Method for detecting and quantifying exosomes

Abstract

The present invention relates to a method for detecting and / or quantifying exosomes in a sample. The method of the invention comprises the steps of: providing a sample known to comprise or suspected to comprise exosomes; bringing a binding agent that specifically binds to the extracellular vesicular membrane into contact with the sample; and detecting and / or quantifying the exosomes by flow cytometry and / or super-resolution microscopes, fluorophotometers, photometers, and optionally isolating the exosomes. The binding agent comprises a label and a phosphatidylethanolamine-binding lanolin sulfur. The invention also relates to methods for detecting and / or diagnosing cancer and viral infections, methods for monitoring tumor growth or viral diseases, methods for quantifying and / or determining tumor-associated exosomes, and kits comprising a binding agent and instructions for carrying out said methods.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of exosomes, in particular to a method for detecting and quantifying exosomes in a sample using a binding agent that specifically binds to phosphatidylethanolamine in the membrane of exosomes. The invention further relates to a method for detecting and / or diagnosing cancer and viral infections, a method for monitoring tumor growth or viral diseases, a method for quantifying and / or qualifying tumor-related exosomes, and a kit comprising the binding agent and instructions for carrying out the method. BACKGROUND

[0002] Exosomes were first reported in 1983 when immature red blood cells were cultured with a labeled transferrin receptor to trace the transferrin receptor from the plasma membrane into reticulocytes. It was observed that the labeled transferrin receptor was internalized into the reticulocytes and then re-packaged into small vesicles within them (Harding 1983; Pan 1983). These vesicles were later termed “exosomes” (Johnstone 1989).

[0003] Exosomes belong to the large family of membrane vesicles called extracellular vesicles (EVs), which generally include microvesicles (approximately 100-1000 nm in diameter), apoptotic bodies (approximately 500-1000 nm in diameter) and exosomes (approximately 30-150 nm in diameter) (Li 2017). Exosomes are thus the smallest type of extracellular microvesicles, arising from inward budding multivesicular bodies (MVBs), forming intraluminal vesicles (ILVs). If the MVBs fuse with the cell surface (plasma membrane), these ILVs are released into the extracellular medium as exosomes by exocytosis. Exosomes can also be produced by the Golgi apparatus. These exosomes will be released through late endosomes (McAndrews and Kallun 2019). Exosomes have a characteristic lipid bilayer with an average thickness of about 5 nm. The lipid composition of exosomes includes ceramides, cholesterol, sphingolipids, and phosphoglycerides with long-chain and saturated fatty acyl chains. The outer surface of exosomes is rich in sugar chains, such as mannose, poly-lactosamine, a-2,6 sialic acid and N-linked glycans (summarized in Li 2017).

[0004] In addition to performing many biological functions, in particular cell-to-cell communication, accumulating evidence suggests that several biological entities in exosomes, such as proteins and microRNAs, are closely related to the pathogenesis of most human malignancies (Li 2017).

[0005] As mentioned above, exosomes are produced in different compartments of the cell, such as endosomes and the Golgi apparatus. The different processes leading to exosome production are regulated by Rab GTPases. Among these GTPases, Rab4 seems to be involved in the recycling of exosomes from early endosomes, while Rabl 1 regulates the slow transport of endosomal compartments from the perinuclear recycling. It was further proposed that Rabl 1 regulates the transport of microvesicular endosomes to the plasma membrane, thereby regulating the release of exosomes (Blanc and Vidal 2018). It was shown that overexpression of Rabl 1 stimulates exosome release in K562 cells, while inhibition of Rabl 1 function reduces exosome release (Savina 1997).

[0006] Exosomes are secreted by normal cells (platelets, immune cells, etc.) and tumor cells. Exosomes can carry DNA, RNA, microRNA, proteins, and lipids. Exosome-associated proteins currently used as biomarkers include tetraspanins (CD9, CD63, CD81), immunomodulatory molecules (HLA-G, MHC I / II), membrane trafficking and fusion proteins (Rab5). It is hypothesized that the molecular composition of exosomes reflects (patho)physiological changes in their cell or tissue of origin (Jia 2017).

[0007] Viruses enter cells via the endocytic pathway. Viruses that enter via endocytosis can hijack and exploit the exosomal pathway for their own benefit. Exosomes share several features with certain viruses. These features include biogenesis, uptake by cells, and intercellular transfer of functional RNA, mRNA, and proteins. It was shown that exosomes secreted by virus-infected cells differ from their viral counterparts but can contain viral RNA and viral proteins (Crenshaw 2018). Such exosomes are referred to as “viroosomes”. Thus, the identification of exosomes (viroosomes) released by cells after viral infection, as well as the identification of viral proteins contained in said virosomes, will enable the diagnosis of viral infections with high sensitivity and independent of DNA or RNA analysis.

[0008] However, isolation, enrichment and detection of exosomes has proven to be complex (van der Pol 2012; Thind 2016; Jia 2016). Due to the complexity of body fluids, it has proven challenging to physically separate exosomes from particles of similar size and cellular origin. Various methods have been applied to isolate exosomes, such as immunocapture using antibodies, ultracentrifugation, precipitation with PEG6000, dehydration, etc. It was found that isolation of exosomes using differential ultracentrifugation resulted in co-isolation of proteins and other contaminants and incomplete separation of vesicles from lipoproteins. It was suggested to combine ultracentrifugation with microfiltration or gradients to improve purity (Tauro 2012; van Deun 2014). Furthermore, it was demonstrated that single-step isolation of extracellular vesicles by size-exclusion chromatography recovers intact vesicles more efficiently than centrifugation methods (Böing 2014), although the use of size-based techniques alone does not allow to distinguish exosomes from other vesicle types. Moreover, when applying these conventional methods, a mixed population of exosomes of different intracellular origin and additional vesicles is usually co-purified.

[0009] EVs have been used to study the phenotype of these particles by flow cytometry by means of antibodies against certain marker molecules, or alternatively by immunoblot analysis after ultracentrifugation (Willms 2016). However, such bulk analysis does not allow to study individual EVs (Willms 2016), thus, information conveyed by EVs, such as information reflecting the status of the donor cell, is lost. This severely hampers the ability to study the application of EVs in detecting specific changes within a cell, e.g. after viral infection or cancerous disease.

[0010] Commercially available exosome enrichment and / or isolation kits include the "Total Exosome Isolation Reagent" from Invitrogen (distributed by ThermoFisher Scientific), the "Exo-spin kit" from Cell Guidance Systems, the "exoEasy Maxi Kit" from Qiagen and the Exosome Isolation Pan Kit from Miltenyi Biotec.

[0011] WO 2019 / 099955 discloses a method for isolating exosomes from cultured placenta by affinity chromatography using binding agents against Rab family GTPases, etc.

[0012] WO 2021 / 209622 discloses a method for enriching exosomes targeting the ecto-part of Rab4 and / or Rabl l on the exosome membrane to identify and isolate exosomes.

[0013] However, despite the progress made in this field, the known methods do not satisfactorily detect and quantify essentially all exosomes contained in a sample, but only a fraction or a subpopulation of exosomes contained in such a sample. Therefore, there is a long-felt need in the art for a reliable, specific and sensitive method for detecting and quantifying exosomes. As exosomes originate from different intracellular sources, it would be advantageous to identify a marker that can be used to detect and quantify essentially all exosomes in a sample. SUMMARY

[0014] In a first aspect, the present application provides a method for detecting and / or quantifying exosomes in a sample, said method comprising the steps of: i) providing a sample known to contain or suspected to contain exosomes; ii) contacting a binding agent that specifically binds to the membrane of extracellular vesicles with said sample; and iii) detecting and / or quantifying exosomes, optionally isolating exosomes, by flow cytometry and / or super resolution microscopy. The binding agent comprises a label and a phosphatidyl ethanolamine binding lantibiotic. The label is preferably selected from the group consisting of an enzymatic label, a peptide label, a fluorochrome, a ligand, a magnetic or paramagnetic label and a quantum dot. The lantibiotic is preferably selected from the group consisting of duramycin and cinnamycin. According to a particularly preferred embodiment, the lantibiotic is cinnamycin.

[0015] According to one embodiment, the binding agent is bound by another binding agent that specifically binds to said binding agent or a label thereof.

[0016] According to another embodiment, the binding agent is bound to a solid surface.

[0017] According to one embodiment, the sample is a cell culture supernatant, a cell preparation obtained by lysing and / or centrifuging cells or a body fluid. The body fluid is preferably selected from the group consisting of plasma, serum, ascites, cerebrospinal fluid, bone marrow, urine, feces and bronchoalveolar lavage fluid.

[0018] According to yet another embodiment, the method further comprises one or more than one of the following steps before step i): a) suspending and / or dissolving the sample; b) subjecting the sample to ultrafiltration and / or centrifugation; and / or c) enriching the concentration of exosomes in the sample by size centrifugation and / or density centrifugation.

[0019] According to one embodiment, the sample is obtained from a subject known to have or suspected to have a disease.

[0020] According to another embodiment, the method further comprises comparing the number of exosomes in a sample of a subject known to have or suspected to have a disease to the number of exosomes known to be present in a sample of a healthy subject, wherein an increase in the number of exosomes in the sample of a subject known to have or suspected to have a disease is indicative of the presence or stage of said disease. Preferably, an increase in the number of exosomes in the sample of a subject known to have or suspected to have a disease is indicative of the presence or stage of cancer.

[0021] According to one embodiment, comparing the number of exosomes comprises applying CD profiling and t-SNE analysis, NTA tracking, and / or Zetasizer and counting beads by flow cytometry.

[0022] According to another embodiment, the method further comprises analyzing surface markers of the exosomes and / or the content of the exosomes. Preferably, the surface markers of the exosomes and / or the content of the exosomes comprise one or more than one peptide, protein, microRNA, DNA, and / or RNA. More preferably, analyzing the content of the exosomes comprises DNA mutation analysis, RNA expression, DNA methylation quantification and / or protein expression.

[0023] According to another aspect, the present application provides a method for diagnosing cancer, said method comprising the steps of: a) detecting and isolating exosomes from a sample by the method of the present application, and b) detecting in the collection of isolated exosomes obtained in step a) those exosomes presenting cancer antigens.

[0024] According to another aspect, the present application provides a method for detecting or diagnosing a viral disease, said method comprising the steps of: a) detecting and isolating exosomes from a sample by the method of the present application, and b) detecting in the collection of isolated exosomes obtained in step a) those exosomes presenting viral antigens.

