• the CD63 sequence in Construct A was replaced by a conventional signal peptide (SP) (MQLLCVFCLVLLWEVGA; SEQ ID NO: 6) derived from the aa sequence of the glycoprotein gH of EBV (https://www.uniprot.org/uniprotkb/P03231/entry).
• SP signal peptide
• the design of the synthetic fusion proteins mimics a type 1 transmembrane protein with its signal peptide. It is cleaved off by a SP peptidase when the protein transverses cellular membranes to be loaded, e.g., onto EVs or localizes to the cellular plasma membrane.
• the coding DNA fragment was cloned into an appropriate expression plasmid (pcDNA3.1 +).
• the enzymatic moiety (aa 31-338) of HRP was molecularly cloned such that it became part of the frame work of the human CD63 protein, an integral membrane protein with four transmembrane domains.
• the largest extracellular moiety of CD63 in between transmembrane domain 3 and 4 was replaced by the enzymatic moiety of HRP flanked by flexible linker peptides similar to those in Construct A.
• the coding DNA fragment was cloned into an appropriate expression plasmid (pcDNA3.1 +).
• Expression plasmids carrying Construct A, B or C were transiently transfected into HEK293 or 293T cells. One day prior to transfection, the cells were plated at a density of 3 x 10 5 cells/ml in a 6-well plate. On the following day, transfection was performed by using TransIT- 293 transfection reagent (Mirus Bio), according to the manufacturer’s protocol. Per well, 2.5 pg of plasmid DNA were used.
• Retroviral vectors encoding Construct A were packaged into a retroviral envelope following standard procedure (Engels et al., Hum Gene Ther (2003), 14(12): 1155-1168.) and the virus stocks were used to transduce a subline of HEK293 cells adapted to grow in suspension in synthetic cell culture medium (LV-MAX Production medium, Thermo Fisher). Briefly, 3 x 10 6 HEK293 cells were mixed wih 2 mL of virus supernatant containing 4 pg/mL protamine sulfate in a 6-well plate. Transduction was performed by spinoculation (centrifugation at 1000 x g for 90 min, 32°C). One week later, HRP-positive cells were sorted by using anti-mouse IgG MicroBeads (130-048-401 , Miltenyi Biotec) with a mouse HRP-specific antibody

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