[0025] According to a preferred embodiment, the viral antigen is a viral surface protein, more preferably a spike protein, most preferably a spike protein of a SARS-CoV-2 virus or a SARS-CoV-1 virus or any other virus such as Respiratory Syncytial Virus (RSV), Cytomegalovirus (CMV), Hepatitis B and Hepatitis D viruses, Influenza virus, Dengue virus, West Nile virus, Epstein-Barr virus.

[0026] According to another aspect, the present application provides a method for quantifying and / or qualifying tumor-related exosomes in a sample, said method comprising the steps of: a) detecting and isolating exosomes from a sample by the method of the present application; and b) detecting in the collection of isolated exosomes obtained in step a) those tumor-related exosomes using at least one binding agent specifically binding to a tumor antigen.

[0027] According to yet another aspect, the present application provides a method for monitoring tumor growth, said method comprising the steps of periodically quantifying the number of tumor-associated exosomes in a sample with the method of the present application, wherein an increase in the number of tumor-associated exosomes between two quantifications is indicative of tumor growth.

[0028] According to yet another aspect, the present application provides a method for monitoring viral diseases, said method comprising the step of periodically quantifying the number of virus-associated exosomes in a sample with the method of the present application.

[0029] According to another aspect, the present application provides a kit for performing the method of the present application, said kit comprising (i) a binding agent comprising a label and a phosphatidyl ethanolamine-binding lanthanolide, wherein the label is preferably selected from the group consisting of an enzymatic label, a peptide label, a fluorescent dye, a radioactive label, a ligand, a magnetic label or a quantum dot, and wherein the lanthanolide is preferably selected from the group consisting of duramycin and cinnamycin; and (ii) instructions for binding the binding agent to exosomes in a sample and applying flow cytometry and / or super resolution microscopy to detect, quantify, enrich or produce exosomes.

[0030] Further aspects and embodiments of the present application can be derived from the following detailed description and examples. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 : Staining of extracellular vesicles derived from MCF-7 cells with FITC-labeled duramycin. Extracellular vesicles in cell culture supernatant were stained with FITC-labeled duramycin and analyzed using flow cytometry. A mixed population of extracellular vesicles was detected between 10 2 and 10 4 FITC-labeled duramycin positive events, representing a mixed population of extracellular vesicles (upper dot plot). As a control (lower dot plot), cell culture supernatant was pre-incubated with 1% Triton to solubilize all extracellular vesicles. Staining with FITC-labeled duramycin did not yield positive events between 10 2 and 10 4 , confirming the accuracy of the detection signal.

[0032] Figure 2 : Titration of FITC-labeled duramycin signal in MCF-7 cell culture supernatant. Extracellular vesicles derived from MCF-7 cell culture supernatant were stained with different concentrations of FITC-labeled duramycin and analyzed by flow cytometry. A concentration-dependent decrease in signal was shown in cell culture supernatant stained with FITC-labeled duramycin (upper dot plots), whereas no signal was detected in MCF-7 cell culture supernatant pre-incubated with 1% Triton (lower dot plots).

[0033] Figure 3 Double staining of EVs derived from MCF-7. Extracellular vesicles produced by MCF-7 cell culture supernatant were stained with FITC-labeled dextran and an antibody detecting the tetraspanin protein CD9 located on the surface of EVs. Most dextran-positive EVs were also positive for CD9 (upper dot plot). Pre-incubation of MCF-7 supernatant with 1% Triton did not yield dextran single-positive events or CD9-dextran double-positive events. CD9 analysis showed some positive events, which can be an artifact caused by antibody aggregation (lower dot plot).

[0034] Figure 4 Double staining of EVs in SKBR 3 cell culture supernatant. EVs derived from SKBR-3 cell culture supernatant were stained for human epidermal growth factor receptor 2 (ERBB2 or Her2neu) and FITC-labeled dextran. HER2neu is overexpressed on the surface of tumor cells and used as a diagnostic tool for breast cancer classification. All Her2neu-positive EVs were also positive for FITC-dextran, while some dextran-positive events were not positive for Her2neu (left dot plot, upper row). Pre-incubation of SKBR-3 cell culture supernatant with 1% Triton did not yield FITC-dextran-positive events and yielded some Her2neu false-positive events, which can represent antibody aggregation (right dot plot; upper row). Incubation of DMEM with both reagents did not yield a signal, confirming specificity (left dot plot; lower row). Pre-incubation of DMEM with 1% Triton did not yield a signal for FITC-dextran, while some Her2neu antibody-positive events were detectable (right dot plot; lower row).

[0035] Figure 5 Double staining of Spike protein 1+ EVs in cell culture supernatant. EVs derived from HEKT293 cells transfected with the SARS-CoV-2 viral spike protein were stained with FITC-labeled dextran and the 35B12 antibody detecting the Spike protein-1 protein on the surface of EVs. Single staining of Spike protein 1+ EVs confirmed functionality of the 35B12 antibody and staining with FITC-dextran confirmed detection of EVs in the Spike protein 1+ supernatant (left dot plot). Pre-incubation of Spike protein-1+ cell culture supernatant with 1% Triton did not yield a signal for FITC-labeled dextran, while the signal for Spike protein-1 was significantly reduced (middle dot plot). Staining of cell culture supernatant derived from HEKT 293 cells (HEK SN) did not yield positive signals for FITC-dextran, which detects EVs independent of their cargo, e.g. Spike protein, and no signal was detected with the 35B12 antibody (right dot plot).

[0036] Figure 6 Enrichment of EVs using ultracentrifugation. MCF-7 cell culture supernatant was centrifuged twice at 1200xg and 20°C for 15 minutes, and the supernatant was centrifuged at 14000xg and 4°C for 35 minutes. The MV pellet was resuspended in PBS and subjected to flow cytometry analysis (MV, microvesicle fraction). The supernatant was ultracentrifuged at 100000xg and 10°C for 90 minutes. The pellet was resuspended in PBS and subjected to flow cytometry analysis (Exo, UZ; exosome fraction). Both samples were stained with FITC-labeled dextrin and signal intensity was compared by flow cytometry. Signal intensity was lower when the sample was centrifuged twice (left dot plot), while the additional ultracentrifugation step significantly improved the FITC-dextrin signal intensity (right dot plot).

[0037] Figure 7 Exosome detection using CD9 signal after sorting exosomes. Sorted fractions were analyzed by flow cytometry.

[0038] Figure 8 Particle size distribution of sorted vesicles. A: Dextrin-FITC + vesicles after sorting using nanoparticle tracking analysis (NTA). + PE + vesicles after sorting using NTA.

[0039] Figure 9 Dextrin / cinnulin structure according to Hullin-Matsuda 2016.

[0040] Figure 10 ELAEXIA marker and ApoE restaining of exosomes from plasma at fasting state and 1h, 3h and 4h after meal.

[0041] Figure 11 ELAEXIA marker and ApoE staining of exosomes from plasma by flow cytometry.

[0042] Figure 12 Flow cytometry results of MCF7 cell culture supernatant stained with FITC-labeled cinnulin (upper row) and subsequently with anti-CD9 antibody (lower row).

[0043] Embodiment

[0044] Before the present application is described in detail below, it is to be understood that this application is not limited to the particular methodology, protocols and reagents described herein as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present application which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0045] The terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", (Leuenberger 1995).

[0046] Throughout this specification the use of the word "comprise" or variations such as "comprises" or "comprising", is not used as a limitation but instead to mean that other items can also be present. Similarly the words "comprise", "comprises" and "comprising" are also used in the context of describing the compositions described herein including compositions which are optional.

[0047] In the following, elements of the present application will be described. These elements list specific embodiments; however, it is to be understood that they can be combined in any way and in any number to create further embodiments. The various examples and preferred embodiments described are not to be interpreted as limiting the application to only the explicitly described embodiments. The description is to be interpreted as supporting and encompassing embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all described elements in the present application is to be considered as disclosed by the specification of the present application, unless context indicates otherwise.

[0048] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to mean the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. As used in this specification and the appended claims, the term "or" is used in its inclusive sense (and not in its exclusive sense) unless the content requires otherwise. DETAILED DESCRIPTION

[0049] Before the present application is described in detail below, it is to be understood that this application is not limited to the particular methodology, protocols and reagents described herein as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present application which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0050] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Klbl, H. Editors. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0051] The practice of the present application will employ, unless otherwise indicated, conventional methods of biochemistry, cell biology, and immunology, which are explained in the literature in the related field (see, e.g., Molecular Cloning: A Laboratory Manual, Second Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0052] In the description and in the appended claims, the word "comprise" and variations such as "comprising" or "comprises" will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps to the subject- matter as defined by the inclusion of the stated member, integer or step or group of members, integers or steps unless the context clearly requires otherwise.

[0053] The term "a" before an ordinal number includes the plural unless the context clearly requires otherwise. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the range, regardless of the effectiveness of the endpoints to yield graduated

[0054] Hereinafter, elements of the present application will be described. These elements are presented with specific application in mind; however, it should be understood that they can be combined in any manner and in any number to create additional implementations. The various examples and preferred implementations described should not be construed as limiting the application to only the explicitly described implementations. This description should be understood as supporting and including implementations that combine the explicitly described implementations with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered as disclosed by the specification of this application unless the context clearly indicates otherwise.

[0055] In the following, definitions of some of the terms frequently used in this specification are provided. These terms will have the respective defined and preferred meanings in each instance of their use in the specification.

[0056] The term "subject" as used herein refers to an individual, for example, a human, a non-human primate (e.g., chimpanzees and other ape and monkey species); a farm animal, such as a bird, fish, cow, sheep, pig, goat, and horse; a domesticated mammal, such as a dog and cat; a laboratory animal including a rodent, such as a mouse, rat, and guinea pig. The term does not indicate a particular age or sex. In a particular meaning, the subject is a mammal. In a preferred meaning, the subject is a human. The subject can be a healthy subject or a subject having or suspected of having one or more diseases. A subject having or suspected of having one or more diseases is also referred to as a patient.

[0057] The term "sample" as used herein refers to a biological material obtained from a subject. The sample can be obtained from any suitable tissue or biological fluid, such as nipple aspirate, blood, serum, plasma, ascites, cerebrospinal fluid, bone marrow, urine, stool or bronchoalveolar lavage. The sample can also be a cell culture supernatant or a cell preparation obtained by lysing and / or centrifuging cells. According to a preferred embodiment, the sample is a body fluid, preferably selected from blood, such as whole blood, (blood) plasma, serum, ascites, cerebrospinal fluid, bone marrow, urine, stool and bronchoalveolar lavage. A particularly preferred sample is blood or plasma. Preferably, the sample is provided in a state selected from the group consisting of native, frozen, lyophilized, preserved, embedded and all possible combinations thereof. Methods for obtaining a sample from a subject are well known to the person skilled in the art. The sample can be pre-processed by methods known in the art to bring the sample into a state that allows carrying out the steps of the inventive method. For example, if the sample is in a solid, semi-solid, or substantially solid or semi-solid state, the sample can be suspended and / or dissolved. The sample can be further pre-processed by removing, for example, cell debris and larger cellular components. Such methods include, but are not limited to, centrifugation techniques, in particular ultrafiltration. According to a preferred embodiment, the sample is centrifuged at about 10,000 g to 15,000 g, preferably at about 12,000 g to separate larger solid particles such as cell debris and to retain the EVs in the supernatant. The sample after pre-processing, prior to applying the inventive method, preferably does not comprise microvesicles having a diameter of more than 1,000 nm, preferably does not comprise microvesicles having a diameter of more than 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 150 nm. Most preferably, the sample after pre-processing, prior to applying the inventive method, does not comprise microvesicles having a diameter of more than 100 nm. It is understood that the diameter of such microvesicles refers to the average diameter of a plurality or population of such microvesicles. The sample can be further pre-processed by enriching the concentration of exosomes in the sample. Suitable methods include size centrifugation and / or density centrifugation. These methods are well known to the person of ordinary skill in the art. However, such exosome enrichment is not essential and the inventive method can be carried out directly on the sample. Thus, according to a preferred embodiment of the present application, the method does not comprise any purification step of the exosomes comprised in the sample prior to the claimed method, i.e. no pre-purification, for example by ultracentrifugation, density centrifugation, fractionation and / or the use of markers for microvesicles or exosomes, such as Rab4 and / or Rabl l, tumor marker signatures or viral markers, which can bind all or a portion of the exosomes comprised in said sample. The inventive method also preferably does not require a prior exosome enrichment using any reagent binding to exosomes or subpopulations of exosomes. According to one embodiment, the exclusion of a pre-purification step comprises the exclusion of cell lysis and / or removal of cell debris in the sample. According to an alternative embodiment, the exclusion of a pre-purification step does not comprise the exclusion of cell lysis and / or removal of cell debris in the sample.According to the present application, the method can thus be performed directly on the respective sample, e.g. on blood plasma.

[0058] The inventors have developed an innovative procedure that enables the analysis of individual EVs, in particular exosomes, isolated from a sample such as human plasma. During the experiments leading to the present application, the inventors found that current markers for EVs such as CD9, CD63 and CD81 do not allow the detection of the entire population of EVs in human blood. The use of CD9, CD63 and CD81 as markers also leads to the detection of EVs from platelets. Other markers such as Calcein or CFSE, which penetrate into exosomes and are activated by esterases, also do not allow the detection of the entire population of EVs, as these activations are ATP-dependent, which can explain the heterogeneity of exosome staining.

[0059] Exosome biosynthesis is known to occur intracellularly and at the molecular level within the multivesicular body (MVB) compartment. Intraluminal vesicles, i.e. precursors of exosomes, are generated inside the MVB compartment (late endosome). The budding of the vesicle membrane is based on the absence of transmembrane asymmetry of the exosome lipid composition and phospholipids. The donor membrane of the budding vesicle is the MVB membrane (Subra 2007). Lipids such as phosphatidylserine and phosphatidylethanolamine, which are usually in the inner leaflet of the cell membrane, form the outer leaflet of the exosome membrane by this inward budding of the MVB. However, it is also known that phosphatidylethanolamine (PE)-binding duramycins and cinnamycin exhibit cytotoxicity and they can even induce PE translocation and membrane deformation at micromolar concentrations (Makino 2003).

[0060] The person skilled in the art of the present application finds that, despite the technical prejudice, PE-binding lantibiotics such as duramycins and cinnamycin can still be used to reliably detect and quantify essentially all exosomes in a sample, not just a fraction or subpopulation thereof. Thus, according to a preferred embodiment, the method of the present application comprises the following steps: i) providing a sample known to comprise or suspected of comprising exosomes; ii) contacting a binding agent that specifically binds to the membrane of extracellular vesicles with the sample; and iii) detecting and / or quantifying exosomes by flow cytometry and / or super-resolution microscopy, and optionally isolating the exosomes. According to the present application, the binding agent comprises a label and a PE-binding lantibiotic. The PE-binding lantibiotic is preferably selected from the group consisting of duramycins and cinnamycin.

[0061] Cinnamycin has an arginine instead of a lysine at amino acid position 2. If embedded in a peptide, the amino group of arginine becomes a guanidino group, and thus this amino group is not a free amino group available for labelling. Therefore, cinnamycin can only be labelled via one amino group at the N-terminus, whereas duramycin has two amino groups available for labelling, one at amino acid position 2 and one at the N-terminus. Thus, cinnamycin can only be labelled at one position compared to duramycin. Therefore, according to a preferred embodiment of the present application, the lantibiotic is cinnamycin.

[0062] The method of the present application enables isolation of exosomes without simultaneously isolating any substantial amount of other microvesicles. Thus, according to one embodiment, the method of the present application does not substantially isolate other microvesicles than exosomes. According to a preferred embodiment, the method of the present application does not isolate other microvesicles than exosomes.

[0063] According to a preferred embodiment, the exosomes in the sample are enriched prior to contacting the sample with the PE-binding agent, based on e.g. size and / or density. Such enrichment of exosomes can be performed using e.g. a Pancoll gradient (PAN-Biotech GmbH) by e.g. density centrifugation or ultracentrifugation. Other suitable gradients include, but are not limited to, Ficoll and Ficoll-Paque (both from GE Healthcare) and Biocoll (Biochrom GmbH). Methods for performing ultracentrifugation are well known to the person skilled in the art and are e.g. disclosed in Li et al., 2017, which is incorporated herein by reference.

[0064] The term "agent" as used herein denotes a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological material.

[0065] The term "binding agent" as used herein relates to any agent capable of specifically and / or selectively binding to a particular biological structure. In the context of the present application, the term "phosphatidylethanolamine-binding" is used to denote an agent that specifically and / or selectively binds to phosphatidylethanolamine (PE), in particular to PE present in the membrane of an exosome.

[0066] According to the present application, the PE-binding agent is a PE-binding lantibiotic. Lantibiotics are a known class of cyclic peptide antibiotics containing the characteristic thioether amino acid lanthionine or methyllanthionine and the unsaturated amino acids dehydroalanine and 2-aminobutyric acid. According to a preferred embodiment, the PE-binding lantibiotic is a duramycin or cinnamycin having a sequence which is at least 60% identical to SEQ ID NO: 1 (duramycin) or SEQ ID NO: 2 (cinnamycin), or which has at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity to SEQ ID NO: 1 or SEQ ID NO: 2, and which is capable of specifically and / or selectively binding to PE. Duramycin and cinnamycin are 19 amino acid long peptides which bind to the head group of PE with high specificity and affinity, as described, for example, in Navarro 1985 and Zhao 2008. According to a preferred embodiment, the PE-binding lantibiotic is duramycin. According to an alternative preferred embodiment, the PE-binding lantibiotic is cinnamycin. Both cinnamycin and duramycin are tetranactins, differing only in one amino acid at position 2: arginine for cinnamycin, lysine for duramycin. They have a high degree of sequence and structural homology. They are produced by Streptomyces sp. and Streptoverticillium cinnamoneus, respectively. The structure of duramycin and cinnamycin according to Hullin-Matsuda et al., Biochimie (2016) doi: 10.1016 / j.biochi.2016.09.020 is shown in Figure 9 Figure 1. The sequences of duramycin and cinnamycin are as follows:

[0067] SEQ ID NO: 1 (duramycin)

[0068] AKQAAAFGPFXFVADGNXL

[0069] and

[0070] SEQ ID NO: 2 (cinnamycin)

[0071] ARQAAAFGPFXFVADGNXL,

[0072] wherein X denotes a-aminobutyric acid.

[0073] According to the present application, the PE-binding lantibiotic comprises a label. The term "label" or "labeled" as used herein refers to the incorporation of a detectable marker, for example by incorporation of a corresponding labeled amino acid or by attachment of a corresponding label to the PE-binding lantibiotic. The term "label" as used herein preferably encompasses a detectable moiety which is attached to the PE-binding lantibiotic, either directly or indirectly. Such direct or indirect attachment can be through one or more covalent or non-covalent bonds. If the label is indirectly attached to the PE-binding lantibiotic, one or more linkers preferably link the label to the PE-binding lantibiotic. The linker can be any type of linker which allows the PE-binding lantibiotic to bind to PE while allowing the label to be detected or bound by another binding agent which specifically binds to the label. The linker is preferably a polypeptide linker. The label used in the context of the present application can be selected from, but is not limited to, an enzyme label, a peptide label, a fluorochrome label, a ligand label, a magnetic or paramagnetic label and a quantum dot. The ligand label can be, for example, biotin which can then be bound by avidin or streptavidin. It will be appreciated that streptavidin or avidin can alternatively be used as the label which is then bound by biotin. It will further be appreciated that a particular label can belong to more than one of the groups of labels used herein to identify different groups of labels. For example, a ligand label can at the same time be a peptide label. One non-limiting example of such a label is the preferred label Strep-tag which belongs to the group of ligand labels and the group of peptide labels. Examples of ligand and peptide labels include, but are not limited to, biotin and avidin / streptavidin and other labels such as His-tag, FLAG-tag and Strep-tag. According to a preferred embodiment, the label is a Strep-tag label, preferably a Strep-tag having the amino acid sequence WSHPQFEK (SEQ ID NO: 3) or a Twin-Strep tag having the sequence WSHPQFEK-GGGSGGGSGG-SA-WSHPQFEK (SEQ ID NO: 4) (IBA Lifesciences, Göttingen). The Strep-tag is known to bind to streptavidin, Streptactin and Steptactin XT with high affinity.

[0074] According to a particularly preferred embodiment, the label is a fluorochrome (also referred to as a fluorophore). The terms "fluorochrome" and "fluorophore" as used herein refer to a non-proteinaceous fluorescent compound that can re-emit light after photoexcitation. The fluorochrome is preferably selected from, but not limited to, the following: xanthene derivatives, such as fluorescein and its derivatives, rhodamine, Oregon Green, Eosin, and Texas Red; cyanine derivatives, such as cyanine, indocyanine, oxonol, thiacyanine, and merocyanine; squarine derivatives and ring- substituted squaraines, including Seta and Square dyes; squarine rotaxane derivatives, such as See Tau dyes; naphthalene derivatives; coumarin derivatives; oxadiazole derivatives, such as pyridyloxadiazole, nitrobenzoxadiazole, and benzoxadiazole; anthracene derivatives, such as anthraquinone, including DRAQ5, DRAQ7, and CyTRAK Orange; pyrene derivatives, such as Cascade Blue; oxazine derivatives, such as Nile Red, Nile Blue, cresyl violet, oxazine 170; acridine derivatives, such as proflavine, acridine orange, and acridine yellow; arylmethane derivatives, such as auramine, crystal violet, and malachite green; tetrapyrrole derivatives, such as porphyrin, phthalocyanine, and bilirubin; and dipyrromethene derivatives, such as BODIPY and aza-BODIPY. Preferred fluorochromes have a size of 200 to 1000 daltons. A preferred fluorochrome is Atto 488. Another particularly preferred fluorochrome according to the present application is fluorescein isothiocyanate (FITC).

[0075] According to a particularly preferred embodiment, the PE-binding lanthanolide is duramycin or cinnamycin, and the label is attached to the first or second amino acid position of duramycin (i.e., the Ala1 or Lys2 position relative to SEQ ID NO: 1), or the label is attached to the first amino acid position of cinnamycin (i.e., the Ala1 position relative to SEQ ID NO: 2). A preferred fluorochrome is Atto 488 or FITC, most preferably FITC. Particularly preferred embodiments are duramycin labeled with FITC at position Ala1 or Lys2, and cinnamycin labeled with FITC at position Ala1. A preferred binding agent in the context of the present application is duramycin labeled with FITC, preferably at position Ala1 or Lys2. Another preferred binding agent in the context of the present application is cinnamycin labeled at position Ala1, preferably with FITC. According to a particularly preferred embodiment, the binding agent is cinnamycin labeled with FITC at position Ala1. According to another preferred embodiment, the binding agent is cinnamycin labeled with a Strep-tag at position Ala1. As mentioned above, there can be a linker (preferably a polypeptide linker) between the PE-binding lanthanolide and the label, e.g., between cinnamycin and the Strep-tag.

[0076] According to the present application, exosomes are detected and / or quantified by flow cytometry and / or super-resolution microscopy. The term "flow cytometry" as used herein refers to a technique used to detect and measure physical and chemical characteristics of cells or particle populations. Flow cytometry is a sorting technique that allows rapid analysis of individual cells or other particles flowing through a single or multiple detectors while suspended in solution. Cell sorting generally describes the process of purifying or enriching a population of cells based on the presence or absence of a particular physical characteristic. Using flow cytometry, exosomes in a sample labeled by a PE-binding agent will be identified based on the corresponding label and will be separated from other components in the sample. Flow cytometry is an established method in the art and a person of ordinary skill will readily identify various possible flow cytometers and corresponding methods for practicing the present application. Flow cytometers that can be used in this context are commercially available and are well recognized in the field of cell biology. A preferred flow cytometry technique is fluorescence flow cytometry, such as fluorescence-activated cell sorting (FACS), which requires a fluorescent label, such as a fluorochrome or quantum dot, on the PE-binding agent. Any suitable flow cytometer or cell sorter can be used to practice the present application, such as but not limited to a spectral cell analyzer or sorter. In flow cytometers with sorting capabilities, the instrument detects cells using parameters such as cell size, morphology, and protein expression. Cells are then sorted and subpopulations are recovered using droplet technology. This principle can also be applied to purify or enrich, for example, cellular components such as exosomes. Other detection methods can be luminometry, fluorimetry, confocal laser microscopy, or super-resolution microscopy analysis and quantification.

[0077] To detect the binding agent, according to one embodiment of the present application, the binding agent can be bound by a further (second) binding agent that specifically binds to the binding agent (first binding agent) bound to the PE. The binding of the further second binding agent to the first binding agent can be at any moiety of the first binding agent, e.g. the PE-binding lantibiotic or a label of the first binding agent. The further (second) binding agent can be, for example, an antigen binding agent, e.g. an antibody or an antigen binding fragment of an antibody. The second binding agent can also comprise a label, which can be selected from the group consisting of an enzymatic label, a fluorescent dye, a magnetic label (e.g. magnetic beads) and a peptide label as disclosed herein. Examples of peptide labels include, but are not limited to, biotin and avidin / streptavidin and other labels such as His-tag, FLAG-tag and Strep-tag. According to a preferred embodiment, the label is a Strep-tag label, preferably a Strep-tag having the amino acid sequence WSHPQFEK (SEQ ID NO: 3) or a Twin-Strep tag having the sequence WSHPQFEK-GGGSGGGSGG-SA-WSHPQFEK (SEQ ID NO: 4) (IBA Lifesciences, Göttingen). The Strep-tag is known to bind to streptavidin, Streptactin and Steptactin XT with high affinity. Commonly used enzymatic labels, fluorescent dyes and magnetic labels can be used in the context of the present application without any specific limitation. Respective examples include, but are not limited to, FITC, rhodamine, lanthanide phosphors for fluorescent labeling; horseradish peroxidase, beta-galactosidase, luciferase and alkaline phosphatase for enzymatic labeling; and chemiluminescent labels. Quantum dots can also be used as labels. Quantum dots are semiconductor nanocrystals with a wide excitation spectrum, a narrow emission spectrum, a tunable emission peak, a long fluorescence lifetime, negligible photobleaching and the ability to bind to proteins. The use of quantum dots in the present application is also not specifically limited.

[0078] According to preferred embodiments, the label is selected from the group consisting of a fluorescent dye, a ligand such as biotin or a Strep-tag peptide, a magnetic or paramagnetic label and a quantum dot. According to a specific embodiment, the label is not an enzymatic or enzymatic label, e.g. horseradish peroxidase. According to another specific embodiment, the label is not a gold label.

[0079] In certain embodiments, the label is connected by a spacer arm of different length to reduce potential steric hindrance. Such spacers can be, for example, chemical spacers or amino acid spacers.

[0080] If the further (second) binding agent is an antigen binding agent, it "specifically binds" to the target antigen when the dissociation constant (K d )≤10 -7 M. When Kd ≤ 5 x 10 -9 M, the binding agent specifically binds its antigen with "high affinity", when Kd≤ 5 x 10 -10 M, with "very high affinity", preferably Kd≤ 5 x 10 -11 M.

[0081] A binding agent is "selective" if it binds one target more tightly than it binds a second target, if the binding agent is an antigen binding agent.

[0082] In an alternative embodiment of the application, the first (PE) binding agent and / or the second binding agent are covalently or non-covalently bound on a solid surface, such as in an array setup. Such an array structure can be used to bind the exosome to the respective probe comprising the binding agent. Thus, the application also provides an array comprising the first binding agent and / or the second binding agent, and the use of such an array for detecting and / or quantifying exosomes in a sample. If bound to the array, the exosome can be released after washing the array to remove any contaminants.

[0083] The conditions allowing the first binding agent and / or the second binding agent to bind to the sample depend inter alia on the type of binding agent used and the type of sample. The person of ordinary skill can easily determine these conditions.

[0084] Detecting the first binding agent and / or the second binding agent can be performed by any method known in the art. If a label is attached, it is envisaged that the label is detected. For example, a biotin moiety can be detected by a labelled avidin (e.g. streptavidin containing a fluorescent marker or enzymatic activity, which can be detected by optical or colorimetric methods), a fluorescent marker can be detected by exciting the fluorophore and detecting the fluorescence emitted by the fluorophore. The respective detection methods are well known to the person skilled in the art.

[0085] According to the present application, exosomes can also be detected and / or quantified by applying super-resolution microscopy (Neice 2010 and Stockert 2017; both incorporated herein by reference). While conventional microscopy techniques like electron microscopy have the ability to resolve individual EVs, they do not easily allow for the detection of different markers and are limited to fixed cells. For conventional optical microscopy techniques, e.g. confocal optical microscopy, many proteins can be labeled, but the small size of EVs means that most of them are well below the resolution limit of optical microscopy, limiting the usefulness of these techniques in identifying different subpopulations of vesicles. Super-resolution microscopy, on the other hand, enables detection and quantification at the sub-vesicular level. This technology enables single-molecule fluorescence microscopy and can be used to track corresponding labeled vesicles in solution. Based on the trajectories of the vesicles and corresponding diffusion measurements, the particle size distribution and concentration of the EV population in the sample can be quickly estimated. Super-resolution microscopy for the detection of extracellular vesicles is described in, e.g. Gebara 2022, incorporated herein by reference.

[0086] The method of the present application is preferably performed with a sample obtained from a subject known or suspected to suffer from one or more diseases. In this case, the method can further comprise the step of comparing the number of exosomes in the sample of the subject known or suspected to suffer from a disease to the number of exosomes known to be present in a sample of a healthy subject. An increase in the number of exosomes in the sample of the subject known or suspected to suffer from a disease thus indicates the presence or stage of the disease. According to a preferred embodiment, an increase in the number of exosomes in the sample of the subject known or suspected to suffer from a disease indicates the presence or stage of a viral infection or cancer.

[0087] According to the present application, when comparing the number of exosomes, this step is preferably performed by applying CD profiling, t-SNE analysis, nanoparticle tracking analysis (NTA) or using a Zetasizer and counting beads by flow cytometry. CD profiling comprises identifying the expression of different cluster of differentiation (CD) proteins on the enriched or isolated exosomes and comparing the CD protein expression of one sample to the CD protein expression of another sample. The CD expression profiles can subsequently be combined in a profile. T-distributed Stochastic Neighbor Embedding (t-SNE) is a machine learning algorithm for visualization. It is a nonlinear dimensionality reduction technique for visualization. It is particularly well suited to the visualization of high-dimensional datasets. Specifically, it models each high-dimensional object by a two- or three-dimensional point such that similar objects are modeled by nearby points and dissimilar objects are modeled by distant points. The t-SNE technique is well known to the person skilled in the art.

[0088] According to a preferred embodiment, the method further comprises analyzing surface markers of the exosomes and / or the content of the exosomes. Surface markers are in particular presented on the surface of the exosomes. The term exosomes "present" or "carry" a molecule such as a peptide, protein or antigen as referred to herein means that the molecule is at least partially accessible from the outside of the exosome. For example, the respective molecule can be located on the outer membrane of the exosome or at least a part of the molecule can be located on the outer membrane of the exosome. The respective presented or carried molecule can also be rotated or extended through the membrane of the exosome.

[0089] The term "extravesicular portion" as used herein denotes a portion of a molecule such as a peptide or protein present in or on an exosome which is accessible from the outside of the exosome.

[0090] The analysis of the surface markers of the exosomes and / or the content of the exosomes allows for further characterizing the exosomes and drawing conclusions, for example, on the health status of the subject of origin of the sample. According to a particularly preferred embodiment, said surface markers of the exosomes and / or said content of the exosomes comprise one or more than one of a peptide, a protein, a microRNA, DNA and / or RNA. The content of the exosomes can be analyzed by applying, for example, DNA mutation analysis, analyzing RNA expression, quantifying DNA methylation and / or quantifying / determining protein expression.

[0091] For example, for the development of a tumor-specific exosome test, it is necessary to detect and purify all or at least most of the exosomes from normal and tumor cells and to isolate and enrich tumor-specific exosomes (oncosomes) in high purity and high quality for subsequent tumor biomarker analysis in clinical diagnostics. By using, for example, blood of a cancer patient as test sample, exosome characterization can serve as a "liquid biopsy" providing an alternative, less invasive sampling modality which can even be applied when tumor tissue is not easily accessible. The corresponding analysis methods also enable screening for cancer, monitoring of therapy, disease progression and recurrence. Thus, the present invention provides a method for diagnosing cancer. The method comprises the steps of a) detecting and isolating exosomes from a sample by the method of the present invention and b) detecting those exosomes presenting a cancer antigen in the isolated exosome collection obtained in step a). The present invention is not particularly limited to the detection of any specific cancer antigen, the person of ordinary skill in the art will select one or more than one suitable cancer antigen for detection. A preferred cancer antigen is GPER-1 (G protein-coupled estrogen receptor 1). Another preferred cancer antigen is HER2. A significant increase in the number of exosomes presenting a cancer antigen and preferably GPER-1 or HER2 compared to a reference sample of a healthy person indicates that the person is at risk of or has cancer. Preferably, GPER-1 or HER2 is detected by using a binding agent that specifically and / or selectively binds to GPER-1 and HER2. Preferably, the binding agent is an antigen binding agent, more preferably an antibody against GPER-1 or HER2. The method for diagnosing cancer can preferably be used for diagnosing breast cancer. In addition to or as an alternative to the detection of GPER-1 or HER2, GPER-5, CD247 (T cell surface glycoprotein CD3 zeta chain; cluster of differentiation 247) and / or phosphatidylserine can also be used to detect such oncosomes. Other cancer surface markers associated with exosomes that can be analyzed according to the present invention are CD49b, CD90 and CD202b.

[0092] According to another aspect, the present invention provides a method for quantifying and / or qualifying tumor-associated exosomes in a sample. The method comprises the steps of a) detecting and isolating exosomes from a sample by the method of the present invention and b) detecting those tumor-associated exosomes in the isolated exosome collection obtained in step a) using at least one binding agent that specifically binds to a tumor antigen.

[0093] Such tumor-associated exosomes (or oncosomes) can be identified by comparing the exosome population of a healthy person to the exosome population of a person suspected of having cancer or known to have cancer. The tumor antigen can be selected from any tumor antigen known in the art. According to a preferred embodiment, the tumor antigen is selected from the group consisting of GPER-1, GPER-5, HER2, CD247 and phosphatidylserine. Other cancer surface markers associated with exosomes that can be analyzed according to the present application are CD49b, CD90, CD274 and CD202b.

[0094] According to another aspect, the present application provides a method for monitoring tumor growth. The method comprises the step of periodically quantifying the number of tumor-associated exosomes in a sample using the method of the present application. An increase in the number of tumor-associated exosomes between two quantifications is indicative of tumor growth.

[0095] According to another aspect, the present application provides a method for diagnosing a viral disease. The method comprises the steps of a) detecting and isolating exosomes from a sample by the method of the present application and b) detecting those exosomes presenting a viral antigen in the collection of isolated exosomes obtained in step a). According to a preferred embodiment, the viral antigen is a viral surface protein. The viral surface protein is preferably a spike protein, more preferably a spike protein of the SARS-CoV-2 virus or the SARS-CoV-1 virus. The viral antigen can also be a subunit of the spike protein, such as subunit 1 or subunit 2, or a derivative peptide thereof. Alternatively, the viral protein can be associated with any other viral infection, such as a hepatitis B virus, hepatitis C virus, hepatitis D virus or hepatitis E virus, RSV, EBV, influenza A / B virus, West Nile virus, Zika virus, Dengue virus or Ebola virus infection. According to a particularly preferred embodiment, the viral antigen is a spike protein. A particularly preferred spike protein is a spike protein of the SARS-CoV-2 virus or the SARS-CoV-1 virus. Alternatively, the spike protein can be from any other virus.

[0096] The present application further provides a method for monitoring a viral disease. The method comprises the step of periodically quantifying the number of virus-associated exosomes in a sample using the method of the present application. The presence of virus-associated exosomes containing virus-derived proteins allows the diagnosis and monitoring of the viral disease. The virus-derived proteins are preferably viral antigens. According to embodiments, the viral antigens are viral surface proteins. The viral surface proteins are preferably spike proteins, more preferably spike proteins of the Riboviria, most preferably spike proteins of the SARS-CoV-2 virus or the SARS-CoV-1 virus. The viral antigens can also be subunits of spike proteins, such as subunit 1 or subunit 2, or derived peptides thereof. Alternatively, the viral proteins can be associated with any other viral infection, such as hepatitis B virus, hepatitis C virus, hepatitis D virus or hepatitis E virus, RSV, EBV, influenza A / B virus, West Nile virus, Zika virus, Dengue virus or Ebola virus infection.

[0097] Any of the methods described herein can further comprise the step of comparing the number of exosomes, preferably disease-associated exosomes, in a sample of a subject known or suspected to have a disease, to the number of similar exosomes known to be present in a sample of a healthy subject. An increase in the number of exosomes in a sample of a subject known or suspected to have a disease thus indicates the presence or stage of the disease. The term "disease-associated exosomes" is intended to refer to exosomes comprising peptides associated with a disease, such as viral peptides and antigens and cancer peptides and antigens described herein. The disease to be detected or monitored according to the present application is preferably a cancer. In such methods, comparing the number of exosomes can comprise applying CD profiling and t-SNE analysis, NTA tracking, and the use of Zetasizer, such as by flow cytometry applying counting beads.

[0098] In addition to those markers associated with a disease (e.g. viral or cancer peptides and / or antigens), additional analysis of other markers such as proteins, peptides and / or antigens can allow localizing the disease to a specific organ or tissue. For example, disease-associated exosomes can additionally carry or comprise one or more markers of a specific organ or tissue, e.g. cardiac troponin of the heart. Thus, additional identification of organ- or tissue-specific markers or markers associated with a group of tissues or organs in a disease-associated exosome population allows associating the disease-associated exosomes with a tissue or organ, thereby associating the disease with that tissue or organ. Thus, for further investigation of the enriched or isolated exosome population of a sample, other surface markers of exosomes and / or the content of exosomes can be analyzed. Such other surface markers include, but are not limited to, beta-amyloid, 14-3-3 protein, actin, ADAM10, Alix, alpha-enolase, alpha-synuclein, aminopeptidase N, annexin 5A, annexin A2, AP-1, ATP citrate lyase, ATPase, Basigin, Caveolin-1, Clathrin, Occludin-1, Sortilin-1, EGFR, Ep-CAM, ICAM, HLA-ABC, Prostate-specific antigen, Rab-14, Rab-7, Syndecan, Tumor-associated glycoprotein, Tetraspanin-8, Tsg101, Vps35, CD2, CD3, CD5, CD8, CD9, CD11a, CD11b, CD11c, CD13, CD29, CD37, CD41, CD44, CD49d, CD49f, CD62L, CD63, CD68, CD80, CD81, CD86, CD90, CD142, CD146, CD163, CD192, and CD202b.

[0099] Specific examples of markers that can associate a disease such as a viral infection with a specific organ or tissue include surfactant-associated protein A (SP-A) and surfactant-associated protein B (SP-B) for the lung, cardiac troponin for the heart, von Willebrand factor and CD31 / PECAM-1 for endothelial cells, enolase-2 (ENO2) and neuron-specific enolase (NSE) for the brain or neural tissue, asialoglycoprotein receptor 1 (ASGR-1) for the liver, and aquaporin 6 for the kidney. These markers are particularly suitable for associating a viral infection with a specific organ or tissue as a source of viral replication, more preferably for associating a SARS-CoV or influenza A / B virus infection with a specific organ or tissue, most preferably for associating a SARS-CoV-2 virus infection with a specific organ or tissue.

[0100] Analysis of the contents of the exosomes can include lysis of the exosomes. Contents to be analyzed include, but are not limited to, peptides, proteins, microRNAs, DNA and / or RNA such as mRNA. Proteins to be analyzed typically include, but are not limited to, platelet-derived growth factor receptors, lactadherins, transmembrane proteins and lysosome-associated membrane protein 2B, membrane transport and fusion proteins such as annexins, flotillins, GTPases, heat shock proteins, tetraspanins, proteins involved in multivesicular body biogenesis, and lipid-associated proteins and phospholipases. Analysis of the contents can include DNA mutation analysis, RNA expression, DNA methylation quantification and / or protein expression, as well as fluorescent flow cytometry. In the case of nucleic acids, these can be quantified, for example, by methods known in the art, for example involving RT-PCR, to identify their profile. Exosomes released from virus-infected cells contain Rabl 1, which enables virion recognition and application of specific secondary antibodies against specific viral proteins to diagnose and monitor viral diseases. Analysis of exosome contents and surface proteins and peptides is not limited to detection and monitoring of cancer or viral infection, but can be used universally to detect or characterize many medical conditions and diseases.

[0101] According to another aspect, the present application provides a kit for carrying out any of the methods of the present application described herein. The kit comprises a binding agent comprising a label and a phosphatidyl ethanolamine-binding lanthanolide, and instructions for binding said binding agent to exosomes in a sample and applying flow cytometry and / or super resolution microscopy to detect, quantify, enrich or produce exosomes. The binding agent is as defined above. The label is preferably selected from the group of enzymatic labels, peptide labels, fluorochromes, ligands, magnetic labels or quantum dots, and the lanthanolide is preferably selected from the group of duramycins and cinnamycin. The kit can further comprise a further (second) binding agent as defined herein above, which second binding agent can comprise a label as defined above. The kit can also comprise means and / or instructions for preparing the sample prior to adding the binding agent to the sample.

[0102] The present application is further described by the following examples, which are to be construed as merely illustrative, and not limitative of the scope of the application.

[0103] Clauses of the present application

[0104] The present application also relates to the following clauses:

[0105] Clause 1 : A method for producing an enriched fraction of exosomes from a sample, the method comprising the steps of:

[0106] i) providing a sample known to comprise or suspected of comprising exosomes;

[0107] ii) contacting a binding agent that specifically binds to the membrane of extracellular vesicles with the sample; and

[0108] iii) detecting and / or quantifying exosomes by flow cytometry and / or super resolution microscopy, and optionally isolating exosomes;

[0109] wherein the binding agent comprises a label and a phosphatidyl ethanolamine binding lantibiotic,

[0110] wherein the label is preferably selected from the group consisting of an enzymatic label, a peptide label, a fluorescent dye, a radioactive label, a ligand, a magnetic or paramagnetic label and a quantum dot, and

[0111] wherein the lantibiotic is preferably selected from the group consisting of duramycin and cinnamycin.

[0112] Clause 2: The method according to clause 1, wherein:

[0113] (a) the binding agent is specifically bound by another binding agent that specifically binds to the binding agent or a label thereof; or

[0114] (b) the binding agent is bound to a solid surface.

[0115] Clause 3: The method according to clause 1 or 2, wherein the sample is a cell culture supernatant, a cell preparation obtained by lysing and / or centrifuging cells, or a body fluid.

[0116] Clause 4: The method according to clause 3, wherein the body fluid is selected from the group consisting of plasma, ascites, cerebrospinal fluid, bone marrow, urine, feces and bronchoalveolar lavage fluid.

[0117] Clause 5: The method according to any one of clauses 1 to 4, wherein the method further comprises one or more than one of the following steps prior to step i):

[0118] a) suspending and / or dissolving the sample;

[0119] b) ultrafiltrating and / or centrifuging the sample; and / or

[0120] c) enriching the concentration of exosomes in the sample by size centrifugation and / or density centrifugation.

[0121] Clause 6: The method according to any one of clauses 1 to 5,

[0122] wherein the sample is obtained from a subject known to have or suspected to have a disease, wherein the method further comprises comparing the number of exosomes in the sample of the subject known to have or suspected to have a disease to the number of exosomes known to be present in a sample of a healthy subject, wherein an increase in the number of exosomes in the sample of the subject known to have or suspected to have a disease is indicative of the presence or stage of the disease,

[0123] Preferably, wherein an increase in the number of exosomes in the sample of a subject known to have or suspected to have a disease is indicative of the presence or stage of cancer.

[0124] Clause 7: The method according to clause 6, wherein comparing the number of exosomes comprises applying CD profiling and t-SNE analysis, NTA tracking, Zetasizer and applying counting beads by flow cytometry.

[0125] Clause 8: The method according to any one of clauses 1 to 7, further comprising analyzing surface markers of exosomes and / or contents of exosomes,

[0126] Preferably, wherein the surface markers of exosomes and / or contents of exosomes comprise one or more than one of a peptide, a protein, a microRNA, DNA and / or RNA, more preferably wherein analyzing the contents of exosomes comprises DNA mutation analysis, RNA expression, DNA methylation quantification and / or protein expression.

[0127] Clause 9: A method for diagnosing cancer, comprising:

[0128] a) detecting and isolating exosomes from a sample by the method according to any one of clauses 1 to 8, and

[0129] b) detecting in the collection of isolated exosomes obtained in step a) those exosomes presenting cancer antigens.

[0130] Clause 10: A method for detecting or diagnosing a viral disease, comprising:

[0131] a) detecting and isolating exosomes from a sample by the method according to any one of clauses 1 to 8, and

[0132] b) detecting in the collection of isolated exosomes obtained in step a) those exosomes presenting viral antigens,

[0133] Preferably, wherein the viral antigens are viral surface proteins, more preferably are spike proteins, most preferably are spike proteins of SARS-CoV-2 virus or SARS-CoV-1 virus or spike proteins of any other virus.

[0134] Clause 11: A method for quantifying and / or qualifying tumor-related exosomes in a sample, the method comprising the steps of:

[0135] a) detecting and isolating exosomes from a sample by the method according to any one of clauses 1 to 8; and

[0136] b) detecting in the collection of isolated exosomes obtained in step a) those exosomes related to a tumor using at least one binding agent specifically binding to a tumor antigen.

[0137] Clause 12: A method for monitoring tumor growth, the method comprising the steps of:

[0138] periodically quantifying the number of tumor-associated exosomes in the sample using the method according to clause 11, wherein an increase in the number of tumor-associated exosomes between two quantifications is indicative of tumor growth.

[0139] Clause 13: A method for monitoring a viral disease, the method comprising the steps of:

[0140] periodically quantifying the number of virus-associated exosomes in the sample using the method according to any one of clauses 1 to 8 or clause 10.

[0141] Clause 14: A kit for carrying out the method according to any one of clauses 1 to 13, comprising:

[0142] (i) a binding agent comprising a label and a phosphatidyl ethanolamine binding lantibiotic,

[0143] wherein the label is preferably selected from an enzymatic label, a fluorochrome, a peptide label, a radioactive label, a ligand, a magnetic label or a quantum dot, and wherein the lantibiotic is preferably selected from duramycin and cinnamycin, more preferably wherein the lantibiotic is duramycin; and

[0144] (ii) instructions for binding the binding agent to exosomes in a sample and applying flow cytometry and / or super resolution microscopy to detect, quantify, enrich or produce exosomes.

[0145] Examples

[0146] Equipment and reagents

[0147] In the examples of the present application, the following equipment and reagents were used:

[0148] FACS equipment:

[0149] Sony SP6800 Spectral Analyser

[0150] Pancoll gradient:

[0151] PAN Biotech, density: 1.077 g / ml

[0152] PEG6000:

[0153] Molecular Biology Grade, Merck

[0154] Centrifuge:

[0155] Heraeus Multifuge X3 R (Article number: 75004515; Serial number: 41615170 Thermo Scientific)

[0156] Heraeus Fresco 17 (Article number 75002420; Serial number: 41284997; Thermo Scientific)

[0157] Optima LE-80K (Beckman Coulter)

[0158] Antibodies:

[0159] CD9 PE (anti-human CD9 antibody labeled with phycoerythrin) (#312106; 20 pg / ml; Biolegend)

[0160] Her2neu PE (anti-human CD340 antibody labeled with phycoerythrin) (#324406; 50 pg / ml; Biolegend)

[0161] Antibodies with the indicated labels against HLA-ABC-PE-Cy5, CD5-FITC, CD8-PE-Cy7, CD13-BV421, CD81-PE-Dazzle 594, CD41-PacBlue, CD68-FITC, CD86-BV650, GPER-1-DyeLight 405, Rab5-PE.

[0162] Other reagents:

[0163] Triton-X100 (PanReac AppliChem)

[0164] Counting beads (Count Bright Absolute Counting Beads, ThermoFisher, Article number: #C36950, Lot.: 2466363)

[0165] NaCl (0.9%; sterile solution; Braun)

[0166] RPMI (+) L-glutamine cell culture medium (Reference number: 21875-034; Gibco)

[0167] DMEM high glucose medium (Article number: D5796; 500 ml; Sigma Life Science)

[0168] Flow cytometer:

[0169] SA 3800 (Sony)

[0170] Settings used (exosome settings)

[0171]

[0172] Example 1

[0173] FITC labelling of duramycin using:

[0174] FITC labelling of duramycin was performed using a column kit from abcam (EZLabel Protein FITC labelling kit: ab288089).

[0175] Labeling reaction:

[0176] 1. One vial of EZLabel FITC is sufficient to label 1 mg of protein per vial. Before use, reconstitute one vial of EZLabel FITC with 10 μΐ of ethanol.

[0177] 2. For reconstitution, completely dissolve the material by repeated pipetting, then dilute (1 :5).

[0178] 3. Transfer 100 μΐ of prepared duramycin to a 1.5 ml microcentrifuge tube.

[0179] 4. Add 10 μΐ of reconstituted and diluted EZLabel FITC solution and mix by repeated pipetting.

[0180] 5. Incubate the mixed solution for 1 hour at room temperature on a rotating shaker or mixer.

[0181] 6. After incubation, add 20 μΐ of EZLabel quenching buffer to quench the reaction. Incubate the reaction solution for a further 30 minutes at room temperature.

[0182] 7. Use HPLC to separate different components containing a single label at amino acid position 1, amino acid position 2, and both position 1 and position 2.

[0183] Analysis of duramycin components by flow cytometry:

[0184] MCF-7 cell culture supernatant and RPMI cell culture medium without supplements (control) were incubated with 1% Triton (30 min; 400 rpm; 22°C). The samples were stained with Duramycin-FITC (1 ng / μΐ). Incubation for 30 min in the dark at 22°C. The samples were diluted 1:100 with 0.9% NaCl and analyzed using a flow cytometer (SA3800; Sony).

[0185] The results are shown in Figure 1. Duramycin-FITC binds to exosomes in the MCF-7 breast cancer cell culture supernatant (top panel). Triton treatment leads to exosome destruction / dissolution, followed by no binding (bottom panel, negative control). Figure 1 Figure 1 The results are shown in Figure 1. Duramycin-FITC binds to exosomes in the MCF-7 breast cancer cell culture supernatant (top panel). Triton treatment leads to exosome destruction / dissolution, followed by no binding (bottom panel, negative control). Figure 1

[0186] Example 2 Duramycin was FITC-labeled as described in Example 1. MCF-7 supernatant and RPMI as control were pre-incubated with 1% Triton, shaken on a Thermomixer (1 h; 22°C; 450 rpm). 10 μΐ of the Triton-treated sample and 10 μΐ of the supernatant or RPMI were stained with different concentrations of Duramycin. The samples were incubated for 30 min in the dark at room temperature. Finally, the samples were diluted 1:100 with 0.9% NaCl. The samples were measured using a SA3800 flow cytometer.

[0187] The results are shown in Figure 2. Duramycin-FITC stains exosomes in the MCF-7 breast cancer cell culture supernatant in a concentration-dependent manner (top row). Triton treatment leads to exosome destruction / dissolution, followed by no binding (bottom row, negative control).

[0188] Figure 2 The results are shown in Figure 2. Duramycin-FITC stains exosomes in the MCF-7 breast cancer cell culture supernatant in a concentration-dependent manner (top row). Triton treatment leads to exosome destruction / dissolution, followed by no binding (bottom row, negative control). Figure 2 Figure 2

[0189] Example 3 Duramycin was FITC-labeled as described in Example 1. MCF-7 cell culture supernatant and RPMI medium (control) were incubated with 1% Triton (30 min; 400 rpm; 22°C). The samples were stained with FITC-Duramycin (1 ng / μΐ) and CD9 PE antibody (1 ng / μΐ). Incubation for 30 min in the dark at 22°C. The samples were diluted 1:100 with 0.9% NaCl and analyzed using a flow cytometer (SA3800; Sony).

[0190] The results are shown in Figure 3. Duramycin-FITC binds to exosomes in the MCF-7 breast cancer cell culture supernatant (top panel). Triton treatment leads to exosome destruction / dissolution, followed by no binding (bottom panel, negative control).

[0191] ​​​The results of double staining of exosomes with duramycin-FITC and exosome marker CD9 are shown (upper panel) for SKBR-3 cells (HER2 receptor positive cell line). The exosome population positive for CD9 and duramycin-FITC is shown in the upper right quadrant. Triton treatment leads to exosome destruction / dissolution, followed by no binding (lower panel, negative control, where the upper right quadrant is empty). Figure 3 Figure 3

[0192] Example 4

[0193] Duramycin was FITC-labeled as described in Example 1. Cell culture supernatant or plasma of SKBR-3 (HER2 receptor positive cell line) cells as well as control medium were incubated with 1% Triton (1 h; 500 rpm; 22°C). The samples were stained with FITC-duramycin and HER2 antibody for double staining. Cell culture supernatant was diluted 1:100 with 0.9% NaCl; plasma was diluted 1:1000 with 0.9% NaCl. Sample analysis was performed using flow cytometry (SA3800; Sony).

[0194] The results are shown in Figure 4 . HER2 positive exosomes were double stained with HER2 antibody and FITC-duramycin (upper left panel, upper right quadrant). Notably, some exosomes were stained with FITC-duramycin but not with HER2 antibody (upper left panel, upper left quadrant), indicating the presence of a subpopulation of exosomes that do not carry HER2 on their surface. If treated with Triton, which dissolves exosomes, no signal was detected (upper right panel). No staining was observed in medium alone (DMEM) and in DMEM treated with Triton (lower left and right panels, respectively). Figure 4 Figure 4 Figure 4 Figure 4

[0195] Example 5

[0196] Duramycin was FITC-labeled as described in Example 1. To generate EVs containing high levels of SARS-CoV-2 spike protein FL (D614G), a HEK293 cell line constitutively expressing the spike protein (CID4618, Helmholtz Zentrum München) was constructed and transfected with the S expression plasmid (#7413, Helmholtz Zentrum München) prior to isolation of EVs from the conditioned medium. EVs were purified and concentrated by ultrafiltration, density gradient ultracentrifugation, followed by characterization by WB, NTA, vesicle flow cytometry and ELISA.​​​​​​

[0197] HEK293-spike protein 1 + Cell culture supernatant and HEK SN (HEK293 supernatant containing unmodified exosomes) were incubated with 1% Triton (1 hour; 500 rpm; 22°C). Samples were stained with FITC-duramycin or spike protein 1 specific monoclonal antibody 35B12. Cell culture supernatant was diluted 1:100 with 0.9% NaCl; plasma was diluted 1:1000 with 0.9% NaCl. Sample analysis was performed using flow cytometry (exosome settings; SA3800; Sony).

[0198] Results are shown in Figure 5 SARS-CoV-2 spike protein antibodies stained exosomes from HEK293 spike protein 1 + , Triton treatment significantly reduced staining (Triton lysed exosomes), while no staining was observed in HEK unmodified cells negative for spike protein 1 Figure 5 , upper panel). FITC-duramycin stained unmodified exosomes and spike protein 1 + exosomes, no staining in Triton treated exosomes Figure 5 , lower panel).

[0199] Example 6

[0200] FITC labeling of duramycin was performed as described in Example 1. EVs were enriched using ultracentrifugation. In a first method, MCF-7 cell culture supernatant was subjected to two centrifugations: the first centrifugation was performed at 1200 x g, 20°C for 15 minutes. The supernatant was then centrifuged at 14000 x g, 4°C for 35 minutes. The resulting microvesicles pellet was resuspended in PBS and subjected to flow cytometry analysis Figure 6 , left panel; MV: microvesicle fraction). In a second method, the supernatant of the second centrifugation step of the first method was subjected to ultracentrifugation (100000 x g, 10°C, 90 minutes). The pellet was resuspended in PBS and subjected to flow cytometry analysis Figure 6 , right panel; Exo, UZ: exosome fraction).

[0201] Samples of both methods were stained with FITC labeled duramycin and signal intensity was compared using flow cytometry.

[0202] Results are shown in Figure 6The signal intensity in the microvesicle fraction (MV) was lower when the sample was centrifuged twice (left panel). Additional ultracentrifugation of the supernatant purified the exosomes, which significantly increased the signal intensity of FITC-duramycin in the flow cytometry analysis (right panel), indicating that the degree of staining of exosomes with FITC-duramycin was significantly higher than for other microvesicles.

[0203] Example 7

[0204] Extracellular vesicles (EVs) from MCF-7 cell culture supernatant were stained with FITC-duramycin and CD9-PE. While duramycin stains all vesicles, the tetraspanin CD9 is not expressed on the cell surface of all cells and thus not all vesicles are positive for CD9. The stained samples were subjected to flow cytometry analysis, analyzed using dot plots and individual populations were identified by gating. In theory, up to three populations can be distinguished during sorting: CD9-PE single positive vesicles (CD9 + ), duramycin single positive vesicles (duramycin + ) and CD9 + duramycin+ double positive vesicles. A "two-way sorting" was performed, i.e. only two populations were sorted: CD9 + single positive vesicles and CD9 + duramycin+ double positive vesicles. The sorted samples were analyzed again using a flow cytometer (SA3800; Sony). Here, the authenticity of the signal was checked by pre-incubating the samples with Triton before flow cytometry analysis, which solubilizes the EVs, resulting in a decrease of the signal. If the sorted events are authentic vesicles, no signal can be detected anymore after Triton incubation.

[0205] Analysis of the CD9-PE signal in all samples showed that the amount of CD9 + counts / ml was high in the double positive sorted sample, which was abolished by triton treatment. The sample that was only sorted for CD9 + signal showed counts / ml that were roughly similar to the triton treated sample, indicating the presence of antibody aggregates.

[0206] Figure 7 It is shown that exosomes detected with FITC-duramycin are also positive for CD9-PE. No significant amount of exosomes was detected that were only positive for CD9-PE and not duramycin. Triton treatment, which solubilizes the exosomes, did not result in any (significant) detection (negative control), proving the specificity of the results. A detailed description of the experimental setup is provided below.

[0207] The sorted samples were diluted in filtered PBS and particle size distribution analysis was performed using nanoparticle tracking analysis (NTA) with ZetaView PMX110 from Particle Metrix. Each sample was measured six times as technical replicates. The chart shows the particle size distribution of Durablemycin + Sorting of vesicles Figure 8 A) and Durablemycin + CD9 + Sorting of vesicles Figure 8 B) with an average diameter in the range of 75 nm to 200 nm for both samples.

[0208] 1) Preparation of EVs from MCF-7 cell culture supernatant

[0209] The complete medium (RPMI + 10% FCS + 1% Pen / Strep) was removed from the cells and replaced by RPMI medium without supplements. After 24 hours of exchange, the cell culture supernatant was removed from the cells. While adding the complete medium to the cultured cells again, the removed cell culture supernatant (total of 10 ml) was centrifuged at 2000 x g, 21 °C for 15 minutes. The supernatant was transferred to 2 ml reaction tubes and centrifuged at 10000 x g, 4 °C for 10 minutes.

[0210] 2) Staining of samples for sorting

[0211] 10 ml of MCF-7 cell culture supernatant were divided into two samples (2 x 5 ml). Sample 1 contained 5 ml of MCF-7 cell culture supernatant, which was stained with 100 ng / μl FITC-Durablemycin (batch 9) and CD9 PE (1 ng / μl). Sample 2 contained 5 ml of MCF-7 cell culture supernatant, which was stained with 100 ng / μl FITC-Durablemycin (batch 9). Both samples were incubated for 30 minutes at room temperature in the dark. Three centrifuge tubes with 100 μl PBS (PAN Technology) were prepared.

[0212] 3) Sorting

[0213] Sorting was performed using Astrios Sorter (MoFLo) from Beckman Coulter. The samples were sorted in "Purification mode".

[0214] Sample 1 (for FITC-Durablemycin and CD9 PE staining):

[0215] Falcon 1 : for sorting CD9 PE + Events

[0216] Falcon 2: for sorting FITC-duramycin + CD9 PE + Events

[0217] Sample 2 (stained for FITC-duramycin):

[0218] Falcon 3: for sorting FITC-duramycin + Events

[0219] The results are shown in the following table:

[0220]

[0221] 4) Triton treatment

[0222] Sorted samples as well as unsorted samples were incubated with Triton. 18 mΐ of each sorted sample was incubated with 2 mΐ of 10% Triton or as a control with 2 mΐ of 0.9% NaCI at room temperature (22°C) for 1 hour on a shaker (500 rpm). For unsorted samples, 45 mΐ of MCF-7 cell culture supernatant was incubated with 5 mΐ of 10% Triton at room temperature (22°C) for 1 hour on a shaker (500 rpm).

[0223] 5) Staining of samples with antibodies

[0224] 20 mΐ of unsorted samples were stained with 100 ng / mΐ FITC-duramycin and CD9 PE (1 ng / mΐ) as single and double staining. 20 mΐ of FITC-duramycin + CD9 PE + Sorted samples were not stained; CD9 PE + Sorted samples were stained with FITC-duramycin (100 ng / mΐ); FITC-duramycin + sorted samples were stained with CD9 PE (1 ng / mΐ). Samples were incubated for 30 minutes at room temperature in the dark. Unsorted samples were finally diluted 1 : 100 with 0.9% NaCI by a two-step method: 90 mΐ of 0.9% NaCI was added to each sample, then the samples were resuspended and 20 mΐ was added to 180 mΐ of 0.9% NaCI and counting beads (beads / 50 mΐ: 0.5 x 10 5 Sorted samples were diluted 1 : 10 with 0.9% NaCI by adding 180 mΐ of NaCI and counting beads to each sample. Sample measurements were performed using a flow cytometer SA3800 (Sony).

[0225] 6) NTA (nanoparticle tracking analysis) measurement

[0226] All measurements were performed in a total volume of 1 ml. The following samples were measured using Particle Metrix's Zetaviewer.

[0227] a) PBS buffer (137mM sodium chloride, 2.7mM potassium chloride and 12mM phosphate buffer, pH 7.4 (PANBiotec)); undiluted

[0228] b) Undiluted RPMI serum without supplementation

[0229] c) MCF-7 cell culture supernatant (unsorted); diluted 1:5 with PBS

[0230] d) Sorting samples (adjust each sample to a total volume of 1 ml with PBS)

[0231] CD9 PE+: 100µl sample + 900µl PBS

[0232] FITC-Noperycin+: 82.6 µl sample + 9174 µl PBS

[0233] CD9 PE+ FITC-Nopermycin+: 400µl sample + 600µl PBS

[0234] Each sample was measured six times to calculate the average. Results are as follows: Figure 7 and Figure 8 As shown.

[0235] Example 8

[0236] Labeling and ApoE staining of plasma-derived exosomes

[0237] Plasma was obtained from healthy donors at 1 hour, 3 hours, and 4 hours after consuming a high-fat diet, as well as from fasted donors. Serum samples were prepared by centrifuging the serum samples at 2000 xg at 20°C for 15 minutes. Plasma samples were obtained by centrifuging the samples at 2000 xg at 20°C for 15 minutes; transferring the samples to new reaction tubes and centrifuging them at 2000 xg at 20°C for 15 minutes; transferring the samples to 2 ml reaction tubes and centrifuging the 2 ml reaction tubes at 10,000 xg at 4°C for 10 minutes.

[0238] The table below shows the detection reagents used for staining.

[0239]

[0240] All plasma samples were pre-diluted 1 :10, followed by a Triton incubation step for one hour: 90 mI pre-diluted plasma + 10 mI 10% Triton or 10 mI NaCI were centrifuged at 450 rpm, 21 °C. Samples were stained with 1 ng / mI ApoE binding 10 ng / mI ELAEXIA (Exosome Selective Label for Identification and Analysis; in this example Dursban and FITC) and incubated for 30 minutes in the dark at 21 °C. Subsequently, samples were diluted 1 :100, resulting in a final dilution of the plasma of 1 :1000 (1 :100 dilution by two steps; NaCI in the second step contained Count Bright Absolute Counting Beads, ThermoFisher, Cat. #C36950, Lot.: 2466363) to quantify exosomes. Figure 10 Results of the analysis by flow cytometry are shown (F = fasting; 1 hour, 3 hours, 4 hours = 1 hour, 3 hours and 4 hours after intake of a high fat meal, respectively). The upper panel shows ApoE and ELAEXIA staining of plasma in the fasting state and 1 hour, 3 hours and 4 hours after intake of food. Exosomes positive for ELAEXIA are not stained with ApoE (the upper right quadrant is empty), the lower panel shows the negative control, i.e. samples treated with Triton to solubilize exosomes.

[0241] Figure 11 ELAEXIA staining of plasma in the fasting state and after intake of food (non-fasting) of one healthy donor is shown. NaCI = negative control staining; Donor 2_nF = ELAEXIA staining of exosomes in the non-fasting state; Donor 2_F = ELAEXIA staining of exosomes in the fasting state; Donor 2_nF + Triton and Donor 2_F + Triton = negative control of the same samples, but pre-treated with Triton to solubilize exosomes, which interferes with the staining.

[0242] Example 9

[0243] Cinnamycin staining of exosomes

[0244] Cinnamycin was FITC labeled as described in Example 1. The samples were prepared from MCF-7 cell supernatant cultured in RPMI. The MCF-7 cell culture supernatant containing EVs was prepared by removing the complete culture medium (RPMI + supplements + 10% FCS + 1% penicillin / streptomycin) from the cells and replacing it with RPMI medium without any supplements. After 24 hours of replacement, the cell culture supernatant was removed from the cells. The removed cell culture supernatant was centrifuged at 2000 x g, 21 °C for 15 minutes. The centrifuged supernatant was transferred to 2 ml reaction tubes and centrifuged at 10000 x g, 4 °C for 10 minutes. 225 mI of cell culture supernatant or cell culture medium was incubated with 25 mI of 10% Triton at room temperature (22 °C) on a shaker (500 rpm) for 1 hour. The samples were stained as follows: Cinnamycin batch: 1 ng / mI or 10 ng / mI; CD9 PE: 1 ng / mI. While staining, the samples were incubated at 22 °C in the dark for 30 minutes and diluted 1 : 100 before flow cytometry measurement using a flow cytometer SA3800 (Sony, exosome settings).

[0245] Figure 12 Results of staining with FITC labeled cinnamycin (ELAEXIA) are shown Figure 12 The upper row; 10 ng / ml cinnamycin left panel, 1 ng / ml cinnamycin right panel). Subsequently, the cinnamycin positively stained exosomes were additionally stained with an anti-CD9 antibody to show that the method purifies exosomes, since the exosomes of the cell line MCF-7 are CD9 + positive Figure 12 (lower row). The results show that the degree of staining of exosomes with fluorescently labeled cinnamycin is similar to that of fluorescently labeled duramycin.

[0246] References

[0247]

[0248]

[0249]

Claims

1. A method for detecting and / or quantifying exosomes in a sample, the method comprising the steps of: i) providing a sample known to comprise or suspected of comprising exosomes; ii) contacting a binding agent that specifically binds to the membrane of extracellular vesicles with the sample; and iii) detecting and / or quantifying exosomes and optionally isolating exosomes by flow cytometry and / or super resolution microscopy; wherein the binding agent comprises a label and a phosphatidyl ethanolamine binding lantibiotic, wherein the label is preferably selected from the group consisting of an enzymatic label, a fluorochrome, a peptide label, a radioactive label, a ligand, a magnetic or paramagnetic label and a quantum dot, and wherein the lantibiotic is preferably selected from the group consisting of duramycin and cinnamycin.

2. The method according to claim 1, wherein the lantibiotic is cinnamycin.

3. The method according to claim 1 or 2, wherein: (a) the binding agent is bound by another binding agent that specifically binds to the binding agent or a label thereof; or (b) the binding agent is bound to a solid surface.

4. The method according to any one of claims 1 to 3, wherein the sample is a cell culture supernatant, a cell preparation obtained by lysing and / or centrifuging cells, or a body fluid.

5. The method according to claim 4, wherein the body fluid is selected from the group consisting of plasma, serum, ascites, cerebrospinal fluid, bone marrow, urine, feces and bronchoalveolar lavage fluid.

6. The method according to any one of claims 1 to 5, wherein the method further comprises one or more than one of the following steps prior to step i): a) suspending and / or dissolving the sample; b) ultrafiltrating and / or centrifuging the sample; and / or c) enriching the concentration of exosomes in the sample by size centrifugation and / or density centrifugation.

7. The method according to any one of claims 1 to 6, wherein the sample is obtained from a subject known to have or suspected of having a disease, wherein the method further comprises comparing the number of exosomes in the sample of the subject known to have or suspected of having a disease to the number of exosomes known to be present in a sample of a healthy subject, wherein an increase in the number of exosomes in the sample of the subject known to have or suspected of having a disease is indicative of the presence or stage of the disease, preferably wherein an increase in the number of exosomes in the sample of the subject known to have or suspected of having a disease is indicative of the presence or stage of cancer.

8. The method according to claim 7, wherein comparing the number of exosomes comprises applying CD profiling and t-SNE analysis, NTA tracking, Zetasizer and applying counting beads by flow cytometry.

9. The method according to any one of claims 1 to 8, further comprising analyzing surface markers of the exosomes and / or contents of the exosomes, preferably wherein the surface markers of the exosomes and / or contents of the exosomes comprise one or more than one of a peptide, a protein, a microRNA, DNA and / or RNA, more preferably wherein analyzing the contents of the exosomes comprises DNA mutation analysis, RNA expression, DNA methylation quantification and / or protein expression.

10. A method for diagnosing cancer, comprising: a) detecting and isolating exosomes from a sample by the method of any one of claims 1 to 9, and b) detecting those exosomes presenting cancer antigens among the collection of isolated exosomes obtained in step a).

11. A method for detecting or diagnosing a viral disease, comprising: a) detecting and isolating exosomes from a sample by the method of any one of claims 1 to 9, and b) detecting those exosomes presenting viral antigens among the collection of isolated exosomes obtained in step a), preferably wherein the viral antigen is a viral surface protein, more preferably a spike protein, most preferably a spike protein of a SARS-CoV-2 virus or a SARS-CoV-1 virus or a spike protein of any other virus.

12. A method for quantifying and / or qualifying tumor-related exosomes in a sample, said method comprising the steps of: a) detecting and isolating exosomes from a sample by the method of any one of claims 1 to 9; and b) detecting those tumor-related exosomes among the collection of isolated exosomes obtained in step a) by using at least one binding agent specifically binding to a tumor antigen.

13. A method for monitoring tumor growth, said method comprising the steps of: periodically quantifying the number of tumor-related exosomes in a sample using the method of claim 12, wherein an increase in the number of tumor-related exosomes between two quantifications is indicative of tumor growth.

14. A method for monitoring a viral disease, said method comprising the steps of: periodically quantifying the number of viral-related exosomes in a sample using the method of any one of claims 1 to 9 or claim 11.

15. A kit for carrying out the method of any one of claims 1 to 14, comprising: (i) a binding agent comprising a label and a phosphatidyl ethanolamine binding lanthanolide, wherein the label is preferably selected from an enzymatic label, a fluorochrome, a peptide label, a radioactive label, a ligand, a magnetic label or a quantum dot, and wherein the lanthanolide is preferably selected from duramycin and cinnamycin, more preferably wherein the lanthanolide is cinnamycin; and (ii) instructions for binding the binding agent to exosomes in a sample and applying flow cytometry and / or super resolution microscopy for detecting, quantifying, enriching or producing exosomes.

Images